GB2443570A

GB2443570A - Identifying parent ions of interest in mass spectrometry

Info

Publication number: GB2443570A
Application number: GB0801092A
Authority: GB
Inventors: Robert Harold Bateman
Original assignee: Micromass UK Ltd
Current assignee: Micromass UK Ltd
Priority date: 2005-11-23
Filing date: 2006-11-23
Publication date: 2008-05-07
Anticipated expiration: 2026-11-23
Also published as: JP4959713B2; GB0816196D0; GB0801092D0; WO2007060437A3; CA2835042A1; EP1952425A2; CA2628927C; JP2009516843A; CA2628927A1; GB0623377D0; GB2435714A; GB2435714B; GB2451963A; GB2443570B; GB2451963B; GB0523811D0; WO2007060437A2

Abstract

A mass spectrometer and method of mass spectrometry are disclosed, wherein an Electron Collision Dissociation (ECD), Electron Transfer Dissociation (ETD), Surface Induced Dissociation (SID), or other type of fragmentation/reaction device is repeatedly switched between a high fragmentation mode and a low fragmentation mode. Parent or precursor ions from a first sample are passed through the device and parent/precursor ion mass spectra and daughter/fragment/adduct/product ion mass spectra are obtained. Precursor or parent ions from a second sample are then passed through the device and a second set of precursor/parent ion mass spectra and product/adduct/fragment/daughter ion mass spectra are obtained. The mass spectra are then compared and if either certain parent ions or certain fragmentation ions in the two samples are expressed differently then further analysis is performed to seek to identify the ions which are expressed differently in the two different samples.

Description

MASS SPECTROMETER

The present invention relates to a method of mass spectrometry and a mass spectrometer It has become common practice to analyse proteins by first enzymatically or chemically digesting the protein and then analysing the peptide products by mass spectrometry. The mass spectrometry analysis of the peptide products normally entails measuring the mass of the peptide products. This method is sometimes referred to as "peptjde mapping" or "peptide fingerprinting..

It is also known to induce parent or precursor peptide ions to fragment and to then measure the mass of one or more fragment or daughter ions as a way of seeking to identify the parent or precursor peptide ion. The fragmentation pattern of a peptide ion has also been shown to be a successful way of distinguishing isobarjc peptide ions. Thus the mass to charge ratio of one or more fragment or daughter ions may be used to identify the parent or precursor peptide ion and hence the protein from which the peptide was derived. In some instances the partial sequence of the peptide can also be determined from the fragment or daughter ion spectrum. This information may be used to determine candidate proteins by searching protein and genomic databases.

Alternatively, a candidate protein may be eliminated or confirmed by comparing the masses of one or more observed fragment or daughter ions with the masses of fragment or daughter ions which might be expected to be observed based upon the peptide sequence of the candidate protein in question. The confidence in the identification increases as more peptide parent or precursor ions are induced to fragment and their fragment masses are shown to match those expected.

It is desired to provide an improved method of mass spectrometry and an improved mass spectrometer.

According to an aspect of the present invention there is provided a method of mass spectrometry comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device comprising an Electron Capture Dissociation fragmentation device; repeatedly switching, altering or varying the Electron Capture Dissociation fragmentation device between a first mode wherein at least some of the parent or precursor ions from the first sample are fragmented upon interacting with electrons to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device comprising an Electron Capture Dissociation fragmentation device; repeatedly switching, altering or varying the Electron Capture Dissociation fragmentation device between a first mode wherein at least some of the parent or precursor ions from the second sample are fragmented upon interacting with electrons to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; automatically determining the intensity of first parentor precursor ions from the first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from the second sample which have the same first mass to charge ratio; and comparing the intensity of the first parent or precursor ions with the intensity of the second parent or precursor ions; wherein if the intensity of the first parent or precursor ions differs from the intensity of the second parent or precursor ions by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest.

According to the preferred embodiment the parent or precursor ions comprise doubly, triply, cjuadruply charged ions or ions having five or more charges.

According to the preferred embodiment the Electron Capture Dissociation fragmenj0 device is Preferably repeatedly switched between the first and second modes during a single experimental run or during a single analysis of a sample.

In the first mode of operation the electrons Preferably have an energy selected from the group Consisting of: (i) < 1. eV; (ii) 1-2 eV; (iii) 2-3 eV; (iv) 3-4 eV; and (v) 4-S eV. In the first mode of operati the relatively low energy electrons are Preferably confined by a relatively strong magnetj field. The ions to be fragmented are Preferably confined within an ion guide. An AC or RF voltage is Preferably applied to the electrodes of the lOfl guide in order to create a radial pseudo_potential field or well which Preferably acts to confine IOflS radially Within the ion guide.

The relatively low energy electrons are Preferably confined by a magnetj field which Preferably overlaps or superimposes the ion guiding region of the ion guide so that multiply charged analyte ions are caused to interact with the relatively low energy electrons.

Fragmentaj0 of ions by Electron Capture Dissociation Preferably does not involve causing internal vibrational energy to be introduced to the ions.

The method Preferably further comprises providing an electron Source. In the first mode of operation the electron source Preferably generates a Plurality of electrons which are arranged to interact with the parent or precursor ions. In the second mode of operation the electron source is Preferably Switched OFF so that analyte ions Preferably do not interact with any electrons and hence Preferably are not caused to fragment.

According to an aspect of the present invention there is provided method of mass spectrometry comprising: passing parent or precursor ions from a first sample to a collision fragmentaj0 or reaction device comprising an Electron Transfer Dissociation fragmentaj0 device; repeatedly switching, altering or varying the Electron Transfer Dissociation fragmentatj0 device between a first mode wherein at least some of the parent or precursor ions from the first sample are fragmented upon interacting with reagent ions to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device comprising an Electron Transfer Dissociation fragmentation device; repeatedly switching, altering or varying the Electron Transfer Dissociation fragmentation device between a first mode wherein at least some of the parent or precursor ions from the second sample are fragmented upon interacting with reagent ions to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; automatically determining the intensity of first parent or precursor ions from the first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from the second sample which have the same first mass to charge ratio; and comparing the intensity of the first parent or precursor ions with the intensity of the second parent or precursor ions; wherein if the intensity of the first parent or precursor ions differs from the intensity of the second parent or precursor ions by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest.

According to the preferred embodiment the parent or precursor ions comprise doubly. triply, quadruply charged ions or ions having five or more charges.

According to the preferred embodiment the Electron Transfer Dissociation fragmentation device is preferably repeatedly switched between the first and second modes during a single experimental run or during a single analysis of a sample.

According to an aspect of the present invention there is provided a method of mass spectrornetry comprising: passing parent or precursor ions from a first sample to a collisj0, fragmentaj0 or reaction device comprising a Surface Induced Dissociation fragmentj0 device; repeatedly Switching, altering or varying the Surface Induced Dissociation fragmenttj0 device between a first mode wherein at least some of the parent or precursor ions from the first sample are fragmen upon impinging upon a surface or target plate to produce fragme or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmen.

Passing parent or precursor ions from a second sample to a collision fragmentaj0 or reaction device comprising a Surface Induced Dissociation fragmenj0 device; repeatedly SWitching, altering or varying the Surface Induced Dissociation fragmentj0 device between a first mode wherein at least Some of the parent or precursor ions from the second sample are fragmented upon impingjg upon a surface or target plate to produce fragme or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented.

automatically determining the intensity of first parent or precursor IOns from the first Sample which have a first ma to charge ratio; automatically determining the intensity of second parent or precursor ions from the second sample Which have the same first mass to charge ratio; and comparing the intensity of the first parent or precursor ions with t1e intensity of the second parent or precursor Ions; Wherein if the intensity of the first parent or precursor ions differs from the intensity of the second parent or precursor ions by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are Considered to be parent or precursor loris of interest.

According to the preferred embodiment the parent or precursor ions comprise doubly, triply, uadruply charged Ions or ions having five or more charges.

According to the preferred embodiment the Surface Induced Dissociation fragmentation device is preferably repeatedly switched between the first and second modes during a single experimental run or during a single analysis of a sample.

In the first mode of operation the parent or precursor ions are preferably directed, diverted or deflected on to the surface or target plate. In the second mode of operation the parent or precursor ions are preferably not directed, diverted or deflected on to the surface or target plate.

The surface or target plate preferably comprises a self-assembled monolayer. The surface or target plate preferably comprises a fluorocarbon or hydrocarbon monolayer.

The surface or target plane is preferably arranged in a plane which is substantially parallel to the direction of travel of the parent or precursor ions in the second mode of operation i.e. when ions are preferably transmitted past the surface or target plate without being directed on to the surface or target plate.

According to another aspect of the present invention there is provided a method of mass spectrometry comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying the collision, fragmentation or reaction device between a first mode wherein at least some of the parent or precursor ions from the first sample are fragmented or reacted to produce fragment, daughter, product or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying the collision, fragmentation or reaction device between a first mode wherein at least some of the parent or precursor ions from the second sample are fragmented or reacted to produce fragment, daughter, product or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; automatically determining the intensity of first parent or precursor ions from the first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from the second sample which have the same first mass to charge ratio; and Comparing the intensity of the first parent or precursor ions with the intensity of the second parent or precursor ions; wherein if the intensity of the first parent or precursor ions differs from the intensity of the second parent or precursor ions by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest; wherein the collision, fragmentation or reaction device selected from the group Consisting of: (i) an Electron Collision or Impact Dissociation fragmentation device; (ii) a Photo Induced Dissociation ("PID") fragmentation device; (iii) A Laser Induced Dissociation fragmentation device; (iv) an infrared radiation induced dissociation device; (v) an ultraviolet radiation induced dissociation device; (vi) a nozzle-skimmer interface fragmentation device; (vii) an in-source fragmentation device; (viii) an ion-source Collision Induced Dissociation fragmentation device; (ix) a thermal or temperature source fragmentation device; Cx) an electric field induced fragmentation device; (xi) a magnetic field induced fragmentation device; (xii) an enzyme digestion or enzyme degradation fragmentation device; (xiii) an ion-ion reaction fragmentation device; (xiv) an ion-molecule reaction fragmentation device; (xv) an ion-atom reaction fragmentation device; (xvi) an ion-metastable ion reaction fragmentation device; (xvii) an ion-metastable molecule reaction fragmentation device; (xviii) an ion-metastable atom reaction fragmentation device; (xix) an ion-ion reaction device for reacting ions to form adduct or product ions; (xx) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxi) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxii) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; and (xxiv) an ionmetastable atom reaction device for reacting ions to form adduct or product ions.

According to the preferred embodiment the parent or precursor ions comprise doubly, triply, quadruply charged ions or ions having five or more charges.

According to the preferred embodiment the collision, fragmentation or reaction device is preferably repeatedly switched between the first and second modes during a single experimental run or during a single analysis of a sample.

According to another aspect of the present invention there is provided a method of mass spectrometry comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device comprising an Electron Capture Dissociation fragmentation device; repeatedly switching, altering or varying the Electron Capture Dissociation fragmentation device between a first mode wherein at least some of the parent or precursor ions from the first sample are fragmented upon interacting with electrons to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device comprising an Electron Capture Dissociation fragmentation device; repeatedly switching, altering or varying the Electron Capture Dissociation fragmentation device between a first mode wherein at least some of the parent or precursor ions from the second sample are fragmented upon interacting with electrons to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; automatically determining the intensity of first parent or precursor ions from the first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from the second sample Which have the same first mass to charge ratio; determining a first ratio of the intensity of the first parent S or precursor ions to the intensity of other parent or precursor ions in the first sample; determining a second ratio of the intensity of the second parent or precursor ions to the intensity of other parent or precursor ions in the second sample; and comparing the first ratio with the second ratio.; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor 1Os are Considered to be parent or precursor ions of interest According to the preferred embodiment the parent or precursor ions comprise doubly, triply Quadruply charged ions or ions having five or more charges.

According to the preferred embodiment the Electron Capture Dissociation fragmentj0 device is Preferably repeatedly switched between the first and second modes during a Single experimental run or during a single analysis of a sample.

In the first mode of operation the electrons preferably have an energy selected from the group consisting of: (1) < 1 eV; (ii) 1-2 eV; (iii) 2-3 eV; (iv) 3-4 eV; and (v) 4-5 eV.

In the first mode of operation the electrons are Preferably

confined by a magnetj field.

The method Preferably further comprises Providing an electron Source In the first mode of operation the electron Source Preferably genera5 a Plurality of electrons Which are arranged to interact with the parent or precursor ions. In the second mode of operation the electron source is Preferably Switched OFF.

According to another aspect of the present invention there is provided a method of mass spectromet comprising.

-10 -passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device comprising an Electron Transfer Dissociation fragmentation device; repeatedly switching, altering or varying the Electron Transfer Dissociation fragmentation device between a first mode wherein at least some of the parent or precursor ions from the first sample are fragmented upon interacting with reagent ions to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device comprising an Electron Transfer Dissociation fragmentation dvice; repeatedly switching, altering or varying the Electron Transfer Dissociation fragmentation device between a first mode wherein at least some of the parent or precursor ions from the second sample are tragmented upon interacting with reagent ions to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; automatically determining the intensity of first parent or precursor ions from the first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from the second sample which have the same first mass to charge ratio; determining a first ratio of the intensity of the first parent or precursor ions to the intensity of other parent or precursor ions in the first sample; determining a second ratio of the intensity of the second parent or precursor ions to the intensity of other parent or precursor ions in the second sample; and comparing the first ratio with the second ratio; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest.

-11 -According to the preferred embodiment the parent or precursor ions comprise doubly, triply, quadruply charged ions or ions having five or more charges.

According to the preferred embodiment the Electron Transfer Dissociation fragmentaj0 device is Preferably repeatedly switched between the first and second modes during a single experimental run or during a single analysis of a sample.

According to another aspect of the present invention there is provided amethod of mass spectrometry comprising: Passing parent or precursor ions from a first sample to a collision fragmentatj0 or reaction device comprising a Surface Induced Dissociation fragmenaj0 device; repeatedly Switching, altering or varying the Surface Induced Dissociation fragmentatj0 device between a first mode wherein at least some of the parent or precursor ions from the first sample are fragmented upon impinging upon a surface or target plate to produce fragment or daught ions and a second mode wherein substantially fewer parent or precursor ions are fragment.

Passing parent or precursor ions from a second sample to a collisj0, fragmentaj0 or reaction device comprising a Surface Induced Dissociation fragmentatj0 device; repeatedly switching, altering or varying the Surface Induced Dissociation fragmentaj0 device between a first mode wherein at least some of the parent or precursor ions from the second sample are tragmene upon impinging upon a surface or target plate to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmefl; automatically determining the intensity of first parent or precursor ions from the first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from the second sample which have the same first mass to charge ratio; determining a first ratio of the intensity of the first parent or precursor ions to the intensity of other parent or precursor ioflS in the first sample; determining a second ratio of the intensity of the second parent or precursor ions to the intensity of other parent or precursor ions in the second Sample; and comparing the first ratio with the second ratio; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or precursor ions and/or the Second parent or precursor ions are Considered to be parent or precursor ions of interest.

According to the preferred embodiment the Suface Induced Dissociation fragmentajo device is Preferably repeatedly switched between the first and second modes during a single experjmen run or during a single analysis of a sample.

The surface or target plate Preferably comprises a self-assembled monolayer The surface or target plate Preferably comprises a fluorocarbon or hydrocarbon monolayer.

The surface or target plane iS preferably arranged in a plane which is substantially parallel to the direction of travel of the parent or precursor ions in the second mode of operation.

According to another aspect of the present invention there is provided a method of mass spectrometry comprising: passing parent or precursor ions from a first sample to a collision, fragmentaj0 or reaction device; repeatedly Switching, altering or varying the Collision, fragmenajo or reaction device between a first mode wherein at -13 -least some of the parent or precursor ions from the first sample are fragmented or reacted to produce fragment, daughter, product or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying the collision, fragmentation or reaction device between a first mode wherein at least some of the parent or precursor ions from the second sample are fragmented or reacted to produce fragment, daughter, product or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; automatically determining the intensity of first parent or precursor ions from the first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from the second sample which have the same first mass to charge ratio; determining a first ratio of the intensity of the first parent or precursor ions to the intensity of other parent or precursor ions in the first sample; determining a second ratio of the intensity of the second parent or precursor ions to the intensity of other parent or precursor ions in the second sample; and comparing the first ratio with the second ratio; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest; wherein the collision, fragmentation or reaction device selected from the group consisting of: (i) an Electron Collision or Impact Dissociation fragmentation device; (ii) a Photo Induced Dissociation ("PID") fragmentation device; (iii) a Laser Induced Dissociation fragmentation device; (iv) an infrared radiation induced dissociation device; (v) an ultraviolet radiation induced -14 -dissociation device; (vi) a flozz1e-skjjer interface fragmentatj device; (vii) an in-source fragmentj0 device; (viii) an ion-source Collision Induced Dissociation fragmentation device; (ix) a thermal or temperature source fragmenaj0 device; (x) an electric field induced fragmentaj device; (xi) a magnetic field induced fragmentation device; (xii) an enzyme digestion or enzyme degradation fragmentation device; (xiii) an ion-ion reaction device; (xiv) an ion-molecule reaction fragmentj0 device; (xv) an ion-atom reaction fragmentat0 device; (xvi) an ion-metastable ion reaction fragmentation device; (xvii) an ion-metastable molecule reaction fragmentaejo device; (Xviii) an iOn-rnetastable atom reaction fragmentaejo device; (xix) an ion-ion reaction device for reacting ions to form adduct or product ions; (xx) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxi) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxii) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-metastable molecule reaction device for reacting ions to form adduct or product OflS; arid (Xxiv) an iOn-metastable atom reaction device for reacting ions to form adduct or product ions.

According to the pref erred embodiment the collision, fragmentajo or reaction device i preferably repeatedly switched between the first and second modes during a single experimental run or during a single analysis of a sample.

A reaction device should be understood as comprising a device wherein ions, atoms or molecules are rearranged or reacted so as to form a new species of ion, atom or molecule. An X-Y reaction fragmentj device should be understood as meaning a device wherein X and I combine to form a product which then fragments. This is different to a fragmenajo device per se wherein ions may be caused to fragment without first forming a product. An X-Y reaction device should be understood as meaning a device wherein X and I combine to form a product and wherein the product does not necessarily then fragment.

According to the present invention ions are collided, fragmented or reacted in a device other than a Collision Induced Dissociation fragmentation device. According to a particularly preferred embodiment an Electron Capture Dissociation (ECD") or an Electron Transfer Dissociation (ETD") fragmentation device are used to fragment analyte ions.

Polypeptjde chains are made up of amino acid residues which havecertain masses. There are three different bonds along a peptide backbone and when a bond is broken the charge may remain either at the N-terminal part of the structure or the C-terminal part of the structure. When a polypeptide is fragmented there are six possible fragmentation series which are commonly referred to as: a, b, c and x, y, z. With Collision Induced Dissociation the most common fragmentation route is for fragmentation to occur through the amide bond (II). If the charge remains on the N-terminal then the ion is referred to as a b series ion. If the charge remains on the C-terminal then the ion is referred to as a y series ion.

Subscripts may be used to indicate how many amino acids residues are contained in the fragment. For example, b3 is the fragment ion resulting from cleavage of the amide bond (II) such that charge remains on the N-terminal and wherein there are 3 amino acid residues in the fragment.

According to an embodiment of the present invention when an Electron Capture Dissociation (ECD") or an Electron Transfer Dissociation (ETDN) fragmentation device is used to fragment ions then the polypeptide chain can be fragmented at different positions to those positions where fragmentation would be expected to occur if the polypeptide were fragmented by Collision Induced Dissociation.

In particular, an Electron Capture Dissociation (ECD") or an Electron Transfer Dissociation VETIY) device enable x and c series fragment ions predominantly to be produced. In certain circumstances it is particularly advantageous to cause ions to fragment into x and -16 -c series fragment ions rather than b and y series fragment ions (as would be the case by Collision Induced Dissociation). In some situations a more complete sequence is possible using ECD or ETD and there can also be less ambiguity in identifying fragment ions. This can make the process of sequencing the peptide easier.

Polypepticles may also be modified by Post Translational Modifications such as phosphorylation, The use of an ECD or ETD fragmentation device and the resulting fragmentation series which are produced enables Post Translational Modifications such as phosphory]. atjon to be more easily observed, it is also possible to make a determination as to where the modification occurs along the length of the polypeptide.

