JP4959712B2 - Mass spectrometer - Google Patents

Description

  The present invention relates to a mass spectrometry method and a mass spectrometer.

  It has become common practice to analyze a protein by first digesting the protein enzymatically or chemically and then analyzing the peptide product by mass spectrometry. Mass spectrometry of peptide products usually involves measuring the mass of the peptide product. This method is sometimes referred to as “peptide mapping” or “peptide fingerprinting”.

  Also known as a method to try to identify a parent or precursor peptide ion is to induce the parent or precursor peptide ion to fragment and then measure the mass of one or more fragment or daughter ions . In addition, the fragmentation pattern of peptide ions has been shown to be a method of successful identification of isobaric peptide ions. Thus, the mass to charge ratio of one or more fragment or daughter ions can be used to identify the parent or precursor peptide ion, thereby identifying the protein from which the peptide is derived. Also, in some cases, the partial sequence of the peptide can be determined from the fragment or daughter ion spectrum. This information can be used to determine candidate proteins by searching protein and genomic databases.

  Alternatively, the candidate protein compares the mass of one or more observed fragments or daughter ions with the mass of fragments or daughter ions that can be expected to be observed based on the peptide sequence of the candidate protein of interest. Can be excluded or confirmed. The confidence of identification increases as more peptide parent or precursor ions are induced to fragment and their fragment masses are shown to match what is expected.

  It would be desirable to provide improved mass spectrometry methods and mass spectrometers.

According to one aspect of the present invention, a method of mass spectrometry comprising:
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device comprising an electron capture dissociation fragmentation device;
Repeatedly switching, modifying or changing the electron capture dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the first sample At least some of which are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode, 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, modifying or changing the electron capture dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the second sample At least some of which are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented;
Recognizing a first parent or precursor ion of interest from a first sample;
Automatically determining the intensity of a first parent or precursor ion of interest, wherein the first parent or precursor ion of interest has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
Comparing the intensity of the first parent or precursor ion of interest with the intensity of the second parent or precursor ion.

    According to the preferred embodiment, the parent or precursor ion comprises a divalent, trivalent, tetravalent or pentavalent or higher ion.

  According to the preferred embodiment, the electron capture dissociation fragmentation device is preferably switched repeatedly between the first and second modes during one experimental trial or one sample analysis.

  In the first mode of operation, the electrons are preferably from the group consisting of (i) <1 eV, (ii) 1-2 eV, (iii) 2-3 eV, (iv) 3-4 eV, and (v) 4-5 eV. Have energy selected.

  In the first mode of operation, relatively low energy electrons are preferably confined by a relatively strong magnetic field.

  The ions to be fragmented are preferably confined within an ion guide. An AC or RF voltage is preferably applied to the electrode of the ion guide to create a radial pseudopotential field or well that preferably acts to confine ions radially within the ion guide.

  Preferably, the relatively low energy electrons are preferably confined by a magnetic field that overlaps or overlaps the ion guide region of the ion guide so that the multivalent analyte ions interact with the relatively low energy electrons. Fragmentation of ions by electron capture dissociation preferably does not include introducing internal vibrational energy into the ions.

  The method preferably further comprises the step of providing an electron source. In the first mode of operation, the electron source generates a plurality of electrons that are preferably configured to interact with a parent or precursor ion.

  In the second mode of operation, the electron source is preferably switched off so that the analyte ions preferably do not interact with the electrons and therefore preferably do not fragment.

According to one aspect of the present invention, a method of mass spectrometry comprising:
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device comprising an electron transfer dissociation fragmentation device;
Repeatedly switching, modifying or changing the electron transfer dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the first sample At least some of which are fragmented as they interact with reagent ions to produce fragment or daughter ions, and in the second mode, 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, modifying or changing the electron transfer dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the second sample At least some of which are fragmented as they interact with reagent ions to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented;
Recognizing a first parent or precursor ion of interest from a first sample;
Automatically determining the intensity of a first parent or precursor ion of interest, wherein the first parent or precursor ion of interest has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
Comparing the intensity of the first parent or precursor ion of interest with the intensity of the second parent or precursor ion.