According to another embodiment the collision, fragmentation or reaction device may comprise a Surface Induced Dissociation fragmentation device. Collision Induced Dissociation can be viewed as being a relatively slow process in that fragmentation is often the result of multiple Collisions between ions and gas molecules. As a result fragmentation tends to be averaged out and a relatively broad range of fragmentation products are typically observed. In contrast, Surface Induced Dissociation can be viewed as being a relatively sudden or instantaneous process. As a result a polypeptide may fragment in a very specific manner. In certain situations this can be particularly useful since it can reveal certain useful information about the structure of the polypeptide.

It will therefore be appreciated that the present invention is particularly advantageous in that parent or precursor ions are preferably fragmented via different fragmentation routes to those that may be obtained by Collision Induced Dissociation. Furthermore, the present invention also enables Post Translational Modifications of peptides to be observed and a determination to be made as to where the modification sits in the peptide. The present invention is also particularly advantageous compared to conventional approaches to fragmenting analyte ions and attempting to elucidate structural information relating to the analyte ions by analysing the corresponding fragment ions.

-17 -The present invention is therefore particularly advantageous compared to conventional arrangements.

According to the preferred embodiment the method preferably further comprises automatically switching, altering or varying the collision, fragmentation or reaction device between at least the first mode and the second mode at least once every 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 seconds. -Other arrangements are also contemplated wherein instead of determining a first ratio of first parent or precursor ions to other parent or precursor ions, a first ratio of first parent or precursor ions to certain fragment, product, daughter or adduct ions may be determined. Similarly, a second ratio of second parent or precursor ions to certain fragment, product, daughter or adduct ions may be determined and the first and second ratios compared.

The other parent or precursor ions present in the first sample and/or the other parent or precursor ions present in the second sample may either be endogenous or exogenous to the sample. The other parent or precursor ions present in the first sample and/or the other parent or precursor ions present ih the second sample may additionally be used as a chromatographic retention time standard.

According to one embodiment parent or precursor ions, preferably peptide ions, from two different samples are analysed in separate experimental runs. In each experimental run parent or precursor ions are passed to a collision, fragmentation or reaction device. The collision, fragmentation or reaction device is preferably repeatedly switched between a fragmentation or reaction mode and a substantially non-fragmentation or reaction mode. The ions emerging from the collision, fragmentation or reaction device or which have been transmitted through the collision, fragmentation or reaction device are then preferably mass analysed. The intensity of parent or precursor ions having a certain mass to charge ratio in one sample are then compared with the intensity of parent or precursor ions having the same certain mass to charge ratio in the other sample. A direct comparison of the parent or precursor ion expression level may be made or the intensity of parent or precursor ions in a sample may first be compared with an internal standard. An indirect comparison may therefore be made between the ratio of parent or precursor ions in one sample relative to the intensity of parent or precursor ions relating to an internal standard and the ratio of parent or precursor ions in the other sample relative to the intensity of parent or precursor ions relating to preferably the same internal standard. A comparison of the two ratios may then be made.

Although the preferred embodiment is described as relating to comparing the parent or precursor ion expression level in two samples, it is apparent.that the expression level of parent or precursor ions in three or more samples may be compared.

Parent or precursor ions may be considered to be expressed significantly differently in two samples if their expression level differs by more than 1%, 10%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 1000%, 5000% or 10000%.

The collision, fragmentation or reaction device is Preferably maintained at a pressure selected from the group Consisting of: Ci) greater than or equal to 0.0001 mbar; (ii) greater than or equal to 0.001 nibar; (iii) greater than or equal to 0.005 nibar; (iv) greater than or equal to 0.01 mbar; (v) between 0.0001 and 100 mbar; and (vi) between o.ooi and 10 znbar. Preferably, the collision, fragmentation or reaction device is maintained at a pressure selected from the group consisting of: (i) greater than or equal to 0.0001 mbar; (ii) greater than or equal to 0.0005 mbar; (iii) greater than or equal to 0.001 mbar; (iv) greater than or equal to 0.005 mbar; Cv) greater than or equal to 0.01 mbar; (vi) greater than or equal to 0.05 mbar; (vii) greater than or equal to 0.1 mbar; (viii) greater than or equal to 0.5 mbar; (ix) greater than or equal to 1 mbar; (x) greater than or equal to 5 mbar and (xi) greater than or equal to lOmbar.

Preferably, the collision, fragmentaio or reaction device is maintained at a pressure selected from the group consisting of: (1) less than or equal to 10 rnbar; (ii) less than or equal to 5 mbar; (iii) less than or equal to 1 mbar; (iv) less than or equal to 0.5 mbar; Cv) less than or equal, to 0.1 rnbar; (vi) *less than or equal to -19 - 0.05 mbar; (vii) less than or equal to 0.01 mbar; (viii) less than or equal to 0.005 mbar; (ix) less than or equal to 0.001 mbar; (x) less than or equal to 0.0005 mbar; and (xi) less than or equal to 0.0001 mbar.

According to a less preferred embodiment, gas in the collision, fragmentation or reaction device may be maintained at a first pressure when the collision, fragmentation or reaction device is in the high fragmentation or reaction mode and at a second lower pressure when the collision, fragmentation or reaction device is in the low fragmentation or reaction mode. According to another less preferred embodiment, gas in the collision, fragmentation or reaction device may compris a first gas or a first mixture of gases when the collision, fragmentation or reaction device is in the high fragmentation or reaction mode and a second different gas or a second different mixture of gases when the collision, fragmentation or reaction device is in the low fragmentation or reaction mode.

Parent or precursor ions which are considered to be parent or precursor ions of interest are preferably identified.. This may comprise determining the mass to charge ratio of the parent or precursor ions of interest, preferably accurately to less than or equal to 20 ppm, 15 ppm, 10 ppm or 5 ppm. The determined mass to charge ratio of the parent or precursor ions of interest may then be compared with a database of ions and their corresponding mass to charge ratios and hence the identity of the parent or precursor ions of interest can be established.

According to the preferred embodiment the step of identifying the parent or precursor ions of interest comprises identifying one or more fragment, product, daughter or adduct ions which are determined to result from fragmentation of the parent or precursor ions of interest. Preferably, the step of identifying one or more fragment, product, daughter or adduct ions further comprises determining the mass to charge ratio of the one or more fragment, product, daughter or adduct ions to less than or equal to 20 ppm, 15 ppm, 10 ppm or 5 ppm.

-20 -The step of identifying first parent or precursor ions of interest may comprise determining whether parent or precursor ions are observed in a mass spectrum obtained when the collision, fragmentation or reaction device is in the low fragmentation or reaction mode for a certain time period and the first fragment, product, daughter or adduct ions are observed in a mass spectrum obtained either immediately before the certain time period, when the collision, fragmentation or reaction device is in the high fragmentation or reaction mode, or immediately after the certain time period, when the collision, fragmentation or reaction device is in the high fragmentation or reaction mode.

The step of identifying first parent or precursor ions of interest may comprise comparing the elution times of parent or precursor ions with the pseudo-elutjon time of first fragment, product, daughter or adduct ions. The fragment, product, daughter or adduct ions are referred to as having a pseudo-elutjon time since fragment, product, daughter or adduct ions do not actually physically elute from a chromatography column. However, since at least some of the fragment, product, daughter or adduct ions are fairly uniq.e to particular parent or precursor ions, and the parent or precursor ions may elute from the chromatography column only at particular times, then the corresponding fragment, product, daughter or adduct ions may similarly only be observed at substantially the same elution time as their related parent or precursor ions. Similarly, the step of identifying first parent or precursor ions of interest may comprise comparing the elution profiles of parent or precursor ions with the pseudo-elutjon profile of first fragment, product, daughter or adduct ions. Again, although fragment, product, daughter or adduct ions do not actually physically elute from a chromatography column, they can be considered to have an effective elution profile since they will tend to be observed only when specific parent or precursor ions elute from the column and as the intensity of the eluting parent or precursor ions varies over a few seconds so similarly the intensity of characteristic fragment, product, daughter or adduct ions will also vary in a similar manner.

-21 -Ions may be determined to be parent or precursor ions by comparing two mass spectra obtained one after the other, a first mass spectrum being obtained when the collision, fragmentation or reaction device was in a high fragmentation or reaction mode and a second mass spectrum obtained when the collision, fragmentation or reaction device was in a low fragmentation or reaction mode, wherein ions are determined to be parent or precursor ions if a peak corresponding to the ions in the second mass spectrum is more intense than a peak corresponding to the ions in the first mass spectrum. Similarly, ions may be determined to be fragment, product, daughter or adduct ions if a peak corresponding to the ions in the first mass spectrum is more intense than a peak corresponding to the ions in the second mass spectru. According to another embodiment, a mass filter may be provided upstream of the collision, fragmentation or reaction device wherein the mass filter is arranged to transmit ions having mass to charge ratios within a first range but to substantially attenuate ions having mass to charge ratios within a second range and wherein ions are determined to be fragment, product, daughter or adduct ions if' they are determined to have a mass to charge ratio falling within the second range.

The first parent or precursor ions and the second parent or precursor ions are preferably determined to have mass to charge ratios which differ by less than or equal to 40 ppm, 35 ppm, 30 ppm, ppm, 20 ppm, 15 ppm, 10 ppm or 5 ppm. The first parent or precursor ions and the second parent or precursor ions may have been determIned to have eluted from a chromatography column after substantially the same elution time. The first parent or precursor ions may also have been determined to have given rise to one or more first fragment, product, daughter or adduct ions and the second parent or precursor ions may have been determined to have given rise to one or more second fragment, product, daughter or adduct ions, wherein the one or more first fragment, product, daughter or adduct ions and the one or more second fragment, product, daughter or adduct ions have substantially the same mass to charge ratio. The mass to charge ratio of the one or more first fragment, product, daughter or -22 -adduct ions and the one or more second fragment, product, daughter or adduct ions may be determined to differ by less than or equal to 40 ppm, 35 ppm. 30 ppm, 25 ppm, 20 ppm, 15 ppm, 10 ppm or 5 ppm.

The first parent or precursor ions may also be determined to have given rise to one or more first fragment, product, daughter or adduct ions and the second parent or precursor ions may have been determined to have given rise to one or more second fragment, product, daughter or adduct ions and wherein the first parent or precursor ions and the second parent or precursor ions are observed in mass spectra relating todata obtained in the low fragmentation or reaction mode at a certain point in time and the one or more first and second fragment, product, daughter or adduct ions are observed in mass spectra relating to data obtained either immediately before the certain point in time, when the collision, fragmentation or reaction device is in the high fragmentation or reaction mode, or immediately after the certain point in time, when the collision, fragmentation or reaction device is in the high fragmentation or reaction mode.

The first parent or precursor ions may be determined to have given rise to one or more first fragment, product, daughter or adduct ions and the second parent or precursor ions may be determined to have given rise to one or more second fragment, product, daughter or adduct ions if the first fragment, product, daughter or adduct ions have substantially the same pseudo-elution time as the second fragment, product, daughter or adduct ions.

The first parent or precursor ions may be determined to have given rise to one or more first fragment, product, daughter or adduct ions and the second parent or precursor ions may be determined to have given rise to one or more second fragment, product, daughter or adduct ions and wherein the first parent or precursor ions are determined to have an elution profile which correlates with a pseudo-elution profile of a first fragment, product, daughter or adduct ion and wherein the corresponding second parent or precursor ions are determined to have an elution profile which correlates with a pseudo-elution profile of a second fragment, product, daughter or adduce ion.

-23 -According to another embodiment the first parent or precursor ions and the second parent or precursor ions which are being compared may be determined to be multiply charged. Phis may rule out a number of fragment, product, daughter or adduct ions which quite often tend to be singly charged. The first parent or precursor ions and the second parent or precursor ions may according to a more preferred embodiment be determined to have the same charge state. According to another embodiment, the parent or precursor ions being compared in the two different samples may be determined to give rise to fragment, product, daughter or adduct ions which have the same charge state.

The first sample and/or the second sample may comprise a plurality of different biopolymers, proteins, peptides, polypeptides, oligionuc].eot ides, oligionucleos ides, amino acids, carbohydrates, sugars, lipids, fatty acids, vitamins, hormones, portions or fragments of DNA, portions or fragments of cDNA, portions or fragments of RNA, portions or fragments *of mRNA, portions or fragments of tRNA, polyclonal antibodies, monoclonal antibodies, ribonucleases, enzymes, metabolites, polysaccharides, phosphorylated peptides, phosphorylated proteins, glycopeptides, glycoproteins or steroids. The first sample and/or the second sample may also comprise at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 molecules having different identities.

the first sample may be taken from a diseased organism and the second sample may be taken from a non-diseased organism.

Alternatively, the first sample may be taken from a treated organism and the second sample may be taken from a non-treated organism.

According to another embodiment the first sample may be taken from a mutant organism and the second sample may be taken from a wild type organism.

Molecules from the first and/or second samples are preferably separated from a mixture of other molecules prior to being ionised by High Performance Liquid Chromatography (HPLC), anion exchange, anion exchange chromatography, cation exchange, cation exchange chromatography, ion pair reversed-phase chromatography, -24 -chromatography, single dimensional electrophoresis, multi-dimensional electrophoresjs, size exclusion, affinity, reverse phase chromatography, Capillary Electrophoresis Chromatography V CEC"), electrophoresjs, ion mobility separation, Field Asymmetric Ion S Mobility Separation (FAIMS) or capillary electrophoresis.

According to a particularly preferred embodiment the first and second sample ions may comprise peptide ions. The peptide ions preferably comprise the digest products of one or more proteins. An attempt may be made to identify a protein which correlates with parent peptide ions of interest. Preferably, a determination is made as to which peptide products are predicted to be formed when a protein is digested and it is then determined whether any predicted peptide product(s) correlate with parent or precursor ions of interest. A determination may also be made as to whether the parent or precursor ions of interest correlate with one or more proteins.

The first and second samples may be taken from the same organism or from different organisms.

A check may be made to confirm that the first and second parent or precursor ions being compared really are parent or precursor ions rather than fragment, product, daughter or adduct ions. A high fragmentation mass spectrum relating to data obtained in the high fragmentation or reaction mode may be compared with a low fragmentation mass spectrum relating to data obtained in the low fragmentation or reaction mode wherein the mass spectra were obtained at substantially the same time. A determination may be made that the first and/or the second parent or precursor ions are not fragment, product, daughter or adduct ions if the first and/or the second parent or precursor ions have a greater intensity in the low fragmentation mass spectrum relative to the high fragmentation mass spectrum. Similarly, fragment, product, daughter or adduct ions may be recognised by noting ions having a greater intensity in the high fragmentation mass spectrum relative to the low fragmentation mass spectrum.

Parent or precursor ions from the first sample and parent or precursor ions from the second sample are preferably passed to the -25 -same collision, fragmentation or reaction device. However, according to a less preferred embodiment, parent or precursor ions from the first sample and parent or precursor ions from the second sample may be passed to different collision, fragmentation or reaction devices.

According to another aspect of the present invention there is provided a mass spectrometer comprising: an Electron Capture Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon interacting with electrons to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have the same first mass to charge ratio; and (iii) compares the intensity of the first parent or precursor ions with the intensity of the second parent or precursor ions; wherein if the intensity of the first parent or precursor ions differs from the intensity of the second parent or precursor ions by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest.

According to another aspect of the present invention there is provided a mass spectrometer comprising: an Electron Transfer Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon interacting with negatively charged reagent ions to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; a mass analyser; and -26 -a control system which in use: Ci) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have the same first mass to charge ratio; and (iii) compares the intensity of the first parent or precursor ions with the intensity of the second parent or precursor ions; wherein if the intensity of the first parent or precursor ions differs from the intensity of the second parent or precursor ions by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest.

According to another aspect of the present invention there is provided a mass spectrometer comprising: a Surface Induced Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon impinging upon a surface or target plate to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have the same first mass to charge ratio; and (iii) compares the intensity of the first parent or precursor ions with the intensity of the second parent or precursor OflS; wherein if the intensity of the first parent or precursor ions differs from the intensity of the second parent or precursor ions by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest.

-27 -According to an aspect of the present invention there is provided a mass spectrometer comprising: a collision, fragmentation or reaction device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented or reacted into one or more fragment, product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have the same first mass to charge ratio; and (iii) compares the intensity of the first parent or precursor ions with the intensity of the second parent or precursor ions; wherein if the intensity of the first parent or precursor ions differs from the intensity of the second parent or precursor ions by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest; wherein the collision, fragmentation or reaction device selected from the group consisting of: (i) an Electron Collision or Impact Dissociation fragmentation device; (ii) a Photo Induced Dissociation (PIDw) fragmentation device; (iii) a Laser Induced Dissociation fragmentation device; (iv) an infrared radiation induced dissociation device; (v) an ultraviolet radiation induced dissociation device; Cvi) a nozzle-skimmer interface fragmentation device; (vii) an in-source fragmentation device; (viii) an ion-source Collision Induced Dissociation fragmentation device; (ix) a thermal or temperature source fragmentation device; Cx) an electric field induced fragmentation device; (xi) a magnetic field induced fragmentation device; (xii) an enzyme digestion or enzyme degradation fragmentation device; (xiii) an ion-ion reaction fragmentation -28 -device; (xiv) an ion-molecule reaction fragmentation device; (xv) an ion-atom reaction fragmentation device; (xvi) an ion-metastable ion reaction fragmentation device; (xvii) an ion-metastable molecule reaction fragmentation device; (xviii) an ion-metastable atom reaction fragmentation device; (xix) an ion-ion reaction device for reacting ions to form adduct or product IOns; (xx) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxi) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxii) an ion-metastabje ion reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-metastable molecule reaction device forreacting ions to form adduct or product ions; and (xxiv) an ion-metastab].e atom reaction device for reacting ions to form adduct or product ions.

According to another aspect Of the present invention there is provided a mass spectrometer comprising: an Electron Capture Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon interacting with electrons to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have the same first mass to charge ratio; (iii) determines a first ratio of the intensity of the first parent or precursor ions to the intensity of other parent or precursor ions in the first sample; (iv) determines a second ratio of the intensity of the second parent or precursor ions to the intensity of other parent or precursor ions in the second sample; -29 - (v) compares the first parent ratio with the second ratio; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest.

According to an aspect of the present invention there is provided a mass spectrometer comprising: an Electron Transfer Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon interacting with negatively charged reagent ions to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity Of second parent or precursor ions from a second sample which have the same first mass to charge ratio; (iii) determines a first ratio of the intensity of the first parent or precursor ions to the intensity of other parent or precursor ions in the first sample; (iv) determines a second ratio of the intensity of the second parent or precursor ions to the intensity of other parent or precursor ions in the second sample; (v) compares the first ratio with the second ratio; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest.

According to an aspect of the present invention there is provided a mass spectrometer comprising: a Surface Induced Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon impinging upon a surface or target plate to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; S a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have the same first mass to charge ratio; (iii) determines a first ratio of the intensity of the first parent or precursor ions to the intensity of other parent or precursor ions in the first sample; (iv) determines a second ratio of the intensity of the second parent or precursor ions to the intensity of other parent or precursor ions in the second sample; (v) compares the first ratio with the second ratio; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest.

According to an aspect of the present invention there is provided a mass spectrometer comprising: a collision, fragmentation or reaction device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented or reacted into one or more fragment, product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; a mass analyser; and a control system which in use: (1) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; 31 - (ii) determines the intensity of second parent or precursor ions from a second sample which have the same first mass to charge ratio; (iii) determines a first ratio of the intensity of the first parent or precursor ions to the intensity of other parent or precursor ions in the first sample; (iv) determines a second ratio of the intensity of the second parent or precursor ions to the intensity of other parent or precursor ions in the second sample; (v) compares the first ratio with the second ratio; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor ions are considered to be parent or precursor ions of interest; wherein the collision, fragmentation or reaction device selected from the group consisting of: (i) an Electron Collision or Impact Dissociation fragmentation device; (ii) a Photo Induced Dissociation (PID) fragmentation device; (iii) a Laser Induced Dissociation fragmentation device; (iv) an infrared radiation induced dissociation device; (v) an iltravjolet radiation induced dissociation device; (vi) a nozzle-skimmer interface fragmentation device; (vii) an in--source fragmentation device; (viii) an ion-source Collision Induced Dissociation fragmentation device; (ix) a thermal or temperature source fragmentation device; (x) an electric field induced fragmentation device; (xi) a magnetic field induced fragmentation device; (xii) an enzyme digestion or enzyme degradation fragmentation device; (xiii) an ion-ion reaction fragmentation device; (xiv) an ion-molecule reaction fragmentjo device; (xv) an ion-atom reaction fragmentajon device; (xvi) an ion-metastable ion reaction fragmentation device; (xvii) an ionmetastable molecule reaction fragmentation device; (xviii) an ion-znetastable atom reaction fragmentation device; (xix) an ion-ion reaction device for reacting ions to form adduct or product ions; (xx) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxi) an ion-atom reaction device for reacting ions to form adduct or -32 -product IOflS; (xxii) an iOfl.-metastable ion reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; and (xxiv) an iofl-metastable atom reaction device for reacting ions to form adduct or product ions.

The mass spectrometer Preferably further comprises an ion source. The ion Source is preferably selected from the group COnSisting of: (1) an Electrospray ionisatjon (uEsI) ion source; (ii) an Atmospheric Pressure Photo IonjSatjon ("APPI") ion source; (iii) an Atmospheric Pressure Chemical Ionisatjon (APCI") ion source; (iv) a Matrix Assisted Laser Desorptjo Ionisation ("MALDI") ion source; (v) a Laser Desorptjo Ionjsatjon ("LDI") ion source; (vi) an Atmospheric Pressure Ionjsatjon ("API") ion source; (vii) a Desorpt Ionjsatjon on Silicon ("DIOS") ion source; (viii) an Electron Impact ("El") ion Source; (ix) a Chemical iOflisatjon ("CI") ion source; (x) a Field lonisation (FI") ion source; (xi) a Field Desorptjon ("FD") ion source; (xii) an Inductively Coupled Plasma ("ICp") ion source; (xiii) a Fast Atom Bombarcent ("FAB") ion Source; (xiv) a Liquid Secondary Ion Mass Spectrometry ("LSIMS") ion source; (xv) a Desorptjon Electrospray Ionisatjon ("DESI") ion source; (Xvi) a Nickel_63 radioactive ion source; (xvii) an Atmospheric Pressure Matrix Assisted Laser Desorptjon lonisation ion source; and (xviii) a Thermospray ion source.

The ion source may comprise a pulsed or a continuous ion source.

According to a Particularly pref erred embodiment the mass spectrometer may comprise an Electrospray Atmospheric Pressure Chemical Ionisatjon ("APCI-), Atmospheric Pressure Photo lonisation ("APPI"), Matrix Assisted Laser Desorption Iorii$atjon ("MALDI"), Laser Desorption lonisation ("LDI"), Inductively Coupled Plasma ("Icp"), Fast Atom Bon1barent ("FAB") or Liquid Secondary Ions Mass Spectrometry ("LSIMS") ion Source. Such ion sources may be provided with an eluent over a period of time, the eluent having been separated from a mixture by means of liquid chromatography or capillary electrophoresis -33 -Alternatively the mass spectrometer may comprise an Electron Impact ("EI) ChemjcalIonjsatjo ("Cii or Field IOfljSatjon (F1) ion Source. Such ion sources may be provided with an eluent over a period of time, the eluent having been separated from a mixture by means of gas chromatOgrpy The mass analyser Preferably comprises a quadrupo mass filter, a Time of Flight ("T0F) mass analyser (an orthogon acceleration Time of Flight mass analyser is Particularly Preferred), a 2D (linear) or 3D (doughnut shaped electrode with two endcap electrodes) Ion trap, a magnetj sector analyser or a Fourier Transfo Ion Cyclotron Resonance ("FTICR-) mass analyser.

According to an embodiment the mass analyser IS Preferably selected from the group consisting of: (I) a quadrupol mass analyser; (ii) a 2D or linear quadrupol mass analyser; (iii) a Paul or 3D cp.adrupole mass analyser; (iv) a Penning trap mass analyser; (v) an ion trap mass analyser; (vi) a magnej sector mass analyser; (Vii) Io Cyclotron Resonance (JcR) mass analyser; (Viii) a Fourier Transf0 Ion Cyc1otro Resonance (FTICR") mass analyser; (ix) an electrostatic or orbitrap mass analyser; (x) a Fourier Transfoz-rn electrostatic or orbitrap mass analyser; and (xi) a Fourier Pransfo mass analyser; (xii) a Time of Flight mass analyser; (xiii) an orthogo5 acceleration Time of Flight mass analyser; (xiv) an axial acceleration Time of Flight mass analyser; and (xv) a quadrupo rod set mass filter or maSs analyser.

The mass Spectreter Preferably further comprises an ion trap or IOfl guide arranged Upstre and/or downstream of the collision fraginenj0 or reaction device.

The ion trap or ion guide is Preferably selected from the group consisting of: (1) a multjpole rod set or a segmen multjpole rod set ion trap or ion guide comprisjg a quadrupole rod set, a hexapoje rod set, an Octapole rod set or a rod set comprising more.tja eight rods; (ii) an ion tunnel or on funnel ion trap or ion guide comprising a Plurality of electrodes or at least 2, 5, 10, 20, 30, -34 - 40, 50, 60, 70, 80, 90 or 100 electrodes having apertures through which ions are transmitted in use, wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the electrodes have apertures which are of substantially the same Size or area or which have apertures which become Progressively larger and/or smaller in size or in area; (iii) a stack or array of planar, plate or mesh electrodes, wherein the stack or array of planar, plate or mesh electrodes comprises a Plurality or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 planar, plate or mesh electrodes and wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the planar, plate or mesh electrodes are arranged generally in the plane in which ions travel in use; and (iv) an ion trap or ion guide comprising a Plurality of groups of electrodes arranged axially along the length of the ion trap or ion guide, wherein each group of electrodes comprises: (a) a first and a second electrode and means for applying a DC voltage or potential to the first and second electrodes in order to confine ions in a first radial direction within the ion guide; and (b) a third and a fourth electrode and means for applying an AC or RF voltage to the third and fourth electrodes in order to confine ions in a second radial direction within the Ofl guide.

The ion trap or ion guide Preferably comprises an ion tunnel or ion funnel ion trap or lOfl guide wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the electrodes have internal diameters or dimensions selected from the group Consisting of: Ii) = 1.0 mm; (ii) = 2.0 mm; (iii) = 3.0 mm; (iv) = 4.0 mm; lv) = 5.0 mm; (vi) = 6.0 mm; (vii) = 7.0 nun; (viii) = 8.0 mm; (ix) = 9.0 mm; (x) = 10.0 mm; and (xi) > 10.0 mm.

The ion trap or ion guide Preferably further comprises first AC or RF voltage means arranged and adapted to apply an AC or RF voltage to at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the Plurality of -35 -electrodes of the ion trap or ion guide in order to confine ions radially Within the ion trap or ion guide.

The first AC or RF voltage means IS Preferably arranged and adapted to apply an AC or RF Voltage having an 8flPlitude selected from the group COflSistjng of: (I) < 50 V peak to peak; (ii) 50-100 V peak to peak; (iii) 100-150 V peak to peak; (iv) 150-200 V peak to peak; Cv) 200-250 V peak to peak; (vi) 250-300 V peak to peak; (vii) 300-350 V peak to peak; (Viii) 350-400 V peak to peak; (ix) 40O-45 V peak to peak; Cx) 450-500 V peak to peak; and (xi) > 500 V peak to peak.

The first AC or RF Voltage means is Preferably arranged and adapted to apply an AC or RF voltage having a frequency selected from the group COflSIstiflg of: (i) < 100 kHz; (ii) 100-200 kHz (iii) 200- 300 kflz; (iv) 300-400 kHz; (v) 400-500 kflz; (vi) 0.5-1.0 MHz; (vii) 1.0-15 MHz; (viji) 1.5-20 {Z; (ix) 2.0-25 MHz; (x) 2.5-30 MHz; (xi) 3.0-35 Z; (Xii) *5-4 Z; (xlii) 4.0-4.5 Z; (xiv) 4.5- 5.0 Z; (xv) 5.0-iz; (xvi) 5.5-6.0 Z; (xVij) 6.0-65 MHz; (XVIII) 6.5-70 MHz; (Xix) 7.0-7.5 MHz; (xx) 7.5-8 MHz; (xxi) 8.0- 8.5 Z; (XXII) 8.5-90 z; (XXijj) 9.0-95 Z; (XXjv) 9.5-i MHz; and (xxv) > 10.0 MHz.

The ion trap or ion guide is Preferably arranged and adapted to receive a beam or group of Ions and to Convert or partition the beam or group of ions such that a Plurality or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 separate packets of ion5 are confined and/or isolated in the ion trap or ion guide at any particu time, and wherein each packet of ions is separately Confined and/or isolated in a separate axial potentj well formed Within the ion trap or Ion guide.

The mass spec trometer Preferably further comprises means arranged and adapted to urge at least some ions upstre and/or dowristre through or along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of the ion trap or IOn guide in a mode of Operation -36 -The mass spectrometer Preferably further comprises first transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or One or more transient DC voltage or potential waveforms to the electrodes forming the ion trap or io guide in order to urge at least some ions upstream and/or downstream along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of the ion trap or ion guide.

The mass Spectromet Preferably further comprises AC or RF voltage means arranged and adapted to apply two or more phase_shifted AC or RF voltages to electrodes forming the ion trap or ion guide in order to urge at least some ions upstre and/or downstream along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of the ion trap or ion guide.

The mass spectrometer Preferably further comprises means arranged and adapted in a mode of operation to maintain at least a portion of the ion trap or ion guide at a pressure selected from the group Consisting of: (i) > 0.0001 mbar; (ii) > 0.001 mbar; (iii) > 0. 01 ar; (iv) > 0.1 mbar; (v) > 1 mbar; (Vi) > 10 mbar; (vjj) > 1 mbar; (viii) 0.O001_1 mbar; and (ix) 0.001.-lO mbar.

The collision, fragmenaj0 or reaction device may comprise a quadrupo1 rod set, an hexapole rod set, an oCtopoje or higher order rod set or an ion tunnel Comprising a Plurality of electrodes having apertures through which ions are transmitted. The apertures are Preferably substantialiy the same Size. The colljs].on fragmentj0 or reaction device may, more generally, comprise a Plurality of electrodes connected to an AC or RF voltage supply for radially Confining ions within the collision, fragmentj0 or reaction device. An axial DC voltage gradjen may or may not be applied along at least a portion of the length of the ion tunnel Collision, fragmentj0 or reaction device The collision, fragmentation or reaction device may be housed in a housing or otherwise arranged so that a substantially gas-tight enclosure is formed around the collis0, fragmentj0 or reaction device apart from an option -37 -aperture to admit ions and an aperture for ions to exit from and optionally a port for introducing gas. A gas such as helium, argon, nitrog air or methane may be introduced into the collision, fragmentaj0 or reaction device Other arrangem5 are also Contemplated wherein the Collision, fragmentj0 or reaction device is not repeatedly Switched, altered or Varied between a high fragmentaj0 or reaction mode and a low fragmentj0 or reaction mode. For example, the collision fragmentj0 or reaction device may be left Permanently ou and arranged to fragment or react ions received within the collision, fragmentj0 or reaction device. An electrode or Other device may be provided upstream of the collisjo, fraginenj0 or reaction device. A high fragmentj0 or reaction mode of operation would occur when the electrode or other device allowed ions to pass to the co1lj0 fragmentj0 or reaction device. A low fragmentj0 or reaction mode of operation would Occur when the electrode or other device caused ions to by-pass the col1jj0 fragmentaj0 or reaction device and hence notbe fragmen or reacted therein.

Other embodiments are also contemplated which would be useful where particular parent or precursor ions could not be easily observed since they co-eluted with other Commonly observed peptide ions. In such circumstances the expression level of fragment, product, daughter or adduct ions is compared between two samples, According to another aspect of the present invention there is provided a method of mass spectromet comprising: Passing parent or precursor ions from a first sample to a co1].jj0 or reaction device; repeatedly switching, altering or varying the colli0 fragmentj0 or reaction device between a first mode wherein at least some of the parent or precursor ions from the first sample are fragmen or reacted into one or more fragme product daughter or adduct ions and a second mode wherein substantialiy fewer parent Or precursor ions are fragmen or reacted; passing parent or precursor ions from a Second Sample to a co].ljsjofl fragmentj0 or reaction device; -38 -repeatedly Switching, altering or varying the coil isbn, fragmentj0 or reaction device between a first mode wherein at least Some of the parent or precursor ions from the second sample are fragmefl or reacted into one or more fragment, product daughter or adduce ions and a Second mode wherein substantially fewer parent or precursor Ions are fragmefl or reacted; automatically determining the intensity of first fragment, product, daughter or adduct ions derived from first parent or precursor iOfls from the first sample, the first fragment product, daughter or adduct ions having a first mass to charge ratio; automatically detenninjng the Intensity of second fragment, product daughter or adduct ions derived from second parent or precursor Ions from the Second sample the second fragment, product, daughter or adduct ions having the same first mass to charge ratio; and comparing the intensity of the first fragment, product, daught or adduct ions with the intensity of the second fragment, product, daughter or adduct ions; wherein if the intensity of the first fragment, product daughter or adduct ions differs from the intensity of the second.

fragme product daughter or adduct ions by more than a predetermined amount then either the first parent or precursor ions and/or the Second parent or precursor ions are considered to be parent or precursor ions of interest; and wherein the co1lij0 fragmenj0 or reaction device is selected from the group consisting of: Ii) a Surface Induced Dissociation (SID-) fragmentaj0 device; (ii) an Electron Transfer Dissociation fragmenaj0 device; (iii) an Electron Capture Dissociation fragmenj0 device; (iv) an Electron Collision or Impact Dissociation fragmenatj0 device; (v) a Photo Induced Dissociation fragmentaj0 device; (vi) a Laser Induced Dissociation fragmentaj0 device; (vii) an infrared radiation induced dissociation device; (viii) an ultravjoiet radiation induced dissociation device; (ix) a nozzle_skjer interface fragmentaj0 device; (xi an 1-source fragmentaj0 device; (xi) an Ion-source -39 -Col1jj0 Induced Dissociation fragmentj0 device; (Xii) a thermal or temperature Source fragmentj0 device; (Xiii) an electric field induced fragmenj0 device; (xiv) a magnetic field induced fraginentj0 device; (xv) an enzyme digestion or enzyme degradaj0 fragmenj0 device; (xvi) an 1Ofl- jo reaction fragmentaj0 device; (Xvii) an Ion-molecule reaction fraentatjo device; (Xviij) an ion-atom reaction fragmentj0 device; (xjx) an ion_metastable ion reaction fragmentj0 device; (xx) an ion..metastable molecule reaction fraentatjon device; (xxi) an ion_metastable atom reaction fraginentj0 device; (xxii) an IOn-jOfl reaction device for reacting loris to form adduct or product Ions; (xxiii) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxiv) an ion-atom reaction device for reacting ions to form adduct or product Ions; (xxv) an lon-metastable ion reaction device for reacting ions to form adduct or product Ions; (xj) an ion_metastable molecule reaction device for reacting ions to form adduct or product ions; and (xxvii) an lon-metastable atom reaction device for reacting Ions to form adduct or product Ions.

According to another aspect of the present invention there is provided a method of mass SPectrometry Comprising: passing parent or precursor ions from a first Sample to a collision, fragmentaj0 or reaction device; repeatedly Switching, altering or varying the Collision fragmenj0 or reaction device between a first mode wherein at least Some of the parent or precursor ions from the first Sample are fragmen or reacted into one or more fragment product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmefl or reacted; Passing parent or precursor ions from a Second sample to a Collision, fragmenj0 or reaction device; repeatedly Switching, altering or varying the co11ii0 fragmenta0 or reaction device between a first mode wherein at least some of the parent or precursor Ions from the second sample are fragment or reacted into one or more fragment product daughter or -40 -adduct ions and a second mode wherein substantialiy fewer parent or precursor ions are fragmen or reacted; automatically determining the intensity of first fragment, product, daughter or adduct ions derived from first parent or precursor ions from the first sample, the first fragment product daught or adduct ion5 having a first mass to charge ratio; automatically determining the intensity of Second fragment, product daughter or adduct ions derived from second parent or precursor ions from the second sample the second fragment, product daughter or adduct ions having the same first mass to charge ratio; determining a first ratio of the intensity of the first fragment product daughter or adduct ions to the intensity of other parent or precursor ions in the first Sample or with the intensity of other fragment, product daughter or adduct ions derived from other parent or precursor ions in the first sample; determining a second ratio of the intensity of the second fragmen product, daughter or adduct ions to the intensity of other parent or precursor loris in the Second sample or with the intensity of other fragment product daught or adduct ions derived from other parent or precursor ions in the seco sample; comparing the first ratio with the second ratio; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or precursor ions and/or the second parent or precursor Ions are Considered to be parent or precursor ions of interest; and wherein the co1lj.0 fragmentj0 or reaction device is selected from the group COnsisting of: (1) a Surface Induced Dissociation (MSID) fragmentj0 device; (ii) an Electron Transfer Dissociation fragmentj0 device; (iii) an Electron Capture Dissocjat0 fragmentj0 device; (iv) an Electron Collij or Impact Dissociation fragmentj0 device; (v) a Photo Induced Dissociation (UPIDN) fragmenttj0 device; (vi) a Laser Induced Dissociation fragmentj0 device; (vii) an infrared radiation induced dissociation device; (viii) an ultraviolet radiation induced dissociation device; (ix) a nozzle_skjer interface fragmenj0 device; (x) an In-source fragmentaj0 device; (xi) an ion-source Collision Induced Dissociation fragmentaj0 device; (xii) a thermal or temperature Source fragmenj0 device; (xiii) an electric field induced fraginentj0 device; (xiv) a magnej field induced fraginentj0 device; (xv) an enzyme digestjo or enzyme degradation fragmentj0 device; (xvj) an IOfl-jO reaction fragmenaj0 device; (xvii) an ion-molecule reaction fragmentaj0 device; (xviii) an iOn-atom reaction fragmentaj0 device; (xix) an ion-metastable ion reaction fragmenaj0 device; (xx) an lon-metastable molecule reaction fragmentaj0 device; (xxi) an lon-metastable atom reaction fragmentaj0 device; (Xxii) an ion-ion reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-molecule reaction device for reacting ions to form adduct or product Ions; (xxiv) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxv) ion_metastable ion reaction device for reacting ions to form adduct or product ions; (xxvi) an ion-metastable molecule reaction device for reacting ions to form adductor product ions; and (XXVjj) an ion_metastable atom reaction device for reacting ions to form adduct or product ions.

According to another aspect of the present invention there is provided a mass spectrometer comprising: a colij fragmentaj0 or reaction device which is arranged and adapted to be repeatedly switched altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented or reacted into one or more fragment product, daughter or adduct Iâ and a second mode wherein substantially fewer parent or precursor ions are fragmen or reacted.; a mass analyser; and a control system which in use: (1) determines the intensity of first fragment product, daughter or adduct ions derived from first parent or precursor ions from a first sample, the first fragment product, daughter or adduct ions having a first mass to charge ratio; (ii) determines the intensity of second fragment, product, daughter or adduct ions derived from second parent or precursor ions -42 -from a second Sample, the secoj fragment product daughter or adduct loris having the same first mass to charge ratio; and (hi) compares the intensity of the first fragme product, daugh or adduct ions with the intensity of the second fragment product daught oradduct Ions; wherein if the intensity of the first fragme product, daugh or adduct ions differs from the intensity of the second fragine product daugh or adduct Ions by more than a predetermined amount then either the first parent or precursor 1Ofl and/or the Second parent or precursor ions are considered to be parent or precursor ions of interest; and wherein the collI0, fragmentatj0 or reaction device is selected from the group COnSisting of: (i) a Surface Induced Dissociation (SID fragmentj0 device; (ii) an Electron Transfer Dissociation fragmentaj0 device; (Iii) an Electron Capture Dissociation fragmenj0 device; (iv) an Electron Collision or Impact Dissociation fragmentj0 device; (v) a Photo Induced Dissociation (PID') fragmentaj0 device; (vi) a Laser Induced Dissociation fragmenttj0 device; (vu) an infrared radiation Induced dissocjt0 device; (viii) an ultraviolet radiation induced dissociation device; (ix) a nozzle_skjer interface fragmentej0 device; (x) an Iri-Source fragmentj0 device; (xi) an IOn-source Collision Induced Dissociation fragmentaj0 device; (Xii) a thermal or temperature source fragmenta0 device; (Xijj) an electric field induced fragmentj0 device; (Xiv) a magnetic field induced fragmentat0 device; (xv) an enzyme digesj0 or enzyme degradaj0 fragmen8j0 device; (xvi) an IOfl-j0 reaction fragmentaj0 device; (XVII) an ion_molecule reaction fragmentj0 device; (XVIji) an ion-atom reaction fraginentaj0 device; (xix) an ion_metastable ion reaction fragmenj0 device; (x.x) an ion-metastable molecule reaction fragmentaj0 device; (XXI) an ion_metastable atom reaction fragmenj0 device; (xxii) an ion-ion reaction device for reacting ions to form adduct or product ions; (XXij) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxiv) an ion-atom reaction device for reacting ions to form adduct or product -43 -ions; (xxv) an iOfl-metastable ion reaction device for reacting ions to form adduct or product ions; (Xxvi) an iOn-metastable molecule reaction device for reacting ions to form adduct or product ions; and (xxvii) an iofl-rnetastabje atom reactjon device for reacting ions to form adduct or product ions.