  According to the preferred embodiment, the parent or precursor ion comprises a divalent, trivalent, tetravalent or pentavalent or higher ion.

  According to the preferred embodiment, the electron transfer dissociation fragmentation device is preferably repeatedly switched between the first and second modes during one experimental trial or one sample analysis.

According to one aspect of the present invention, 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, modifying or changing the surface-induced dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the first sample At least some of which are fragmented as they hit the surface or target plate to produce fragment or daughter ions, and in the second mode, 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, modifying or changing the surface-induced dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the second sample At least some of which are fragmented as they hit the surface or target plate to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented;
Recognizing a first parent or precursor ion of interest from a first sample;
Automatically determining the intensity of a first parent or precursor ion of interest, wherein the first parent or precursor ion of interest has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
Comparing the intensity of the first parent or precursor ion of interest with the intensity of the second parent or precursor ion.

  According to the preferred embodiment, the parent or precursor ion comprises a divalent, trivalent, tetravalent or pentavalent or higher ion.

  According to the preferred embodiment, the surface induced dissociation fragmentation device is preferably repeatedly switched between the first and second modes during one experimental trial or one sample analysis.

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

  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 plate is preferably configured in a plane that is substantially parallel to the direction of travel of the parent or precursor ions in the second mode of operation. Note that the second mode of operation is when ions are transported past the surface or target plate, preferably not directed onto the surface or target plate.

According to one aspect of the present invention, a method of mass spectrometry comprising:
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device;
Repeatedly switching, modifying or changing a collision, fragmentation or reaction device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the first sample At least some of which are fragmented or reacted to produce fragment, daughter, product or adduct ions, and in the second mode, substantially less parent or precursor ions are fragmented or reacted, step When,
Passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device;
Repeatedly switching, modifying or changing a collision, fragmentation or reaction device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the second sample At least some of which are fragmented or reacted to produce fragment, daughter, product or adduct ions, and in the second mode, substantially less parent or precursor ions are fragmented or reacted, step When,
Recognizing a first parent or precursor ion of interest from a first sample;
Automatically determining the intensity of a first parent or precursor ion of interest, wherein the first parent or precursor ion of interest has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
Comparing the intensity of the first parent or precursor ion of interest with the intensity of the second parent or precursor ion;
Collision, fragmentation or reaction devices are: (i) electron impact or impact dissociation fragmentation device, (ii) photo-induced dissociation (“PID”) fragmentation device, (iii) laser induced dissociation fragmentation device, (iv) infrared radiation induced dissociation Devices, (v) ultraviolet radiation induced dissociation devices, (vi) nozzle-skim interface fragmentation devices, (vii) in-source fragmentation devices, (viii) ion source collision induced dissociation fragmentation devices, (ix) thermal or temperature source fragmentation devices (X) electric field induced fragmentation device, (xi) magnetic field induced fragmentation device, (xii) enzymatic digestion or enzymatic degradation fragmentation device Chair, (xiii) ion-ion reaction fragmentation device, (xiv) ion-molecule reaction fragmentation device, (xv) ion-atom reaction fragmentation device, (xvi) ion-metastable ion reaction fragmentation device, (xvii) ion-metas Table molecular reaction fragmentation device, (xviii) ion-metastable atomic reaction fragmentation device, (xix) ion-ion reaction device for reacting ions to form adducts or product ions, (xx) reacting ions Ion-molecule reaction devices for forming adducts or product ions, (xxi) ion-atom reactions for reacting ions to form adducts or product ions Device, (xxii) Ion for reacting ions to form adduct or product ions-Metastable ion reaction device, (xxiii) Ion for reacting ions to form adduct or product ions- Selected from the group consisting of metastable molecular reaction devices, and ion-metastable atomic reaction devices for reacting (xxiv) ions to form adducts or product ions,
A method is provided.