According to another aspect of the present invention there is provided a mass spectrometer comprising: a Collision, fragmentatj0 or reaction device repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented into one or more fragment, product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; a mass analyser; and a control system which in use: (i) determines the intensity of first fragment, product, daughter or adduct ions derived from first parent or precursor ions from a first sample, the first fragment, product, daughter or adduct ions having a first mass to charge ratio; (ii) determines the intensity of second fragment, product, daughter or adduct ions derived from second parent or precursor ions from a second sample, the second fragment, product, daughter or adduct ions having the same first mass to charge ratio; (iii) determines a first ratio of the intensity of the first fragment, product, daughter or adduct ions to the intensity of other parent or precursor ions in the first sample or with the intensity of other fragment, product, daughter or adduct iOnS derived from other parent or precursor ions in the first sample; (iv) determines a second ratio of the intensity of the second fragment, product, daughter or adduct ions to the intensity of other parent or precursor ions in the second sample or with the intensity of other fragment, product, daughter or adduct ions derived from other parent or precursor ions in the second sample; and Cv) compares the first ratio with the second ratio; wherein if the first ratio differs from the second ratio by more than a predetermined amount then either the first parent or -44 -precursor Iofl and/or the second parent or precursor ions are Considered to be parent or precursor IOfl of interest, and wherein the co1lj0 fraentati or reacti0 device is selected from the group COnsisting of: (I) a Surface Induced Dissociation (SxD) fraentatjo device; (Ii) an Electron Transfer Dissociatlo fraentatj device; (Iii) an Electron Capture Dissociation fraentatj0 device; (iv) an Electron CO1 Sion or Impact Dissociation fraentati device; (v) a Photo Induced Dissociation (PID) fraentatjon device; (VI) a Laser Iflduced Dissociation fraentatjo device; (vii) an infrared radiation induced dissociation device; (Vljj) an ultraviolet radiation Induced dissociation device; (ix) a flOzzle_ski interface fraentatjo device; (x) an 1flsource fraentatj device; (xi) an ion_source Collision Induced Dissociation fraefltati device; (xii) a theaj or temperature Source fraentatj0 device; (XIII) an electric field induced fraentatjon device; (xiv) a magnetj field induc fraentatjon device; (xv) an enze digesj0 or enze degradj0 fraentatj device; (xvi) an IOfl-j reaction fraentatj device; (XVII) an ion_molecule reacti0 fraentatjo device; (XVIII) an ion-atom reaction fraentation device; (Xix) an ionmetaStab1e ion reaction fragmej0 device; (xx) an ionmetastab1e molecule reacti0 fraentatjo device; (XxI) an ion_metastable atom reaction fraentation device; (xxii) an reaction device for reacting IOfl to fo adduct or product Ions; (XXIII) an ion_molecule reaction device for reacting ions to fo adduct or product Ions; (xxiv) a ion-atom reaction device for reacting IOflS to adduct or product Ions; (xxv) an ion_meta$tabl ion reaction device for reacting Iofl to fo adduct or product ions; (X)Wj) an ion-metastable molecule react10 device for reacting IOnS to fo üdduct or product Ions; and (Xjj; an ion_metastable atom reaction device for reacting Ions to foi adduct or PrOdUCt ions.

It wifl be apparent that the above described embodiments which relate to comparing the expression level of frag1 product daugh or adduct IOfls rather than parent or precursor ions either directly or indirectly may employ the method and apparatus relating to the preferred embodiment Therefore, the seine preferred features Which are recited with respect to the Preferred embodiment may also be used with the embodiments which relate to comparing the expressi level of fragme product, daught or adduct ions.

If parent or precursor ions having a particular mass to charge ratj0 are expres5 differentiy in two different samples then according to the preferred embodiment further investigj0 of the parent or precursor ions of interest then Occurs This further investigatlo may comprIse seeking to identify the parent or precursor lOfl of interest Which re expre55 differently in the two different Samples In order to verify that the parent or precursor ions Whose expression levels are being Compared in the two different samples really are the same ions, a number of checks may be made.

Measurements of changes in the abundance of proteins in complex protein mixtures can be extremely infotive ror example, changes to the abundance of protein5 in cells, Often referred to as the protein expression level, could be due to different cellular stresses the effect of stimuli, the effect of disease or the effect of drugs. Such proteins may provide relevant targe5 for Study, screening or inteefltion The identjfjcatjo of such proteins will a1ly be of interest. Such proteins may be identified by the method of the preferred embodiment Therefore according to the Preferred embodiment a new criterion for the discovery of parent or precursor IOS of interest is based on the antifjcatj of proteins in two different Samples ThIs rejres the deteinatjo of the relative abundances of their peptide products in two or more Samp5 However, the determination of relative abundance requj8 that the same peptj ions must be compared in the two (or more) different Samples and ensuring that this happe5 is a non-trivial problem Hence, it is necessa to be able to recognj5 and Preferably identify the peptide ion to the extent that t ca at least be UniJely recogn5 Within the samp'e Such peptide Ions may be adeateiy recognj$ by measurement of the mass of the parent or precursor ion and by measurement of the mass to charge ratio of one or more fraent product daught or adduct -46 -ions derived from that parent or precursor lOfl. The sPecificity with Which the peptides may be recogni$ may be increased by the determination of the accurate mass of the parent or precursor ion and/or the accurate mass of one or more fragment product daughter or adduct ions.

The same method of recognising parent or precursor ions in one sample is also Preferably used to recognj5 the Same parent or precursor ions in another sample and this enables the relative abundances of the parent or precursor ions in the two different samples to be measured.

Measurement of relative abundances allows discove, of proteins with a signjfjc change or difference in expression level of that protein The same data allows identification of that protein by the method already described in Which several or all fragment product, daughter or adduct ions associated with each such peptjde product ion is discovered by closeness of fit of their respective elutjon times.

Again, the accurate measurent of the masses of the parent or precursor ion and associated fragment product daughter or adduct Ions substantially improves the SPecificjt and confidence with which the protein may be identified.

The specificity with which the peptjc1e may be recognis may also be increased by comparjs of retention times. For example, the HPLC or CE retention or elution times will be measured as part of the procedure for associating fragment, product, daughter or adduct ions with parent or precursor ions, and these elution times may also be compared for the two or more samples The elutjon times may be used to reject measurements where they do not fall within a pre-defined time difference of each other. Alternatively retention times may be used to confirm recognjtjo of the same peptjde when they do fall Within a predefifled window of each other. COmmonly there may be some redundancy if the parent or precursor ion accurate mass, one or more fragment, product daughter or adduct ion accurate masses, and the retention times are all measured and comparj In many Instances just two of these measurements will be adequa to recogflj the same peptide parent or precursor Ion in the two or more samples. For -47 -example measurement of just the accurate parent or precursor ion mass to charge ratio and a fragment, product daughter or adduct ion mass to charge ratio, or the accurate parent or precursor ion. mass to charge ratio and the retention time, may well be adequate.

Nevertheless, the additional measurements may be used to confirm the recognltjon of the same parent peptjde ion.

The relative expression levels of the matched parent peptjde ions may be quantifj by measuring the peak areas relative to an internal standard The preferred embodiment does not require any interruption to the acqujsjj0 of data and hence is Particularly Suitable for quantjLaj applications. According to an embodiment one or more endogeno5 peptides coJpjofl to both mixtures Which are not changed by the experimental state of the samples may used as an internal standard or standards for the relative peak area measurements According to another embodiment an internal standard may be added to each sample where no such internal standard is present or can be relied upon. The internal standard whether naturally present or added, may also serve as a chromatograpj retention time standard as well as a mass accuracy standard Ideally more than one peptjde parent or precursor ion may be measured for each protein to be quantified For each peptide the same means of recognjt is Preferably used when comparing intensities in each of the different samples. The measurements of different peptjdes serves to validate the relative abundance measurements Furthermore, the measurements from several peptides a means of determining the average relative abundance, and of determining the relative signjfjca of the measurements.

According to one embodiment all parent or precursor ions may be identified and their relative abundances determined by comparison of their intensities to those of the same identity in one or more other samples.

In another embodiment the relative abundance of all parent or precursor ions of interest, discovered on the basis of their relationship to a predetermined fragment product, daughter or adduct -48 -ion, may be determined by comparison of their intensities to those of the same identity in one or more other samples.

In another embodiment the relative abundance of all parent or precursor ions of interest, discovered on the basis of their giving rise to a predetermined mass loss, may be determined by comparison of their intensities to those of the same identity in one or more other samples.

In another embodiment it may be merely required to quantify a protein already identified. The protein may be in a complex mixture, and the same means for separation and recognition may be used as that already described. Here it is only necessary to recognise the relevant peptide product or products and measure their intensities in one or more samples. The basis for recognition may be that of the peptide parent or precursor ion mass or accurate mass, and that of one or more fragment, product, daughter or adduct ion masses, or accurate masses. Their retention times may also be compared thereby providing a means of confirming the recognition of the same peptide or of rejecting unmatched peptides.

The preferred embodiment is applicable to the study of proteomics. However, the same methods of identification and quantification may be used in other areas of analysis such as the study of metabolomics.

The method is appropriate for the analysis of mixtures where different components of the mixture are first separated or partially separated by a means such as chromatography that causes components to elute sequentially.

The source of ions may preferably yield mainly molecular ions or pseudo-molecular ions and relatively few (if any) fragment, product, daughter or adduct ions. Examples of such sources include atmospheric pressure ionisation sources (e.g. Electrospray and APCI) and Matrix Assisted Laser Desorption lonisation (MALDI).

If the two main operating modes of the collision, fragmentation or reaction device are suitably set, then parent or precursor ions can be recognised by virtue of the fact that they will be relatively more intense in the mass spectrum without substantial fragmentation -49 -or reaction. Similarly, fragment1 product, daughter or adduct ions can be recognised by virtue of the fact that they will be relatively more intense in the mass spectrw with substantial fragmentat0 or reaction The mass analyser may comprise a quadrupole, Time of Flight, ion trap, magnetj sector or FT-ICR mass analyser. According to a preferred embodiment the mass analyser Should be capable of deteinjng the exact or accurate mass to charge value for ions.

This is to maxjmjse selectivity for detection of characteristic fragment, product, daughter or adduct ions or mass losses, and to maximjse specificity for identjfiation of proteins.

The mass analyser Preferably samples or records the whole spectr simultaneously This ensures that the elution times observed for all, the masses are not modified or distorted by the mass analyser, and in turn would allow accurate matching of the elution times of different masses, such as parent and fragment, product, daughter or adduct ions, it also helps to ensure that the quantjtativ measurements are not compromised by the need to measure abundances of transient signals.

A mass filter, Preferably a quadrupoj,e mass filter, may be provided upstre of the collision fragmentaj0 or reaction device.

The mass filter may have a highpass filter characteristic and, for example, be arranged to transmit ions having a mass to charge ratio greater than or equal to 100, 150, 200, 250, 300, 350, 400, 450 or 500. Alternatively the mass filter may have a lowpass or bandpass filter characteristic n ion guide may be provided upstream of the Coll1j, fragmentajo or reaction device. The ion guide may comprise either a hexapole, quadrupol, octopole or higher order multipole rod set.

In another embodiment the ion guide may comprise an ion tunnel ion guide comprising a plurality of electrodes having apertures through which ions are transmitted in use. Preferably, at least 90% of the electrodes have apertures which are substantially the same size.

Alternatively the ion guide may comprise a plurality of ring

-

electrodes having substantially tapering internal diameters ("ion funnel").

Parent or precursor ions that belong to a particular class of parent or precursor ions, and which are recogrjjsa by a characteristic fragment, product, daughter or adduct ion or characteristic neutral loss are traditionally discovered by the methods of parent or precursor ion scanning or constant neutral loss scanning. Previous methods for recording parent or precursor ion scans or constant neutral loss Scans involve scanning one or both quadrupo5 in a triple quadrupo mass spectrometer or Scanning the quadrupoe in a tandem quadrupoe orthogon TOF mass spectrometer or Scanning at least one element in other types of tandem mass spectrometers As a conseque these methods suffer from the low duty cycle associated with scanning instruments As a further conseqIJe information may be discarded and lost whilst the mass spectrometer is Occupied recording a parent or precursor ion scan or a Constant neutral loss scan. As a further conseque these methods are not appropriate for use where the mass spectrometer is required to analyse Substances eluting directly from gas or liquid chromatography equipment According to the preferred embodiment a tandem quadrupole orthogona' TOF mass spectrometer in used in a way in which parent or precursor ions of interest are discovered Using a method in which sequentja low and high colljsj0 energy mass spectra are recorded.

The Switching altering or varying back and forth is Preferably not interrupted. Instead a complete set of data is acquired, and this is then processed afterwards Fragment product daughter or adduct ions may be associated with parent or precursor ions by closeness of fit of their respective elutj.on times. In this way parent or precursor ions of interest may be confirmed or otherwise Without interrupting the acqujsitio of data, and information need not be lost.

According to one embodiment Possible parent or precursor ions of interest may be selected on the basis of their relationship to a predetermined fragment, product, daughter or adduct ion. The -51 -predetennjnj fragment, product, daughter or adduce ion may comprise, for example, immorijum ions from peptides, functional groups including phosphate group P03 ions from phosphorylated peptides or mass tags which are intended to cleave from a specific molecule or class of molecule and to be subsequently identified thus reporting the presence of the specific molecule or class of molecule. A parent or precursor ion may be short listed as a possible parent or precursor ion of interest by generating a mass chromatogr for the predetermined fragment, product, daughter or adduct ion using high fragmentation or reaction mass spectra. The centre of each peak in the mass chromatograin is then determined together with the corresponding predetermined fragment, product, daughter or adduct ion elution time(s). Then for each peak in the predetermined fragment, product, daughter or adduct ion mass chromatogr both the low fragmentation or reaction mass spectrum obtained inunediately before the predetermined fragment, product, daughter or adduct ion èlution time and the low fragmentation or reaction mass spectrum obtained immediately after the predetermined fragment, product, daughter or adduct ion elution time are interrogated for the presence of previously recognised parent or precursor ions. A mass chromatogram for any previously recognised parent or precursor ion found to be present in both the low fragmentatjo or reaction mass spectrum obtained immediately before the predetermined fragment, product, daughter or adduct ion elution time and the low fragmentation or reaction mass spectrum obtained immediately after the predetermined fragment, product, daughter or adduct ion elutiori time is then generated and the centre of each peak in each mass chromatogram is determined together with the corresponding possible parent or precursor ion of interest elutjon time(s). The possible parent or precursor ions of interest may then be ranked according to the closeness of fit of their elution time with the predetermined fragment, product, daughter or adduct ion elution time, and a list of final possible parent or precursor ions of interest may be formed by rejecting possible parent or precursor ions of interest if their elution time precedes or exceeds the predetermined fragment, product, -52 -daughter or adduct ion elutjon time by more than a predetermined amount.

According to an alternative embodiment a parent or precursor ion may be shortljsted as a possible parent or precursor ion of interest on the basis of it giving rise to a predetermined mass loss.

For each low fragmentaj0 or reaction mass spectrun, a list of target fragment product, daughter or adduct ion mass to charge values that would result from the loss of a predetermined ion or neutral particle from each Previously recognised parent or precursor ion present in the low fragmentation or reaction mass spectr is generated, Then both the high fragmentaj0 or reaction mass spectrwn obtained immediately before the low fragmenta or reaction mass spectrum and the high fragmentatj0 or reaction mass spectrum obtained immediately after the low fragmentajo or reaction mass spectrum are interrogated for the presence of fragment, product, daughter or adduct ions having a mass to charge value corresponding with a target fragment, product, daughter or adduct ion mass to charge value. i list of Possible parent or precursor ions of interest (optionally including their corresponding fragment, product, daughter or adduct ions) is then formed by including in the list a parent or precursor ion if a fragment, product, daughter or adduct ion having a mass to charge value corresponding with a target fragment, product, daughter or adduct ion mass to charge value is found to be present in both the high fragmentaj0 or reaction mass spectrum immediately before the low fragmentajo or reaction mass spectrum and the high fragmentj0 or reaction mass spectr immediately after the low fragmentaj0 or reaction mass spectrum. A mass loss chromatogram may then be generated based upon possible candidate parent or precursor ions and their corresponding fragment, product, daughter or adduct IOflS. The centre of each peak in the mass loss chromatogr is determined together with the corresponding mass loss elutjon time(s). Then for each Possible candidate parent or precursor ion a mass chromatogr is generated using the low fragmentation or reaction mass spectra. A Corresponding fragment, product, daughter or adduct ion mass chromatogr is also generated -53 -for the corresponding fragment, product, daughter or adduct ion. The centre of each peak in the Possible candidate parent or precursor ion mass chromatogr and the corresponding fragment, product, daughter or adduct ion mass chrornatogr are then determined together with the corresponding possible candidate parent or precursor ion elution time(s) and correspondjg fragment product, daughter or adduct ion elutjon time(s). A list of final candidate parent or precursor ions may then be formed by rejecting Possible candidate parent or precursor ions if the eluijon time of a possible candidate parent or Precursor ion precedes or exceeds the corresponding fragment, product, daughter or adduct ion elutjon time by more than a predetej amount.

Once a list of parent or precursor ions of interest has been formed (which Preferably comprises only some of the originally recogujs parent or precursor ions and possible parent or precursor ions of interest) then each parent or precursor ion of interest can then be identified.

Identification of parent or precursor ions may be achieved by making use of a combination of information This may include the accurately determined mass or mass to charge ratio of the parent or precursor ion. It may also include the masses or mass to charge ratios of the fragment, product, daughter or adduct ions. In Some instances the accurately determined masses or mass to charge ratios of the fragment product, daughter or adduct ions may be preferred It is known that a protein may be identified from the masses or mass to charge ratios, Preferably the exact masses, of the peptjcie products from proteins that have been enzymatically digested These may be compared to those expected from a library of known proteins It is also known that when the results of this comparison suggest more than one possible protein then the ambiguity can be resolved by analysis of the fragments of one or more of the peptjdes. The preferred embodiment allows a mixture of proteins, which have been enzymaticaj.ly digested, to be identified in a single analysis. The masses or mass to charge ratios, or exact masses or mass to charge ratios, of all the peptides and their associated fragment, product, -54 -daughter or adduct ions may be searched against a library of known proteins Alternatively, the peptide masses or mass to charge ratios or exact masses or mass to charge ratios, may be searched against the library of known proteins, and where more than one protein is sugges the correct protein may be confirmed by searching for fragment, product, daughter or adduct ions which match those to be expected from the relevant peptjdes from each candidate protein.

The step of identifying each parent or precursor ion of interest Preferably comprises recalling the elution time of the parent or precursor ion of interest generating a list of Possible fragment product, daughter or adduct ions which comprises Previously recognjs fragment product, daughter or adduct ions which are present in both the low fragmentatj0 or reaction mass specer obtained immediately before the elutjon time of the parent or precursor ion of interest and the low fragmentaj0 or reaction mass spectr obtained immediately after the elutjon time of the parent or precursor ion of interest, generating a mass chromatogram of each Possible fragment, product, daughter or adduct ion, determining the centre of each peak in each possible fragment, product, daughter or adduct ion mass chromatogr, and determining the corresponding Possible fragment, product, daughter or adduct ion elutjon time(s).

The possible fragment product, daughter or adduct ions may then be ranked according to the closeness of fit of their elution time with the elutjon time of the parent or precursor ion of interest. A list of fragment, product, daughter or adduct ions may then be formed by rejecting fragment, product, daughter or adduct ions if the elutjon time of the fragment, product daughter or adduct ion precedes or exceeds the elutjon time of the parent or precursor ion of interest by more than a predetermined amount.

The list of fragment, product, daughter or adduct ions may be yet further refined or reduced by generating a list of neighbouring parent or precursor ions which are present in the low fragmenaj0 or reaction mass spectr obtained nearest in time to the elution time of the final candidate parent or precursor ion. A mass -55 -chroniatogram of each parent or precursor ion contained in the list is then generated and the centre of each mass chromatogram is determined along with the corresponding neighbouring parent or precursor ion elution time(s). Any fragment, product, daughter or adduct ion having an elution time which corresponds more closely with a neighbouring parent or precursor ion elution time than with the elution time of a parent or precursor ion of interest may then be rejected from the list of fragment, product, daughter or adduct ions.

Fragment, daughter, product or adduct ions may be assigned to a parentor precursor ion according to the closeness of fit of their elution times, and all fragment, product, daughter or adduct ions which have been associated with the parent or precursor ion may be listed.

An alternative embodiment which involves a greater amount of data processing but yet which is intrinsically simpler is also contemplated. Once parent and fragment, product, daughter or adduct ions have been identified, then a parent or precursor ion mass chromatogram for each recognised parent or precursor ion is generated. The centre of each peak in the parent or precursor ion mass chromatogram and the corresponding parent or precursor ion elution time(s) are then determined. Similarly, a fragment, product, daughter or adduct ion mass chromatogram for each recognised fragment, product, daughter or adduct ion is generated, and the centre of each peak in the fragment, product, daughter or adduct ion mass chromatogram and the corresponding fragment, product, daughter or adduct ion elution time(s) are then determined. Rather than then identifying only a sub-set of the recognised parent or precursor ions, all (or nearly all) of the recognised parent or precursor ions are then identified. Fragment ions are assigned to parent or precursor ions according to the closeness of fit of their respective elution times and all fragment, product, daughter or adduct ions which have been associated with a parent or precursor ion may then be listed.

Passing ions through a mass filter, preferably a quadrupole mass filter, prior to being passed to the collision, fragmentation or -56 -reaction device presents an alternative or an additional method of recognising a fragment product, daughter or adduct lOfl. A fragment, product daughter or adduct ion may be recognised by recognising ions in a high fragmentation or reaction mass spectrum which have a mass to charge ratio which is not transmitted by the collisj0 fragmentaj0 or reaction device i.e. fragment, product, daughter or adduct ions are recognised by virtue of their having a mass to charge ratio falling Outside of the transmission window of the mass filter.

If the ions would not be transmitted by the mass filter then they must have been produced in the collision, fragmenj0 or reaction device.

Various embodiments of the present invention will now be described, by way of example only, and with reference to the accompanying drawings in which: Fig. 1 is a schematic drawing of a preferred mass spectrometer; Fig. 2 Shows a schematic of a valve Switching arrangemn during sample loading and desalting and the inset shows desorptjon of a sample from an analytical column; Fig. 3A Shows a fragment or daughter ion mass spectrum and Fig. 3B shows the corresponding parent or precursor ion mass spectrum obtained when a mass filter upstre of a collision cell was arranged so as to transmit ions having a mass to charge ratio > 350 to the collision cell; Fig. 4A shows a mass chromatogram of a parent or precursor ion, Fig. 4B shows a mass chromatogr of a parent or precursor Ofl, Fig. 4C Shows a mass chromatogr of a parent or precursor ion, Fig. 4D Shows a mass chromatogr of a fragment or daughter ion and Fig. 4E shows a mass chromatogram of a fragment or daughter; Fig. 5 shows the mass chromatogrs of Figs. 4A-E superimposed Upon one another; Fig. 6 shows a mass chromatogr of the Asparagjne immonjum ion which has a mass to charge ratio of 87.04; Fig. 7 Shows a mass spectrum of the peptjde ion P5 derived from ADH which has the sequen ANELLINVJ( and a molecular weight of 1012.59; -57 -Fig. 8 shows a mass spectr of a tryptjc digest of 13-Casejn obtained when a collision cell was in a low fragmentj0 mode; Fig. 9 shows a mass spectrn of a tryptjc digest of t3-Casejn obtained when a collision cell was in a high fragmentatj0 mode; Fig. 10 shows a processed and expanded view of the mass spectrum shown in Fig. 9; Fig. 11?. shows a mass chromatogram of an ion from a first sample having a mass to charge ratio of 880.4, Fig. 11B shows a similar mass chromatogram of the same ion from a second sample, Fig. lic shows a mass chromatogr of an ion from a first sample having a mass to charge ratio of 582.3 and Fig. liD shows a similar mass chromatogra of the same ion from a second sample; Fig. 12A shows a mass spectrum recorded from a first Sample and Fig. 12B shows a corresponding mass spectrum recorded from a second sample which is similar to the first sample except that it contains a higher concentration of the digest products of the protein Casein which is COiflIOfl to both samples; Fig. 13 shows the mass spectrum shown in Fig. 12A in more detail and the insert shows an expanded part of the mass spectrum showing isotope peaks at mass to charge ratio 880.4; and Fig. 14 shows the mass spectrum shown in Fig. 12B in more detail and the insert shows an expanded part of the mass spectrun showing Isotope peaks at mass to charge ratio 880.4.

A preferred embodiment will now be described with reference to Fig. 1. A mass spectrometer 6 is shown which comprises an ion source 1. Preferably an Electrospray Ionisatjon source, an ion guide 2 arranged downstream of the ion source i, a quadx-upole mass filter 3, a collision fragmentaj0 or reaction device 4 and an orthogonal acceleration Time of Flight mass analyser 5 incorporating a reflectron The ion guide 2 and mass filter 3 may be omitted if necessary. The mass spectrometer 6 is Preferably interfaced with a chromatograph such as a liquid chromatograp (not shown) so that the sample entering the ion Source 1. may be taken from the eluent of the liquid chromatograp.

The quadrupole mass filter 3 is preferably disposed in an evacuated chamber which is maintained at a relatively low pressure e.g. less than lO mbar. The rod electrodes comprising the mass filter 3 are preferably connected to a power supply which generates both RF and DC potentials which determine the mass to charge value transmission window of the mass filter 3.

The collision, fragmentation or reaction device 4 preferably comprises a Surface Induced Dissociation (S1D) fragmentation device, an Electron Transfer Dissociation fragmentation device or an Electron Capture Dissociation fragmentation device.

According to an embodiment the collision, fragmentation or reaction device 4 may comprise an Electron Capture Dissociation fragmentation device. According to this embodiment multiply charged analyte ions are preferably caused to interact with relatively low energy electrons. The electrons preferably have energies of < i eV or 1-2 eV. The electrons are -preferably confined by a relatively strong magnetic field and are directed so that the electrons collide with the analyte ions which are preferably confined within an RF ion guide which is preferably arranged within the fragmentation device 4.

An AC or RF voltage is preferably applied to the electrodes of the RF ion guide so that a radial pseudo-potential well is preferably created which preferably acts to confine ions radially within the ion guide so that the ions can interact with the low energy electrons.

According to another embodiment the collision, fragmentation or reaction device 4 may comprise an Electron Transfer Dissociation fragmentation device. According to this embodiment positively charged analyte ions are preferably caused to interact with negatively charged reagent ions. The negatively charged reagent ions are preferably injected into an RE' ion guide or ion trap located within the collision, fragmentation or reaction device 4. An AC or RE' voltage is preferably applied to the electrodes of the RF ion guide so that a radial pseudo-potential well is preferably created which preferably acts to confine ions radially within the ion guide so that the ions can interact with the negatively charged reagent ions. According to a less preferred embodiment negatively charged -59 -analyte ions may alternatively be arranged to interact with Positively charged reagent ions.

According to another embodiment the Collision fragmenaj0 or reaction device 4 may comprise a Surface Induced Dissociation fragmentj0 device. According to this embodiment ions are Preferably directed towards a surface or target Plate with a relatively low energy. The ions may, for example, be arranged to have an energy of 1-10 ev. The surface or target plate may comprise stainless steel or more Preferably the surface or target plate may comprise a metallic plate Coated with a monolayer of fluorocarbon or hydrocarbon. The rionolayer Preferably comprises a self-assembled monolayer The surface or target plate may be arranged in a plane which is substantially parallel with the direction of travel of ions through the Surface Induced Dissociation fragmentatj0 device in a mode of operation wherein ions are not fragment In a mode of operation wherein it is desired to fragment ions, the ions may be deflected onto or towards the surface or target plate so that the ions impinge the surface or target plate at a relatively shallow angle with respect to the surface of target plate. Fragment ions are Preferably produced as a result of the analyte ions colliding with the surface or target plate. The fragment ions are Preferably directed of f or away from the surface or target plate at a relatively shallow angle with respect to the surface or target plate. The fragment ions are then Preferably arranged to assu.me a trajectory which Preferably corresponds with the trajectory of ions which are transmitted through or past the Surface Induced Dissociation fragmentaj0 device in a mode of operation wherein ions are not Substantially fragmented The Collision fragmenaj0 or reaction device 4 may comprise an Electron Collision or Impact Dissociation fragmentaj0 device wherein ions are fragmen upon COlljSj with relatively energetic electrons e.g. wherein the electrons have > 5ev.

According to other embodiments the CO1ljjo fragmentatjo or reaction device 4 may comprise a Photo Induced Dissociation ("PID") fragmentaj0 device, a Laser Induced Dissociation fragmentj0 -60 -device, an infrared radiation induced dissociation device, an ultraviolet radiation induced dissociation device, a thermal or temperature source fragmentation device, an electric field induced fragmentation device, a magnetic field induced fragmentation device, an enzyme digestion or enzyme degradation fragmentation device, an ion-ion reaction fragmentation device, an ion-molecule reaction fragmentation device, an ion-atom reaction fragmentation device, an ion-metas table ion reaction fragmentation device, an ion-metastable molecule reaction fragmentation device, an ion-metastable atom reaction fragmentation device, an ion-ion reaction device for reacting ions to form adduct or product ions, an ion-molecule reaction device for reacting ions to form adduct or product ions, an ion-atom reaction device for reacting ions to form adduct or product ions, an ion-metastable ion reaction device for reacting ions to form adduct or product ions, an ion-metastable molecule reaction device for reacting ions to form adduct or product ions or an ion-metastable atom reaction device for reacting ions to form adduct or product ions.

According to an embodiment the collision, fragmentation or reaction device may form part of the ion source 1. For example, the collision, fragmentation or reaction device may comprise a nozzle-skimmer interface fragmentation device, an in-source fragmentation device or an ion-source Collision Induced Dissociation fragmentation device.

The collision, fragmentation or reaction device 4 may comprise either a quadrupole or hexapole rod set which may be enclosed in a substantially gas-tight casing (other than having a small ion entrance and exit orifice) into which a gas such as helium, argon, nitrogen, air or methane may be introduced at a pressure of between iO and l01 mbar, further preferably i0 mbar to l02 mbar.

Suitable AC or RF potentials for the electrodes comprising the collision, fragmentation or reaction device 4 are provided by a power supply (not shown).

Ions generated by the ion source 1 are transmitted by the ion guide 2 and pass via an interchamber orifice 7 into vacuum chamber 8.

-61 -Ion guide 2 is Preferably maintained at a pressure intermediate that of the ion source i and the vacuum chamber 8. In the embodiment shown, ions maybe mass filtered by mass filter 3 before entering the pref erred colljsjon, fragmentaj0 or reaction device 4. However, the mass filter 3 is an optional feature of this embodiment Ions exiting from the collision, fragmentaj0 or reaction device 4 or which have been transmitted through the colljsjo, fragmena5 or reaction device 4 Preferably pass to a mass analyser which preferably comprises a Time of Flight mass analyser 5. Other ion optical components, such as further ion guides and/or electrostatic lenses, may be provided which are not shown in the figures or described herein. Such components may be used to maximj.se ion transmission between various parts or stages of the mass spectrometer Various vacuum pumps (not shown) may be provided for maintaining optimal vacuujn conditions. The Time of Flight mass analyser 5 incorporating a ref lectron operates in a known way by measuring the transit time of the ions comprised in a packet of ions so that their mass to charge ratios can be determined.

A control means (not shown) Preferably provides control signals for the various power supplies (not shown) which respectively provide the necessary operating potentials for the ion source 1, the ion guide 2, the quadrupole mass filter 3, the co1ljjon, fragmentation or reaction device 4 and the Time of Flight mass analyser 5. These control signals Preferably determine the operating parameters of the mass spectrome for example the mass to charge ratios transmitted through the mass filter 3 and the operation of the analyser 5. The control means may comprise a computer (not shown) which may also be used to process the mass spectral data acquired. The computer may also display and store mass spectra produced by the mass analyser 5 and receive and process commands from an operator. The control means may be set to perform automatically various methods and make various determinations without operator intervention, or may optionally require operator input at various stages.

The control means IS Preferably arranged to switch, vary or alter the colljsjo, fragmentatj0 or reaction device 4 back and -62 -forth between at least two different modes. If the collision, fragmentation or reaction device 4 comprises an Electron Capture Dissociation fragmentation device then the electron source or beam may be switched ON in a first mode of operation and may be switched 0FF in a second mode of operation. If the collision, fragmentation or reaction device 4 comprises an Electron Transfer Dissociation fragmentation device 4 then reagent ions may be injected into an ion guide or ion trap comprising analyte ions in a first mode of operation and substantially no reagent ions may be injected into the ion guide or ion trap in a second mode of operation.

If the collision, fragmentation or reaction device 4 comprises a Surface Induced Dissociation fragmentation device then the analyte ions may be directed so that they collide or impinge upon the surface or target plate in a first mode of operation and the analyte ions may be directed straight past the surface or target plate in a second mode of operation so that the analyte ions do not collide or impinge upon the surface of target plate.

In one embodiment the control means may switch, alter. or vary between modes approximately every second. When the mass spectrometer 6 is used in conjunction with an ion source 1 being provided with an eluerit separated from a mixture by means of liquid or gas chromatography, the mass spectrometer 6 may be run for several tens of minutes over which period of time several hundred high and low fragmentation or reaction mass spectra may be obtained.

At the end of the experimental run the data which has been obtained is preferably analysed and parent or precursor ions and fragment, product, daughter or adduct ions can be recognised on the basis of the relative intensity of a peak in a mass spectrum obtained when the collision, fragmentation or reaction device 4 was in one mode compared with the intensity of the same peak in a mass spectrum obtained approximately a second later in time when the collision, fragmentation or reaction device 4 was in the second mode.

According to an embodiment, mass chromatograms for each parent and fragment, product, daughter or adduct ion are generated and -63 -fragment, product, daughter or adduct ions are assigned to parent or precursor ions on the basis of their relative elution times.

An advantage of this method is that Since all the data is acquired and subsequent'y processed then all fragment, product, daughter or adduct ions may be associated with a parent or precursor ion by closeness of fit of their respective elutjon times. This allows all the parent or precursor ions to be identified from their fragment, product, daughter or adduct ions, irrespective of whether or not they have been discovered by the presence of a characteristic fragment, product, daughter or adduct ion or characteristic "neutral loss".

According to another embodiment an attempt maybe made to reduce the number of parent or precursor ions of interest A list of possible (i.e. not yet finaljsed) parent or precursor ions of interest may be formed by looking for parent or precursor ions which may have given rise to a predetermined fragment, product, daughter or adduci ion of interest e.g. an immonium ion from a peptjde.

Alternatively a search may be made for parent and fragment, product, daughter or adduct ions wherein the parent or precursor ion could have fragmented or reacted into a first component comprising a predetermined ion or neutral particle and a second component comprising a fragment, product, daughter or adduct Ofl. Various steps may then be taken to further reduce/refine the list of possible parent or precursor ions of interest to leave a number of parent or precursor ions of interest Which are then Preferably subsequently identified by comparing elution times of the parent or precursor ions of interest and fragment product, daughter or adduct ions; As will be appreciated two ions could have similar mass to charge ratios but different chemical structures and hence would most likely fragment differently enabling a parent or precursor ion to be identified on the basis of a fragme, product, daughter or adduct ion.

A sample introduction system is shown in more detail in Fig. 2.

Samples may be introduced into the mass spectrometer 6 by means of a Micromass (RTM) modular Capr.,c system. For example, samples may be loaded onto a C18 cartridge (0.3 mm x 5 mm) and desalted with 0.1% -64 -HCOOH for 3 minutes at a flow rate of 3OpL per minute. A ten port valve may then switched such that the peptides are eluted onto the analytical column for separation, see inset of Fig. 2. Flow from two pumps A and B may be split to produce a flow rate through the column of approximately 200nh/mjn.

A preferred analytical column is a PicoFrjt (RTM) column packed with Waters (RPM) Symmetry Cl8 set up to spray directly into the mass spectrometer 6. An Electrospray potential (ca. 3kV) may be applied to the liquid via a low dead volume stainless steel union. A small amount e.g. 5 psi (34.48 kPa) of nebulising gas may be introduced around the spray tip to aid the Electrospray process.

Data may be acquired using a mass spectrometer 6 fitted with a Z-spray (RPM) nanof low Electrospray ion source. The mass spectrometer may be operated in the positive ion mode with a source temperature of 80 C and a cone gas flow rate of 401/hr.

The instrument may be calibrated with a multi-point calibration using selected fragment, product, daughter or adduct ions that result, for example, from the fragmentajo of Clu-fibrinopeptide b.

Data may be processed using the MassLymc (RPM) suite of software.

Switching a Collision. Induced Decomposjtj fragmentation cell between two different modes of operation is not intended to fall within the Scope of the present invention. However, experjmenta results which were obtained according to this method will nonetheless be presented since they serve to illustrate aspects of the present invention.

Figs. 3A and 3B show respectively fragment or daughter and parent or precursor ion spectra of a tryptic digest of alcohol dehydrogenase (ADH). The fragment or daughter ion spectrum shown in Fig. 3A was obtained by maintaining a gas collision cell at a relatively high potential around 30V which resulted in signifjcan fragmentation of ions passing therethrough. The parent or precursor ion spectrum shown in Fig. 3B was obtained at low Collision energy e.g. less than or equal to 5V. The data presented in Fig. 3B was obtained using a mass filter 3 arranged upstream of the collision cell and set to transmit ions having a mass to charge value greater -65 -than 350. The mass spectra in this particular example were obtained from a sample eluting from a liquid chromatograph, and the spectra were obtained Sufficiently rapidly and close together in time so that they essentially Correspond to the same component or components eluting from the liquid chromatograph The mass specLru shown in Fig. 3A was obtained using a cOlljj cell to fragment ions by Collision Induced Dissociation.

Such an approach is not intended to fall within the scope of the present invention However, the mass spectra which were obtained and the following description relating to the processing of the mass spectral data illustrate various aspects of the present invention.

In Fig. 3B, there are several high intensity peaks in the parent or precursor ion spectrum, e.g. the peaks at 418.7724 and 568.7813, which are substantially less intense in the corresponding fragment or daughter ion spectrum shown in Fig. 3A. These peaks may therefore be recognised as being parent or precursor ions. Likewise, ions which are more intense in the fragment or daughter ion spectrum shown in Fig. 3A than in the parent or precursor ion spectrum shown in Fig. 3B may be recognised as being fragment or daughter lOris. As will also be apparent, all the loris having a mass to charge value less than 350 in the high fragmentation mass spectrum shown in Fig. 3A can be readily recognised as being fragment or daughter ions on the basis that they have a mass to charge value less than 350 and the fact that only parent or precursor ions having a mass to charge value greater than 350 were transmitted by the mass fi1ter 5 to the Collision cell.

Figs. 4A-E show respectively mass chromatogr for three parent or precursor ions and two fragment or daughter lOriS. The parent or precursor ions were determined to have mass to charge ratios of 406.2 (peak MC1"), 418.7 (peak MC2") and 568.8 (peak MC3) and the two fragment or daughter ions were determined to have mass to charge ratios of 136.1 (peaks MC4" nd MC5") and 120.1 (peak MC6).

It can be seen that parent or precursor ion peak MC]. (mass to charge ratio 406.2) correlates well with fragment or daughter ion 66 - peak MC5 (mass to charge ratio 136.1) i.e. a parent or precursor ion with a mass to charge ratio of 406.2 seems to have fragmefl to produce a fragment or daughter ion with a mass to charge ratio of 136.1. Similarly, parent or precursor ion peaks MC2 and MC3 correlate well with fragment or daughter ion peaks MC4 and MC6, but it is djffj1t to determine which parent or precursor ion corresponds with which fragment or daughter ion.

Fig. 5 Shows the peaks of Figs. 4-E Overlaid on top of one other and redrawn at a different scale. By careful comparison of the peaks of MC2, MC3, MC4 and MC6 it can be seen that in fact parent or precursor ion MC2 and fragment or daughter ion MC4 correlate well whereas parent or precursor ion MC3 correlates well with fragment or daughter ion HC6. This suggests that parent or precursor ions with a mass to charge ratio of 418.7 fragmen to produce fragment or daughter ions with a mass to charge ratio of 136.1 and that parent or precursor ions with mass to charge ratio 568.8 fragmented to produce fragment or daughter ions with a mass to charge ratio of 120.1.

This cross_correlation of mass chromatograms may be Carried out using automatic peak comparison means such as a suitable peak comparison software program running on a suitable computer.

Fig. 6 show the mass chromatogram for the fragment or daughter ion having a mass to charge ratio of 87.04 extracted from a HPLC separation and mass analysis obtained Using mass spectrometer 6. it is known that the immonium ion for the amino acid Asparagjne has a mass to charge value of 87.04. This chromatogram was extracted from all the high energy spectra recorded on the mass spectrometer 6.

Fig. 7 shows the full mass spectrij corresponding to scan number 604.

This was a low energy mass spectr recorded on the mass spectrometer 6, and is the low energy spectrum next to the high energy spectrum at scan 605 that corresponds to the largest peak in the mass chromatogram of mass to charge ratio 87.04. This shows that the parent or precursor ion for the Asparagine immonjum ion at mass to charge ratio 87.04 has a mass of 1012.54 Since it shows the singly charged (M+H)' ion at mass to charge ratio 1013.54, and the doubly charged (M+2n) ion at mass to charge ratio 507.27.

-67 -Fig. 8 shows a mass spectrum from a low energy spectra recorded on a mass spectrometer 6 of a tryptic digest of the protein 3-Casein.

The protein digest products were separated by HPLC and mass analysed.

The mass spectra were recorded on a mass spectrometer 6 operating in a MS mode and alternating between low and high collision energy in a gas collision cell for successive spectra. Fig. 9 shows a mass spectrum from the high energy spectra recorded at substantially the same time that the low energy mass spectrum shown in Fig. 8 relates to. Fig. 10 shows a processed and expanded view of the mass spectrum shown iii Fig. 9 above. For this spectrum, the continuum data has been processed so as to identify peaks and display them as lines with heights proportional to the peak area, and annotated with masses corresponding to.their centroideci masses. The peak at mass to charge ratio 1031.4395 is the doubly charged (M+2H) ion of a peptide, and the peak at mass to charge ratio 982.4515 is a doubly charged fragment or daughter ion. It has to be a fragment or daughter ion since it is not present in the low energy spectrum. The mass difference between these ions is 48.9880. The theoretical mass for H3P04 is 97.9769, and the mass to charge value for the doubly charged H3PO4 ion is 48.9884, a difference of only 8 ppm from that observed.

It is therefore assumed that the peak having a mass to charge ratio of 982.4515 relates to a fragment or daughter ion resulting from a peptide ion having a mass to charge of 1031.4395 losing a H,PO ion.

Some experimental data is now presented which illustrates the ability of the preferred embodiment to quantify the relative abundance of two proteins contained in two different samples which comprise a mixture of proteins.

A first sample contained the tryptic digest products of three proteins BSA, Glycogen Phosphorylase B and Casein. These three proteins were initially present in the ratio 1:1:1. Bach of the three proteins had a concentration of 330 fmol/pl. A second sample contained the tryptic digest products of the same three proteins BSA, Glycogen Phosphorylase B and Casein. However, the proteins were initially present in the ratio 1:l:X. X was uncertain but believed to be in the range 2-3. The concentration of the proteins BSA and -68 -Glycogen Phosphorylase B in the second sample mixture was the same as in the first sample, namely 330 fmol/1.i1.

The experimental protocol which was followed was that 1 p1 of Sample was loaded for separation on to a HPLC column at a flow rate of 4 il/min. The liquid flow was then split such that the flow rate to the nano-electrospray ionisatjon source was approximately 200 nl/mjn.

Mass spectra were recorded on the mass spectrometer 6. Mass spectrawere recorded at alternating low and high Collision energy using nitrogen CO1ljson gas. The low-collision energy mass spectra were recorded at a COlljj0 voltage of 10v and the high-colj5j0 energy mass spectra were recorded at a Collision voltage of 33V. The mass spectrometer was fitted with a Nano-Lock.Spray device which delivered a separate liquid flow to the source which may be occasionally sampled to provide a reference mass from which the mass calibration may be Periodically validated. This ensured that the mass measurements were accurate to within an RMS accuracy of 5 ppm.

Data were recorded and processed using the MaSSLYnX (RTM) data System.

The first sample was initially analysed and the data was used as a reference The first sample was then analysed a further two times. The second sample was analysed twice. The data from these analyses were used to attempt to quantify the (unknown) relative abundance of Casejn in the second sample.

All data files were processed automatically generating a list of ions with associated areas and high-co1ij5j0 energy spectra for each experiment. This list was then searched against the Swiss-prot protein database using the ProtejnLx (RPM) search engine.

Chromatograpj peak areas were obtained using the Waters (RPM) Apex Peak Tracking algorjtj Chromatogr for each charge state found to be present were summed prior to integraj The experimentally determined relative expressi level of various peptjde ions norma]jsed with respect to the reference data for the two samples are given in the following tables.

-69 -BSA peptide ions Sample 1 Sample 1 Sample 2 Sample 2 Run 1 Run 2 Run 1 Run 2 FKDLGEEHFK 0.652 0.433 0.914 0.661 HL,VDEPQNLIK 0.905 0.829 0.641 0.519 KVPQVSTPTLVEVSR 1.162 0.787 0.629 0.635 LVNELTEFAK 1. 049 0.795 0.705 0.813 LGEYGFQNALI'JR 1.278 0.818 0.753 0.753 AEFVEVTK 1.120 0.821 0.834 0.711 Average 1.028 0.747 0.746 0.682 Glycogen Sample 1 Sample 1 Sample 2 Sample 2 Phophorylase B Run 1 Run 2 Run 1 Run 2 peptide ions VLVDIER 1.279 0.751 n/a 0.701 TNFDAFPDK 0.798 0.972 0.691 0.699 EIWGVEPSR 0.734 0.984 1.053 1.054 LIPAIGDVVNHDPVVGDR 1.043 0.704 0.833 0.833 VLPNDNFFEGI< 0. 969 0.864 0. 933 0. 808 QIIEQL,SSGFFSPK 0.691 n/a 1. 428 1.428 VAAAFPGDVDR 1.140 0.739 0.631 0.641 Average 0.951 0.836 0.928 0.881 CASEIN Sample 1 Sample 1 Sample 2 Sample 2 Peptide sequence Run 1 Run 2 Run 1 Run 2 EDVPSER 0.962 0.941 2.198 1.962 HQGLPQEVLNENLLR 0.828 0.701 1.736 2.090 FFVAPFPEVFGK 1.231 0.849 2.175 1.596 Average 1.007 0.830 2. 036 1.883 -70 -Peptjdes whose sequenc were confirmed by high-coj50 energy data are underlined in the above tables. Confirmation means that the Probability of this peptide, given its accurate mass and the corresponding high_co1ljsj0 energy data, is larger than that of any other peptjde in the database given the current fragmentaj0 or reaction model. The remaining peptjdes are believed to be correct based on their retention time and mass compared to those for Confirmed peptides. It was expected that there would be some experimental error in the results due to injection volume errors and other effects.

When Using BSA as an internal reference, the relative abundance of Glycogen Phospho,5 B in the first sample was determined to be 0.925 (first analysis) and 1.119 (second analysis) giving an average of 1.0. The relative abundance of Glycogen Phosphoryla B in the second sample was determined to be 1.244 (first analysis) and 1.292 (second analysis) giving an average of 1.3. These results compare favourably with the expected value of 1.

Similarly, the relative abundance of Casejn in the first sample was determined to be 0.980 (first analysis) and 1.111 (second analysis) giving an average of 1.0. The relative abundance of Casein in the second Sample was determined to be 2.729 (first analysis) and 2.761 (second analysis) giving an average of 2.7. These results compare favourably with the expected values of 1 and 2-3.

The following data relates to chroniatograms and mass spectra obtained from the first and second samples. One peptjde having the sequen HQGLPQEVLNLLR and derived from Casein elutes at almost exactly the same time as the peptide having the sequence IJVNELTEFAK derived from BSA. Although this is an unusual occurrence it provided an opportunity to compare the abundance of Casein in the two different samples Figs. I1A-D show four mass chromatograms, two relating to the first sample and two relating to the second sample. Fig. hA shows a mass chromatogram relating to the first sample for ions having a mass to charge ratio of 880.4 which corresponds with the peptide ion (M+2Jj) having the sequen HQGLPQEVUq and which is derived -71 -from Casein. Fig. 11B shows a mass chromatograin relating to the second sample which corresponds with the same peptide ion having the sequence HQGLPQEVLNENLLR which is derived from Casein.

Fig. 11C shows a mass chromatogram relating to the first sample for ions having a mass to charge ratio of 582.3 which corresponds with the peptide ion (M+2H) having the sequence LVNELTEFAK and which is derived from BSA. Fig. liD shows a mass chromatograxn relating to the second sample which corresponds with the same peptide ion having the sequence LVNErJTEFAK and which is derived from BSA.

The mass chromatograms show that the peptide ions having a mass to charge ratio of mass to charge ratio 582.3 derived from BSA are present in both samples in roughly equal amounts whereas there is approximately a 100% difference in the intensity of peptide ion having a mass to charge ratio of 880.4 derived from Casein.

l5 Fig. 12A show a parent or precursor ion mass spectrum recorded after around 20 minutes from the first sample and Fig. 12B shows a parent or precursor ion mass spectrum recorded after around substantially the same time from the second sample. The mass spectra show that the ions having a mass to charge ratio of 582.3 (derived from BSA) are approximately the same intensity in both mass spectra whereas ions having a mass to charge ratio of 880.4 which relate to a peptide ion from Casein are approximately twice the intensity in the second sample compared with the first sample. This is consistent with expectations.

Fig. 13 shows the parent or precursor ion mass spectrum shown in Fig. 12A in more detail. Peaks corresponding with ESA peptide ions having a mass to charge of 582.3 and peaks corresponding with the Casein peptide ions having a mass to charge ratio of 880.4 can be clearly seen. The insert shows the expanded part of the spectrum showing the isotope peaks of the peptide ion having a mass to charge ratio of 880.4. Similarly, Fig. 14 shows the parent or precursor ion mass spectrum shown in Fig. 12B in more detail. Again, peaks corresponding with BSA peptide ions having a mass to charge ratio of 582.3 and peaks corresponding with the Casein peptide ions having a mass to charge ratio of 880.4 can be clearly seen. The insert shows -72 -the expanded part of the spectrum showing the isotope peaks of the peptjde ion having a mass to charge ratio of 880.4. It is apparent from Figs. 12-14 and from comparing the inserts of Figs. 13 and 14 that the abundance of the peptide ion derived from Casein which has a mass spectral peak of mass to charge ratio 880.4 is approximately twice the abundance in the second sample compared with the first sample.

Although the present invention has been described with reference to preferred embodiments it will be understood by those skilled in the art that various changes in form and detail maybe made without departing from the scope of the invention as set forth in the accompanying claims.

Claims

-73 -8965809c2 Claims 1. A method of mass spectrometry comprising:

passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device comprising an Electron Transfer Dissociation fragmentation device; repeatedly switching, altering or varying said Electron Transfer Dissociation fragmentation device between a first mode wherein at least some of said parent or precursor ions from said first sample are fragmented upon interacting with reagent ions to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device comprising an Electron Transfer Dissociation fragmentation device; repeatedly switching, altering or varying said Electron Transfer Dissociation fragmentation device between a first mode wherein at least some of said parent or precursor ions from said second sample are fragmented upon interacting with reagent ions to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; automatically determining the intensity of first parent or precursor ions from said first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from said second sample which have said same first mass to charge ratio; and comparing the intensity of said first parent or precursor ions with the intensity of said second parent or precursor ions; wherein if the intensity of said first parent or precursor ions differs from the intensity of said second parent or precursor ions by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or a, precursor ions are considered to be parent or precursor ions of interest.
2. A method of mass spectroznetry comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device comprising a Surface Induced Dissociation fragmentation device; repeatedly switching, altering or varying said Surface Induced Dissociation fragmentation device between a first mode wherein at least some of said parent or precursor ions from said' first saile are fragmented upon impinging upon a surface or target plate to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device comprising a Surface Induced Dissociation fragmentation device; repeatedly switching, altering or varying said Surface Induced Dissociation fragmentation device between a first mode wherein at least some of said parent or precursor ions from said second sample are fragmented upon impinging upon a surface or target plate to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; automatically determining the intensity of first parent or precursor ions from said first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from said second sample which have said same first mass to charge ratio; and comparing the intensity of said first parent or precursor ions with the intensity of said second parent or precursor ions; wherein if the intensity of said first parent or precursor ions differs from the intensity of said second parent or precursor ions by more than a predetermined amount then either -75 -said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest.
3. A method as claimed in claim 2, wherein in said first mode of operation said parent or precursor ions are directed, diverted or deflected on to said surface or target plate.
4. A method as claimed in claim 2 or 3, wherein in said second mode of operation said parent or precursor ions are not directed, diverted or deflected on to said surface or target plate.
5. A method as claimed in claim 2, 3 or 4, wherein said surface or target plate comprises a self-assembled monolayer.
6. A method as claimed in any of claims 2-5, wherein said surface or target plate comprises a fluorocarbon or hydrocarbon monolayer.
7. A method as claimed in any of claims 2-6, wherein said surface or target plane is arranged in a plane which is substantially parallel to the direction of travel of said parent or precursor ions in said second mode of operation.
8. A method of mass spectrometry comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying said collision, fragmentation or reaction device between a first mode wherein at least some of said parent or precursor ions from said first sample are fragmented or reacted to produce fragment, daughter, product or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; -76 -passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying said collision, fragmentation or reaction device between a first mode wherein at least some of said parent or precursor ions from said second sample are fragmented or reacted to produce fragment, daughter, product or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; automatically determining the intensity of first parent or precursor ions from said first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from said second sample which have said same first mass to charge ratio; and comparing the intensity of said first parent or precursor ions with the intensity of said second parent or precursor ions; wherein if the intensity of said first parent or precursor ions differs from the intensity of said second parent or precursor ions by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest; wherein said collision, fragmentation or reaction device selected from the group consisting of: (i) an Electron Collision or Impact Dissociation fragmentation device; (ii) a Photo Induced Dissociation (PID") fragmentation device; (iii) a Laser Induced Dissociation fragmentation device; (iv) an infrared radiation induced dissociation device; (v) an ultraviolet radiation induced dissociation device; (vi) a nozzle-skimmer interface fragmentation device; (vii) an in-source fragmentation device; (viii) an ion-source Collision Induced Dissociation fragmentation device; (ix) a thermal or temperature source fragmentation device; (x) an electric field induced fragmentation device; (xi) a magnetic field induced fragmentation device; (xii) an enzyme digestion or enzyme -77 -degradation fragmentatjn device; (Xiii) an ion-ion reaction fragmentation device; (xiv) an ion-molecule reaction fragmentatjon device; (xv) an ion-atom reaction fragmentation device; (xvi) an ion-metastable ion reaction fragmentation device; (xvii) an ion-metastable molecule reaction fragmentation device; (xviii) an ion-metastable atom reaction fragmentation device; (xix) an ion-ion reaction device for reacting ions to form adduct or product ions; (xx) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxi) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxii) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-metastable molecule reaction device for reacting ions to form adduct or product Ions; and (xxiv) an ion-metastable atom reaction device for reacting ions to form adduct or product ions.
9. A method of mass spectrometry comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device comprising an Electron Transfer Dissociation fragmentation device; repeatedly switching, altering or varying said Electron Transfer Dissociation fragmentation device between a first mode wherein at least some of said parent or precursor ions from said first sample are fragmented upon interacting with reagent ions to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device comprising an Electron Transfer Dissociation fragmentation device; repeatedly switching, altering or varying said Electron Transfer Dissociation fragmentation device between a first mode wherein at least some of said parent or precursor ions from said second sample are fragmented upon interacting with reagent ions * to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; automatically determining the intensity of first parent or precursor ions from said first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from said second sample which have said same first mass to charge ratio; determining a first rati9 of the intensity of said first parent or precursor ions to the intensity of other parent or precursor ions in said first sample; determining a second ratio of the intensity of said second parent or precursor ions to the intensity of other parent or precursor ions in said second sample; and comparing said first ratio with said second ratio; * wherein if said first ratio differs from said second ratio by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest.
10. A method of mass spectrometrY comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device comprising a Surface Induced Dissociation fragmentation device; repeatedly switching, altering or varying said Surface Induced Dissociation fragmentation device between a first mode wherein at least some of said parent or precursor ions from said first sample are fragmented UOfl impinging upon a surface or target plate to produce fragment or daughter ions and a second mode wherein substantiallY fewer parent or precursor ions are fragmented; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device comprising a Surface Induced Dissociation fragmentation device; -79 -repeatedly switching, altering or varying said Surface Induced Dissociation fragmentation device between a first mode wherein at least some of said parent or precursor ions from said second sample are fragmented upon impinging upon a surface or target plate to produce fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; automatically determining the intensity of first parent or precursor ions from said first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from said second sample which have said same first mass to charge ratio; determining a first ratio of the intensity of said first parent or precursor ions to the intensity of other parent or precursor ions in said first sample; determining a second ratio of the intensity of said second parent or precursor ions to the intensity of other parent or precursor ions in said second sample; and comparing said first ratio with said second ratio; wherein if said first ratio differs from said second ratio by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest.
11. A method as claimed in claim 10, wherein in said first mode of operation said parent or precursor ions are directed, diverted or deflected on to said surface or target plate.
12. A method as claimed in claim 10 or 11, wherein in said second mode of operation said parent or precursor ions are not directed, diverted or deflected on to said surface or target plate.

-80 -
13. A method as claimed in claim 10, 11 or 12, wherein said suracê or target plate comprises a self-assembled monolayer.
14. A method as claimed in any of claims 10-13, wherein said surface or target plate comprises a fluorocarbon or hydrocarbon monolayer.
15. A method as claimed in any of claims 10-14, wherein said surface or target plane is arranged in a plane which is substantially parallel to the direction of travel of said parent or precursor ions in said second mode of operation.
16. A method of mass spectrometrY comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying said collision, fragmentation or reaction device between a first mode wherein at least some of said parent or precursor ions from said first sample are fragmented or reacted to produce fragment, daughter, product or adduct ions and a second mode wherein substantiallY fewer parent or precursor ions are fragmented or reacted; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying said collision, fragmentation or reaction device between a first mode wherein at least some of said parent or precursor ions from said second sample are fragmented or reacted to produce fragment, daughter, product or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; automatically determining the intensity of first parent or precursor ions from said first sample which have a first mass to charge ratio; automatically determining the intensity of second parent or precursor ions from said second sample which have said same first mass to charge ratio; -81 -determining a first ratio of the intensity of said first parent-or precursor ions to the intensity of other parent or precursor ions in said first sample; determining a second ratio of the intensity of said second parent or precursor ions to the intensity of other parent or precursor ions in said second sample; and comparing said first ratio with said second ratio; wherein if said first ratio differs from said second ratio by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest; wherein said collision, fragmentation or reaction device selected from the group consisting of: (i) an Electron Collision or Impact Dissociation fragmentation device; (ii) a Photo Induced Dissociation (PID') fragmentation device; (iii) a Laser Induced Dissociation fragmentation device; (iv) an infrared radiation induced dissociation device; (it) an ultraviolet radiation induced dissociation device; (vi) a nozzle-skimmer interface fragmentation device; (vii) an in-source fragmentation device; (viii) an ion-source Collision Induced Dissociation fragmentation device; (ix) a thermal or temperature source fragmentation device; (x) an electric field induced fragmentation device; (xi) a magnetic field induced fragmentation device; (xii) an enzyme digestion or enzyme degradation fragmentation device; (xiii) an ion-ion reaction fragmentation device; (xiv) an ion-molecule reaction fragmentation device; (cv) an ion-atom reaction fragmentation device; (xvi) an ion-Inetastable ion reaction fragmentation device; (xvii) an ion-metastable molecule reaction fragmentation device; (xviii) an ion-metastable atom reaction fragmentation device; (xix) an ion-ion reaction device for reacting ions to form adduct or product ions; (xx) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxi) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxii) an ion-metastable ion reaction device for -82 - reacting ions to form adduct or product ions; (xxiii) an ion-metastable molecule reaction device for reacting ions to form adductor product ions; and (xxiv) an ion-metastable atom reaction device for reacting ions to form adduct or product ions.
17. A method as claimed in any of claims 9-16, wherein either said other parent or precursor ions present in said first sample and/or said other parent or precursor ions present in said second sample are endogenous to said sample.
18. A method as claimed in any of claims 9-16, wherein either said other parent or precursor ions present in said first sample and/or said other parent or precursor ions present in said second sample are exogenous to said sample.
19. A method as claimed in any of claims 9-18, wherein said other parent or precursor ions present in said first sample and/or said other parent or precursor ions present in said second sample are additionally used as a chromatographic retention time standard.
20. A method as claimed in any preceding claim, comprising automatically switching, altering or varying said collision, fragmentation or reaction device between at least said first mode and said second mode at least once every 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 seconds.
21. A method as claimed in any preceding claim, wherein said predetermined amount is selected from the group consisting of: (i) 1%; (ii) 10%; (iii) 50%; (iv) 100%; (v) 150%; (vi) 200%; (vii) 250%; (viii) 300%; (ix) 350%; (x) 400%; (xi) 450%; (xii) 500%; (xiii) 1000%; (xiv) 5000%; or (xv) 10000%.

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22. A method as claimed in any preceding claim, wherein said collision, fragmentation or reaction device is maintained at a pressure selected from the group consisting of: (i) greater than or equal to 0.0001 rnbar; (ii) greater than or equal to 0.0005 mbar; (iii) greater than or equal to 0.001 mbar; (iv) greater than or equal to 0.005 mbar; Cv) greater than or equal to 0.01 mbar; (v-i) greater than or equal to 0.05 mbar; (vii) greater than or equal to 0.1 mbar; (viii) greater than or equal to 0.5 mbar; (ix) greater than or equal to 1 mbar; (x) greater than or equal to 5 mbar; and (xi) greater than or equal to 10 mbar.
23. A method as claimed in any preceding claim, wherein said collision, fragmentation or reaction device is maintained at a pressure selected from the group consisting of: (i) less than or equal to 10 mbar; (ii) less than or equal to 5 mbar; (iii) less than or equal to 1 mbar; (iv) less than or equal to 0.5 mbar; Cv) less than or equal to 0.1 mbar; (v-i) less than or equal to 0.05 mbar; (vii) less than or equal to 0.01 mbar; (viii) less than or equal to 0.005 mbar; (ix) less than or equal to 0.001 mbar; (x) less than or equal to 0.0005 mbar; and (xi) less than or equal to 0.0001 mbar.
24. A method as claimed in any preceding claim, wherein gas in said collision, fragmentation or reaction device is maintained at a first pressure when said collision, fragmentation or reaction device is in said first mode and at a second lower pressure when said collision, fragmentation or reaction device is in said second mode.
25. A method as claimed in any preceding claim, wherein gas in said collision, fragmentation or reaction device comprises a first gas or a first mixture of gases when said collision, fragmentation or reaction device is in said first mode and a second different gas or a second different mixture of gases when -84 -said collision, fragmentation or reaction device is in said second mode.
26. A method as claimed in any preceding claim, further comprising the step of identifying said parent or precursor ions of interest.
27. A method as claimed in claim 26, wherein the step of identifying said parent or precursor ions of interest comprises determining the mass to charge ratio of said parent or precursor ions of interest.
28. A method as claimed in claim 27, wherein the mass to charge ratio of said parent or precursor ions of interest is determined to less than or equal to 20 ppm, 15 ppm, 10 ppm or 5 ppm.
29. A method as claimed in claim 27 or 28, further comprising comparing the determined mass to charge ratio of said parent or precursor ions of interest with a database of ions and their corresponding mass to charge ratios.
30. A method as claimed in any of claims 26-29, wherein said step of identifying said parent or precursor ions of interest comprises identifying one or more fragment, product, daughter or adduct ions which are determined to result from fragmentation or reaction of said parent or precursor ions of interest.
31. A method as claimed in claim 30, wherein said step of identifying one or more fragment, product, daughter or adduct ions further comprises determining the mass to charge ratio of said one or more fragment, product, daughter or adduct ions to less than or equal to 20 ppm, 15 ppm, 10 ppm or 5 ppm.
32. A method as claimed in claim 30 or 31, wherein the step of identifying parent or precursor ions of interest comprises -85 -determining whether said parent or precursor ions of interest are observed in a mass spectrum obtained when said collision, fragmentation or reaction device is in said second mode for a certain time period and said fragment, product, daughter or adduct ions are observed in a mass spectrum obtained either immediately before said certain time period, when said collision, fragmentation or reaction device is in said first mode, or immediately after said certain time period, when said collision, fragmentation or reaction device is in said first mode.
33. A method as claimed in claims 30, 31 or 32, wherein the step of identifying said parent or precursor ions of interest comprises determining that the elution time of said parent or precursor ions of interest is substantially the same as the pseudo-elution time of said fragment, product, daughter or adduct ions.
34. A method as claimed in any of claims 30-33, wherein the step of identifying said parent or precursor ions of interest comprises comparing the elution profile of said parent or precursor ions of interest with the pseudo-elution profile of said fragment, product, daughter or adduct ions.
35. A method of mass spectrometry as claimed in any preceding claim, further comprising determining that ions are parent or precursor ions by comparing two mass spectra obtained one after the other, a first mass spectrum being obtained when said collision, fragmentation or reaction device was in said first mode and a second mass spectrum being obtained when said collision, fragmentation or reaction device was in said second mode, wherein ions are determined to be parent or precursor ions if a peak corresponding to said ions in said second mass spectrum is more intense than a peak corresponding to said ions in said first mass spectrum.

-86 -
36. A method as claimed in any preceding claim, further comprising determining that ions are determined to be fragment, product, daughter or adduct ions by comparing two mass spectra obtained one after the other, a first mass spectrun being obtained when said Collision, fragmentation or reaction device was in said first mode and a second mass spectri being obtained when said collision, fragmentation or reaction device was in said second mode, wherein ions are determined to be fragment, product, daughter or adduct ions if a peak corresponding to said ions in said first mass spectr is more intense than a peak Corresponding to said ions in said second mass spectrum.
37. A method as claimed in any Preceding claim, further comprising: Providing a mass filter upstre of said collision, fragmentaj or reaction device wherein said mass filter is arranged to transmit ions having mass to charge ratios within a first range but to substantially attenuate ions having mass to charge ratios within a second range; and wherein ions are determined to be fragment, product, daughter or adduct ions if they are determined to have a mass to charge ratio falling within said second range.
38. A method as claimed in any Preceding claim, wherein said first parent or precursor ions and said second parent or precursor ions are determined to have mass to charge ratios which differ by less than or equal to 40 ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm, 15 ppm, 10 ppm or 5 ppm.
39. A method as claimed in any preceding claim, wherein said first parent or precursor ions and said second parent or precursor ions are determined to have eluted from a chromatography column after substantially the same elution time.

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40. A method as claimed in any preceding claim, wherein said first parent or precursor ions are determined to give rise to one or more first fragment, product, daughter or adduct ions and said second parent or precursor ions are determined to give rise to one or more second fragment, product, daughter or adduct ions, wherein said one or more first fragment, product, daughter or adduct ions and said one or more second fragment, product, daughter or adduct ions have substantially the same mass to charge ratio.
41. A method as claimed in claim 40, wherein the mass to charge ratio of said one or more first fragment, product, daughter or adduct ions and said one or more second fragment, product, daughter or adduct ions are determined to differ by less than or equal to 40 ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm, 15 ppm, 10 ppm or 5 ppm.
42. A method as claimed in any preceding claim, wherein said first parnt or precursor ions are determined to give rise to one or more first fragment, product, daughter or adduct ions and said second parent or precursor ions are determined to give rise to one or more second fragment, product, daughter or adduct ions and wherein said first parent or precursor ions and said second parent or precursor ions are observed in mass spectra relating to data obtained in said second mode at a certain point in time and said one or more first and second fragment, product, daughter or adduct ions are observed in mass spectra relating to data obtained either immediately before said certain point in time when said collision, fragmentation or reaction device is in said first mode or immediately after said certain point in time when said collision, fragmentation or reaction device is in said first mode.
43. A method as claimed in any preceding claim, wherein said first parent or precursor ions are determined to give rise to -88 -one or more first fragment, product, daughter or adduct ions and said second parent or precursor ions are determined to give rise to one or more second fragment, product, daughter or adduct ions and wherein said first fragment, product, daughter or adduct ions have substantially the same pseudo-e].ution time as said second fragment, product, daughter or adduct ions.
44. A method as claimed in any preceding claim, wherein said first parent or precursor ions are determined to give rise to one or more first fragment, product, daughter or adduct ions and said second parent or precursor ions are determined to give rise to one or more second fragment, product, daughter or adduct ions and wherein said first parent or precursor ions are determined to have an elution profile which correlates with a pseudo-elution profile of said first fragment, product, daughter or adduct ions and wherein said second parent or precursor ions are determined to have an elution profile which correlates with a pseudo-elutjon profile of said second fragment, product, daughter or adduct ions.
45. A method as claimed in any preceding claim, wherein said first parent or precursor ions and said second parent or precursor ions are determined to be multiply charged.
46. A method as claimed in any preceding claim, wherein said first parent or precursor ions and said second parent or precursor ions are determined to have the same charge state.
47. A method as claimed in any preceding claim, wherein fragment, product, daughter or adduct ions which are determined to result from the fragmentation or reaction of said first parent or precursor ions aredetermined to have the same charge state as fragment, product, daughter or adduct ions which are determined to result from the fragmentation or reaction of said second parent or precursor ions.

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48. A method as claimed in any preceding claim, wherein said first sample and/or said second sample comprise a plurality of different biopolymers, proteins, peptides, polypeptideS, oligionucleotideS, oligionucleosideS, amino acids, carbohydrates. sugars, lipids, fatty acids, vitamins, hormones, portions or fragments of DNA, portions or fragments of cDNA, portions or fragments of PNA, portions or fragments of mENA, portions or fragments of tRNA, polyc].onal antibodies, monoclonal antibodies, ribonucleases, enzymes, metaboliteS, polysaccharides, phosphorylated pept ides, phosphorylated proteins, glycopeptides, glycoproteins or steroids.
49. A method as claimed in any preceding claim, wherein said first sample and/or said second sample comprise at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 molecules having different identities.
50. A method as claimed in any preceding claim, wherein either; (i) said first sample is taken from a diseased organism and said second sample is taken from a non-diseased organism; (ii) said first sample is taken from a treated organism and said second sample is taken from a non-treated organism; or (iii) said first sample is taken from a mutant organism and said second sample is taken from a wild type organism.
51. A method as claimed in any preceding claim, wherein molecules from said first and/or second samples are separated from a mixture of other molecules prior to being ionised by: (i) High Performance Liquid chromatography ("HPLC"); (ii) anion exchange; (iii) anion exchange chromatography; (iv) cation exchange; (v) cation exchange chromatography; (vi) ion pair reversed-phase chromatography; (vii) chromatography; (viii) single dimensional electrophoreSiS; (ix) multi-dimensional -90 -electrophoresjs; (x) size exclusion; (xi) affinity; (xii) reverse phase chromatography; (xiii) Capillary Electrophoresis Chromatography (CEC); (xiv) electrophoresjs; (xv) ion mobility separation; (xvi) Field Asymmetric Ion Mobility Separation (FAIMS") or (xvi) capillary electrophoresjs.
52. A method as claimed in any preceding claim, wherein said first and second sample ions comprise peptide ions.
53. A method as claimed in claim 52, wherein said peptide ions comprise the digest products of one or more proteins.
54. A method as claimed in claim 52 or 53, further comprising the step of attempting to identify a protein which correlates with said parent or precursor ions of interest.
55. A method as claimed in claim 54, further comprising determining which peptide products are predicted to be formed when a protein is digested and determining whether any predicted peptide product(s) correlate with parent or precursor ions of interest.
56. A method as claimed in claim 54, further comprising determining whether said parent or precursor ions of interest correlate with one or more proteins.
57. A method as claimed in any preceding claim, wherein said first and second samples are taken from the same organism.
58. A method as claimed in any of claims 1-56, wherein said first and second samples are taken from different organisms.
59. A method as claimed in any preceding claim, further comprising the step of confirming that said first parent or precursor ions and/or said second parent or precursor ions are -91 -not fragment, product, daughter or adduct ions caused by fragmentation of parent or precursor ions in said collision, fragmentation or reaction device.
60. A method as claimed in claim 59, further comprising: comparing a first mass spectrum relating to data obtained in said first mode with a second mass spectrum relating to data obtained in said second mode, said mass spectra being obtained at substantially the same time; and determining that said first and/or said second parent or precursor ions are not fragment, product, daughter or adduct ions if said first and/or said second parent or precursor ions have a greater intensity in the second mass spectrum relative to the first mass spectrum.
61. A method as claimed in any preceding claim, wherein parent or precursor ions from said first sample and parent or precursor ions from said second sample are passed to the same collision, fragmentation or reaction device.
62. A method as claimed in any of claims 1-60, wherein parent or precursor ions from said first sample and parent or precursor ions from said second sample are passed to different collision, fragmentation or reaction devices.
63. A mass spectrometer comprising: an Electron Transfer Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon interacting with reagent ions to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; a mass analyser; and a control system which in use: -92 -Ci) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have said same first mass to charge ratio; and (iii) compares the intensity of said first parent or precursor ions with the intensity of said second parent or precursor ions; wherein if the intensity of said first parent or precursor ions differs from the intensity of said second parent or precursor ions by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest.
64. A mass spectrometer comprising: a Surface Induced Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon impinging upon a surface or target plate to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have said same first mass to charge ratio; and (iii) compares the intensity of said first parent or precursor ions with the intensity of said second parent or precursor ions; -93 -wherein if the intensity of said first parent or precursor ions differs from the intensity of said second parent or precursor ions by more than a predetermined amount then either said first parent or precursor ions arid/or said second parent or precursor ions are considered to be parent or precursor ions of interest.
65. A mass spectrometer comprising: a Collision, fragmentation or reaction device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented or reacted into one or more fragment, product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have said same first mass to charge ratio; and (iii) compares the intensity of said first parent or precursor ions with the intensity of second parent or precursor IOnS; wherein if said intensity of said first parent or precursor ions differs from said intensity of said second parent or precursor ions by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest; wherein said collision, fragmentation or reaction device selected from the group Consisting of: (i) an Electron Collision or Impact Dissociation fragmentation device; (ii) a Photo Induced Dissociation (1PID") fragmentation device; (iii) a Laser Induced Dissociation fragmentation device; (iv) an infrared radiation induced dissociation device; (v) an ultraviolet radiation induced dissociation device; (vi) a nozzle-skimmer interface fragmentation device; (vii) an in-source fragmentation device; (viii) an ion-source Collision Induced Dissociation fragmentation device; (ix) a thermal or temperature source fragmentation device; (x) an electric field induced fragmentation device; (xi) a magnetic field induced fragmentation device; (xii) an enzyme digestion or enzyme degradation fragmentation device; (xiii) an ion-ion reaction fragmentation device; (xiv) an ion-molecule reaction fragmentation device; (xv) an ion-atom reaction fragmentation device; (xvi) an ion-metastable ion reaction fragmentation device; (xvii) an ion-metastable molecule reaction fragmentation device; (xviii) an ion-metastable atom reaction fragmentation device; (xix) an ion-ion reaction device for reacting ions to form adduct or product ions; (xx) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxi) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxii) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; and (xxiv) an ion-metastable atom reaction device for reacting ions to form adduct or product ions.
66. Amass spectrometer comprising: an Electron Transfer Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon interacting with reagent ions to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; -95 -a mass analyser; and a control system which in user (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have said same first mass to charge ratio; (iii) determines a first ratio of the intensity of said first parent or precursor ions to the intensity of other parent or precursor ions in said first sample; (iv) determines a second ratio of the intensity of said second parent or precursor ions to the intensity of other parent or precursor ions in said second sample; and (v) compares said first ratio with said second ratio; wherein if said first ratio differs from said second ratio by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest.
67. A mass spectrometer comprising: a Surface Induced Dissociation fragmentation device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented upon impinging upon a surface or target plate to form fragment or daughter ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have said same first mass to charge ratio; (iii) determines a first ratio of the intensity of said first parent or precursor ions to the intensity of other parent or precursor ions in said first sample; (iv) determines a second ratio of the intensity of said second parent or precursor ions to the intensity of other parent or precursor ions in said second sample; and (v) compares said first ratio with said second ratio; wherein if said first ratio differs from said second ratio by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest.
68. A mass spectrometer comprising: a collision, fragmentation or reaction device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented or reacted into àne or more fragment, product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; a mass analyser; and a control system which in use: (i) determines the intensity of first parent or precursor ions from a first sample which have a first mass to charge ratio; (ii) determines the intensity of second parent or precursor ions from a second sample which have said same first mass to charge ratio; (iii) determines a first ratio of the intensity of said first parent or precursor ions to the intensity of other parent or precursor ions in said first sample; (iv) determines a second ratio of the intensity of said second parent or precursor ions to the intensity of-other parent or precursor ions in said second sample; and (v) compares said first ratio with said second ratio; wherein if said first ratio differs from said second ratio by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest; wherein said collision, fragmentatjon0 reaction device selected from the group Consisting of: (i) an Electron Collision or Impact Dissociation fragmentation device; (ii) a Photo Induced Dissociation (IpIDu) fragmentation device; (iii) a Laser Induced Dissociation fragmentajo device; (iv) an infrared radiation induced dissociation device; (v) an ultraviolet radiation induced dissociation device; (vi) a nozzle-skimer interface fragmentj device; (Vii) an in-source fragmentation device; (Viii) an ion-source Collision Induced DissocIation fragmenajo device; (ix) a thermal or temperature source fragmentation device; (x) an electric field induced fragmentation device; (xi) a magnetic field induced fragmentaio device; (xii) an enzyme digestion or enzyme degradation fragmentation device; (xiii) an ion-jon reaction fragmentation device; (xiv) an ion-molecule reaction fragmentatjo device; (xv) an ion-atom reaction fragmentation device; (xvi) an ion-metastable ion reaction fragmentation device; (xvii) an ion-metastable molecule reaction fragmentation device; (xviii) an ion-metastable atom reaction fragmentation device; (xix) an ion-ion reaction device for reacting ions to form adduct or product ions; (xx) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxi) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxii) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; and (xxiv) an ionmetastable atom -98 -reaction device for reacting ions to form adduct or product ions.
69. A mass spectrometer as claimed in any of claims 63-68, further comprising an ion source.
70. A mass spectrometer as claimed in claim 69, wherein said ion source is selected from the group consisting of: (i) an Electrospray ionisation ("ESI') ion source; (ii) an Atmospheric Pressure Photo Ionjsatjon VAPPI") ion source; (iii) an Atmospheric Pressure Chemical Ionisatjon (APCI) ion source; (iv) a Matrix Assisted Laser Desorptjon lonisation (MALDI) ion source; Cv) a Laser Desorptj.on Ionisatjon ("LDI") ion source; (vi) an Atmospheric Pressure Ionisatjon (API) ion source; (vii) a Desorption lonisation on Silicon VDIOS') ion source; (viii) an Electron Impact (EI) ion source; (ix) a Chemical lonisation (CI) ion source; Cx) a Field Ionjsatjon (F1) ion source; (xi) a Field Desorption ("PD') ion source; (xii) an Inductively Coupled Plasma ("ICP") ion source; (xiii) a Fast Atom Bombardment ("FAB") IOfl Source; (xiv) a Liquid Secondary Ion Mass Spectrometry (LSIMS") ion source; (xv) a Desorption Electrospray Ionisatjon (DESI') ion source; (xvi) a Nickel-63 radioactive ion source; (xvii) an Atmospheric Pressure Matrix Assisted Laser Desorption lonisation ion source; and (xviii) a Thermospray ion source.
71. A mass spectrometer as claimed in claim 69 or 70, wherein said ion source comprises a pulsed or éontinuous ion source.
72. A mass spectrometer as claimed in any of claims 69, 70 or 71, wherein said ion source is provided with an eluent over a period of time, said eluent having been separated from a mixture by means of liquid chromatography or capillary electrophoresjs.

-99 -
73. A mass spectrometer as claimed in any of claims 69, 70 or 71, wherein said ion source is provided with an eluent over a period of time, said eluent having been separated from a mixture by means of gas chromatography.
74. A mass spectrometer as claimed in any of claims 63-73, wherein said mass analyser is selected from the group consisting of: (i) a quadrupo].e mass analyser; (ii) a 2D or linear quadrupole mass analyser; (iii) a Paul or 3D quadrupole mass analyser; (iv) a Penning trap mass analyser; (v) an ion trap mass analyser; (vi) a magnetic sector mass analyser; (vii) Ion Cyclotron Resonance (ICR') mass analyser; (Viii) a Fourier Transform Ion Cyclotron Resonance (FTICR") mass analyser; (ix) an electrostatic or orbitrap mass analyser; Cx) a Fourier Transform electrostatic or orbitrap mass analyser; and (xi) a Fourier Transform mass analyser; (xii) a Time of Flight mass analyser; (xiii) an orthogonal acceleration Time of Flight mass analyser; (xiv) an axial acceleration Time of Flight mass analyser; and (xv) a quadrupole rod set mass filter or mass analyser.
75. Amass spectrometer as claimed in any of claims 63-74, further comprising an ion trap or ion guide arranged upstream and/or downstream of said collision, fragmentation or reaction device.
76. A mass spectrometer as claimed in claim 75, wherein said ion trap or ion guide is selected from the group consisting of: (i) a rnultipo].e rod Set or a segmented multipole rod set ion trap or ion guide comprising a quadrupoj.e rod set, a hexapole rod set, an octapole rod set or a rod set comprising more than eight rods; (ii) an ion tunnel or ion funnel ion trap or ion guide comprising a plurality of electrodes or at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 electrodes having apertures -101) -through which ions are transmitted in use, wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of said electrodes have apertures which are of substantially the same size or area or which have apertures which become progressively larger arid/or smaller in size or in area; (iii) a stack or array of planar, plate or mesh electrodes, wherein said stack or array of planar, plate or mesh electrodes comprises a plurality or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 planar, plate or mesh electrodes and wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of said planar, plate or mesh electrodes are arranged generally in the plane in which ions travel in use; and (iv) an ion trap or ion guide comprising a plurality of groups of electrodes arranged axially along the length of the ion trap or ion guide, wherein each group of electrodes comprises: (a) a first and a second electrode and means for applying a DC voltage or potential to said first and second electrodes in order to confine ions in a first radial direction within said ion guide; and.(b) a third and a fourth electrode and means for applying an AC or RF voltage to said third and fourth electrodes in order to confine ions in a second radial direction within said ion guide.
77. A mass spectrometer as claimed in claim 76, wherein said ion trap or ion guide comprises an ion tunnel or ion funnel ion trap or ion guide wherein at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of said electrodes have internal diameters or dimensions selected from the group consisting of: C1) = 1.0 mm; (ii) = 2.0 nun; (iii) = 3.0 mm; (iv) = 4.0 mm; (v) = 5.0 mm; (vi) = 6.0 mm; (vii) = 7.0 nun; (viii) = 8.0 mm; (ix) =9.0 mm; (x) = 10.0 mm; and (xi) > 10.0 nun. -
78. A mass spectrometer as claimed in claim 75, 76 or 77, wherein said ion trap or ion guide further comprises first AC or RF voltage means arranged and adapted to apply an AC or RF voltage to at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of said plurality of electrodes of said ion trap or ion guide in order to confine ions radially within said ion trap or ion guide.
79. A mass spectrometer as claimed in claim 78, wherein said first AC or RF voltage means is arranged and adapted to apply an AC or RF voltage having an amplitude selected from the group consisting of: (1) < 50 V peak to peak; (ii) 50-100 V peak to peak; (iii) 100-150 V peak to peak; (iv) 150-200 V peak to peak; (v) 200-250 V peak to peak; (vi) 250-300 V peak to peak; (vii) 300-350 V peak to peak; (viii) 350-400 V peak to peak; (ix) 400- 450 V peak to peak; (x) 450-500 V peak to peak; and (xi) > 500 V peak tO peak.
80. A mass analyser as claimed in claim 78 or 79, wherein said first AC or RF voltage means is arranged and adapted to apply an AC or RF voltage having a frequency selected from the group consisting of: (i) < 100 kHz; (ii) 100-200 kHz; (iii) 200-300 kHz; (iv) 300-400 kllz; (v) 400-500 kHz; (vi) 0.5-1.0 MHz; (vii) 1.0-1.5 MHz; (viii) 1.5-2.0 MHz; (ix) 2.0-2:5 MHz; (x) 2.5-3.0 MHZ; (xi) 3.0-3.5 MHz; (xii) 3.5-4.0 MHz; (xiii) 4.0-4.5 MHz; (xiv) 4.5-5.0 MHz; (xv) 5.0-5.5 MHz; (xvi) 5.5-6.0 MHz; (xvii) 6.0-6.5 MHz; (xviii) 6.5-7.0 MHz; (xix) 7.0-7.5 MHz; (xx) 7.5- 8.0 MHz; (xxi) 8.0-8.5 MHz; (xxii) 8.5-9.0 MHz; (xxiii) 9.0-9.5 MHZ; (xxiv) 9.5-10.0 MHz; and (xxv) > 10.0 MHz.
81. A mass spectrometer as claimed in any of claims 75-80, wherein said ion trap or ion guide is arranged and adapted to receive a beam or group of ions and to convert or partition said beam or group of ions such that a plurality or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, lj, 12, 13, 14, 15, 16, 17, 18, 19 or 20 separate packets of ions are confined and/or isolated in said ion trap or ion guide at any particular time, and wherein each packet of ions is separately confined and/or isolated in a separate axial potential well fonued within said ion trap or ion guide.
82. A mass spectrometer as claimed in any of claims 75-81, further comprising means arranged and adapted to urge at least some ions upstream and/or downstream through or along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of said ion trap or ion guide in a mode of operation.
83. A mass spectrometer as claimed in any of claims 75-82, further comprising first transient DC voltage means arranged and adapted to apply one or more transient DC voltages or potentials or one or more transient DC voltage or potential waveforms to said electrodes forming said ion trap or ion guide in order to urge at least some ions upstream and/or downstream along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of said ion trap or ion guide.
84. Amass spectrometer as claimed in any of claims 75-83, further comprising AC or RF voltage means arranged and adapted to apply two or more phase-shifted AC or RF voltages to electrodes forming said ion trap or ion guide in order to urge at least some ions upstream and/or downstream along at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the axial length of said ion trap or ion guide.
85. A mass spectrometer as claimed in any of claims 75-84, further comprising means arranged and adapted in a mode of operation to maintain at least a portion of said ion trap or ion guide at a pressure selected from the group consisting of: (i) > 0.0001 mbar; (ii) > 0.001 mbar; (iii) > 0.01 rnbar; (iv) > 0.1 mbar; (v) > 1 mbar; (vi) > 10 mbar; (vii) > 1 mbar; (viii) 0.0001-100 mbar; and (ix) 0.001-10 mbar.
86. A mass spectrometer as claimed in any of claims 63-85, further comprising a mass filter arranged upstream and/or downstream of said collision, fragmentation or reaction device.
87. A mass spectrometer as claimed in any of claims 63-86, wherein said collision, fragmentation or reaction device comprises: (i) a quadrupo].e rod set; (ii) an hexapole rod set; (iii) an octopo].e or higher order rod set; (iv) an ion tunnel comprising a plurality of electrodes having apertures through which ions are transmitted; or (v) a plurality of electrodes connected to an AC or RF voltage supply for radially confining ions within said collision, fragmentation or reaction device.
88. A mass spectrometer as claimed in any of claims 63-87, wherein said collision, fragmentation or reaction device forms a substantially gas-tight enclosure apart from an aperture to admit ions and an aperture for ions to exit from and optionally a port for introducing gas.
89. A mass spectrometer as claimed in any of claims 63-88, wherein said collision, fragmentation or reaction device is maintained at a pressure selected from the group consisting of: (1) greater than or equal to 0.0001 mbar; (ii) greater than or equal to 0.0005 mbar; (iii) greater than or equal to 0.001 mbar; (iv) greater than or equal to 0.005 mbar; (v) greater than or equal to 0.01 mbar; (vi) greater than or equal to 0.05 mbar; (vii) greater than or equal to 0.1 inbar; (viii) greater than or equal to 0.5 mbar; (ix) greater than or equal to 1 mbar; (x) -104 -greater than or equal to 5 mbar; and (xi) greater than or equal to 10 mbar.
90. A mass spectrometer as claimed in any of claims 63-89, wherein said collision, fragmentation or reaction device is maintained at a pressure selected from the group consisting of: (i) less than or equal to 10 mbar; (ii) less than or equal to 5 mbar; (iii) less than or equal to 1 mbar; (iv) less than or equal to 0.5 ntbar; (v) less than or equal to 0.1 mbar; (vi) less than or equal to 0.05 mbar; (vii) less than or equal to 0.01 mbar; (viii) less than or equal to 0.005 mbar; (ix) less than or equal to 0.001 mbar; (x) less than or equal to 0.0005 mbar; and (xi) less than or equal to 0.0001 mbar.
91. A mass spectrometer as claimed in any of claims 63-90, wherein gas in said collision, fragmentation or reaction device is maintained at a first pressure when said collision, fragmentation or reaction device is in said first mode and at a second lower pressure when said collision, fragmentation or reaction device is in said second mode.
92. A mass spectrometer as claimed in any of claims 63-91, wherein gas in said collision, fragmentation or reaction device comprises a first gas or a first mixture of gases when said collision, fragmentation or reaction device is in said first mode and a second different gas or a second different mixture of gases when said collision, fragmentation or reaction device is in said second mode.
93. A mass spectrometer as claimed in any of claims 63-92, wherein parent or precursor ions from said first sample and parent or precursor ions from said second sample are passed to the same collision, fragmentation or reaction device.
94. A mass spectrometer as claimed in any of claims 63-93, wherein parent or precursor ions from said first sample and parent or precursorions from said second sample are passed to different collision, fragmentation or reaction devices.
95. A mass spectrometer as claimed in any of claims 63-94, wherein molecules from said first and/or second samples are separated from a mixture of other molecules prior to being ionised by: (i) High Performance Liquid Chromatography (1HPLC"); (ii) anion exchange; (iii) anion exchange chromatography; (iv) cation exchange; Cv) cation exchange chromatography; (vi) ion pair reversed-phase chromatography; (vii) chromatography; (viii) single dimensional electrophoresjs; (ix) multi-dimensional electróphoresjs; (x) size exclusion; (xi) affinity; (xii) reverse phase chromatography; (xiii) Capillary Electrophoresis Chromatography (CEC"); (xiv) electrophoresis; (xv) ion mobility separation; (xvi) Field Asymmetric Ion Mobility Separation ("FAIMS"); or (Xvi) capillary electrophoresjs.
96. A method of mass spectrometry comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying said collision, fragmentation or reaction device between a first mode wherein at least some of said parent or precursor ions from said first sample are fragmented or reacted into one or more fragment.

product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying said collision, fragmentation or reaction device between a first mode wherein at least some of said parent or precursor ions from said second sample are fragmented or reacted into one or more fragment, product, daughter or adduct ions and a second mode wherein -106 --substantially fewer parent or precursor ions are fragmented or reacted; automatically determining the intensity of first fragment, product, daughter or adduct ions derived from first parent or precursor ions from said first sample, said first fragment, product, daughter or adduct ions having a first mass to charge ratio; automatically determining the intensity of second fragment, product, daughter or adduct ions derived from second parent or precursor ions from said second sample, said second fragment, product, daughter or adduct ions having said same first mass to charge ratio; and comparing the intensity of said first fragment, product, daughter or adduct ions with the intensity of said second fragment, product, daughter or adduct ions; wherein if the intensity of said first fragment, product, daughter or adduct ions differs from the intensity of said second fragment, product, daughter or adduct ions by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest; and wherein said collision, fragmentation or reaction device is selected from the group consisting of: (1) a Surface Induced Dissociation (SID") fragmentation device; (ii) an Electron Transfer Dissociation fragmentation device; (iii) an Electron Collision or Impact Dissociation fragmentation device; (iv) a Photo Induced Dissociation ("PID") fragmentation device; (v) a Laser Induced Dissociation fragmentation device; (vi) an infrared radiation induced dissociation device; (vii) an ultraviolet radiation induced dissociation device; (viii) a nozzle-skimmer interface fragmentation device; (ix) an in-source fragmentation device; (x) an ion-source Collision Induced Dissociation fragmentation device; (xi) a thermal or temperature source fragmentation device; (xii) an electric field induced fragmentation device; (xiii) a magnetic field induced -107 -fragmentation device; (xiv) an enzyme digestion or enzyme degradation fragmentation device; (xv) an ion-ion reaction fragmentation device; (xvi) an ion-molecule reaction fragmentation device; (xvii) an ion-atom reaction fragmentation device; (xviii) an ion-metastable ion reaction fragmentation device; (xix) an ion-metastable molecule reaction fragmentation device; (xx) an ion-metastable atom reaction fragmentation device; (xxi) an ion-ion reaction device for reacting ions to form adduct or product ions; (xxii) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxiv) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxv) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; and (xxvi) an ion-rnetastable atom reaction device for reacting ions to form adduct or product ions.
97. A method of mass spectrometry comprising: passing parent or precursor ions from a first sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying said collision, fragmentation or reaction device between a first mode wherein at least some of said parent or precursor ions from said first sample are fragmented or reacted into one or more fragment, product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device; repeatedly switching, altering or varying said collision, fragmentation or reaction device between a first mode wherein at least some of said parent or precursor ions from said second sample are fragmented or reacted into one or more fragment, product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; automatically determining the intensity of first fragment, product, daughter or adduct ions derived from first parent or precursor ions from said first sample, said first fragment, product, daughter or adduct ions having a first mass to charge ratio; automatically determining the intensity of second fragment, product, daughter or adduct ions derived from second parent or precursor ions from said second sample, said second fragment, product, daughter or adduct ions having said same first mass to charge ratio; determining a first ratio of the intensity of said first fragment, product, daughter or adduct ions to the intensity of other parent or precursor ions in said first sample or with the intensity of other fragment, product, daughter or adduct ions derived from other parent or precursor ions in said first sample; determining a second ratio of the intensity of said second fragment, product, daughter or adduct ions to the intensity of other parent or precursor ions in said second sample or with the intensity of other fragment, product, daughter or adduct ions derived from other parent or precursor ions in said second sample; comparing said first ratio with said second ratio; wherein if said first ratio differs from said second ratio by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest; and wherein said collision, fragmentation or reaction device is selected from the group consisting of; (i) a Surface Induced Dissociation (SID') fragmentation device; (ii) an Electron Transfer Dissociation fragmentation device; (iii) an Electron Collision or Impact Dissociation fragmentation device; (iv) a -109 -Photo Induced Dissociation (UPIDU) fragmentation device; (v) a Laser Induced Dissociation fragmentation device; (vi) an infrared radiation induced dissociation device; (vii) an ultraviolet radiation induced dissociation device; (viii) a nozz1e-skier interface fragmentation device; (ix) an in-source fragmentation device; (x) an ion-source Collision Induced Dissociation fragmentation device; (xi) a thermal or temperature source fragmentation device; (xii) an electric field induced fragmentation device; (xiii) a magnetic field induced fragmentation device; (xiv) an enzyme digestion or enzyme degradation fragmentation device; (xv) an ion-ion reaction fragmentation device; (xvi) an ion-molecule reaction fragmentation device; (xvii) an ion-atom reaction fragmentation device; (xviii) an ion-metastable ion reaction fragmentation device; (xix) an ion-inetastable molecule reaction fragmentation device; (xx) an ion-metastable atom reaction fragmentation device; (xxi) an ion-ion reaction device for reacting ions to form adduct or product ions; (xxii) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-atom reaction device for reacting ions to form adduct or product Ions; (xxiv) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxv) an ion-metastab]. e molecule reaction device for reacting ions to form adduct or product ions; and (xxvi) an ion-metastable atom reaction device for reacting ions to form adduct or product ions.
98. A mass spectrometer comprising: a collision, fragmentation or reaction device which is arranged and adapted to be repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented or reacted into one or more fragment, product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented or reacted; -110 -a mass analyser; and a control system which in use: (I) determines the intensity of first fragment, product, daughter or adduct ions derived from first parent or precursor ions from a first sample, said first fragment, product, daughter or adduct ions having a first mass to charge ratio; (ii) determines the intensity of second fragment, product, daughter or adduct ions derived from second parent or precursor ions from a second sample, said second fragment, product, daughter or adduct ions having said same first mass to charge ratio; and (iii) compares the intensity of said first fragment, product, daughter or adduct ions with the intensity of said second fragment, product, daughter or adduct ions; wherein if the intensity of said first fragment, product, daughter or adduct ions differs from the intensity of said second fragment, product, daughter or adduct ions by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are. considered to be parent or precursor ions of interest; and wherein said collision, fragmentation or reaction device is selected from the group consisting of: (i) a Surface Induced Dissociation ("SID) fragmentation device; (ii) an Electron Transfer Dissociation fragmentation device; (iii) an Electron Collision or Impact Dissociation fragmentation device; (iv) a Photo Induced Dissociation (PID") fragmentation device; (v) a Laser Induced Dissociation fragmentation device; (v-i) an infrared radiation induced dissociation device; (vii) an ultraviolet radiation induced dissociation device; (viii) a nozzle-skimmer interface fragmentation device; (ix) an in-source fragmentation device; (x) an ion-source Collision Induced Dissociation fragmentation device; (xi) a thermal or temperature source fragmentation device; (xii) an electric field induced fragmentation device; (xiii) a magnetic field induced fragmentation device; (xiv) an enzyme digestion or enzyme -111 -degradation fragmentation device; (xv) an ion-ion reaction fragmentation device; (xvi) an ion-molecule reaction fragmentation device; (xvii) an ion-atom reaction fragmentation device; (xviii) an ion-metastab].e ion reaction fragmentation device; (xix) an ion-metastab].e molecule reaction fragmentation device; (xx) an ion-metastab].e atom reaction fragmentation device; (xxi) an ion-ion reaction device for reacting ions to form adduct or product ions; (xxii) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxiv) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxv) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; and (xxvi) an ion-metastable atom reaction device for reacting ions to form adduct or product ions.
99. A mass spectrometer comprising: a collision, fragmentation or reaction device repeatedly switched, altered or varied in use between a first mode wherein at least some parent or precursor ions are fragmented into one or more fragment, product, daughter or adduct ions and a second mode wherein substantially fewer parent or precursor ions are fragmented; amass analyser; and a control system which in use: (i) determines the intensity of first fragment, product, daughter or adduct ions derived from first parent or precursor ions from a first sample, said first fragment, product, daughter or adduct ions having a first mass to charge ratio; (ii) determines the intensity of second fragment, prQduct, daughter or adduct ions derived from second parent or precursor ions from a second sample, said second fragment, product, daughter or adduct ions having said same first mass to charge ratio; -112 - (iii) determines a first ratio of the intensity of said first fragment, product, daughter or adduct ions to the intensity of other parent or precursor ions in said first sample or with the intensity of other fragment, product, daughter or adduct ions derived from other parent or precursor ions in said first sample; (iv) determines a second ratio of the intensity of said second fragment, product, daughter or adduct ions to the intensity of other parent or precursor ions in said second sample or with the intensity of other fragment, product, daughter or adduct ions derived from other parent or precursor ions in said second sample; and (v) compares said first ratio with said second ratio; wherein if said first ratio differs from said second ratio by more than a predetermined amount then either said first parent or precursor ions and/or said second parent or precursor ions are considered to be parent or precursor ions of interest; and wherein said collision, fragmentation or reaction device is selected from the group consisting of: (i) a Surface Induced Dissociation (SID') fragmentation device; (ii) an Electron Transfer Dissociation fragmentation device; (iii) an Electron Collision or Impact Dissociation fragmentation device; (iv) a Photo Induced Dissociation (PIB) fragmentation device; (v) a Laser Induced Dissociation fragmentation device; (vi) an infrared radiation induced dissociation device; (vii) an ultraviolet radiation induced dissociation device; (viii) a nozzle-skimmer interface fragmentation device; (ix) an in-source fragmentation device; (x) an ion-source Collision Induced Dissociation fragmentation device; (xi) a thermal or temperature source fragmentation device; (xii) an electric field induced fragmentation device; (xiii) a magnetic field induced fragmentation device; (xiv) an enzyme digestion or enzyme degradation fragmentation device; (xv) an ion-ion reaction fragmentation device; (xvi) an ion-molecule reaction -113 -fragmentation device; (xvii) an ion-atom reaction fragmentation device; (xviii) an ion-metastable ion reaction fragmentation device; (xix) an ion-metastab]. e molecule reaction fragmentation device; (xx) an ion-metastable atom reaction fragmentation device; (xxi) an ion-ion reaction device for reacting ions to form adduct or product ions; (xxii) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxiii) an ion-atom reaction device for reacting ions to form adduct or product ions; (xxiv) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxv) an ion-metastable molecule reaction device for reacting ions to form adductor product ions; and (xxvi) an ion-metastable atom reaction device for reacting ions to form adduct or product ions.