  According to the preferred embodiment, the parent or precursor ion comprises a divalent, trivalent, tetravalent or pentavalent or higher ion.

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

According to one aspect of the present invention, a method of mass spectrometry comprising:
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device comprising an electron capture dissociation fragmentation device;
Repeatedly switching, modifying or changing the electron capture dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the first sample At least some of which are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode, 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, modifying or changing the electron capture dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the second sample At least some of which are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented;
Recognizing a first parent or precursor ion of interest from a first sample;
Automatically determining the intensity of a first parent or precursor ion of interest, wherein the first parent or precursor ion of interest has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
Determining a first ratio of the intensity of the first parent or precursor ion of interest to the intensity of other parent or precursor ions in the first sample;
Determining a second ratio of the intensity of second parent or precursor ions to the intensity of other parent or precursor ions in the second sample;
Comparing the first ratio to the second ratio.

  According to the preferred embodiment, the parent or precursor ion comprises a divalent, trivalent, tetravalent or pentavalent or higher ion.

  According to the preferred embodiment, the electron capture dissociation fragmentation device is preferably switched repeatedly between the first and second modes during one experimental trial or one sample analysis.

  In the first mode of operation, the electrons are preferably from the group consisting of (i) <1 eV, (ii) 1-2 eV, (iii) 2-3 eV, (iv) 3-4 eV, and (v) 4-5 eV. Have energy selected.

  In the first mode of operation, the electrons are preferably confined by a magnetic field.

  The method preferably further comprises the step of providing an electron source. In the first mode of operation, the electron source generates a plurality of electrons that are preferably configured to interact with a parent or precursor ion. In the second mode of operation, the electron source is preferably switched off.

According to another aspect of the invention, a method of mass spectrometry comprising:
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device comprising an electron transfer dissociation fragmentation device;
Repeatedly switching, modifying or changing the electron transfer dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the first sample At least some of which are fragmented as they interact with reagent ions to produce fragment or daughter ions, and in the second mode, 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, modifying or changing the electron transfer dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the second sample At least some of which are fragmented as they interact with reagent ions to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented;
Recognizing a first parent or precursor ion of interest from a first sample;
Automatically determining the intensity of a first parent or precursor ion of interest, wherein the first parent or precursor ion of interest has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
Determining a first ratio of the intensity of the first parent or precursor ion of interest to the intensity of other parent or precursor ions in the first sample;
Determining a second ratio of the intensity of second parent or precursor ions to the intensity of other parent or precursor ions in the second sample;
Comparing the first ratio to the second ratio.

  According to the preferred embodiment, the parent or precursor ion comprises a divalent, trivalent, tetravalent or pentavalent or higher ion.

  According to the preferred embodiment, the electron transfer dissociation fragmentation device is preferably repeatedly switched between the first and second modes during one experimental trial or one sample analysis.

According to one aspect of the present invention, 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, modifying or changing the surface-induced dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the first sample At least some of which are fragmented as they hit the surface or target plate to produce fragment or daughter ions, and in the second mode, 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, modifying or changing the surface-induced dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the second sample At least some of which are fragmented as they hit the surface or target plate to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented;
Recognizing a first parent or precursor ion of interest from a first sample;
Automatically determining the intensity of a first parent or precursor ion of interest, wherein the first parent or precursor ion of interest has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
Determining a first ratio of the intensity of the first parent or precursor ion of interest to the intensity of other parent or precursor ions in the first sample;
Determining a second ratio of the intensity of second parent or precursor ions to the intensity of other parent or precursor ions in the second sample;
Comparing the first ratio to the second ratio.

  According to the preferred embodiment, the parent or precursor ion comprises a divalent, trivalent, tetravalent or pentavalent or higher ion.

  According to the preferred embodiment, the surface induced dissociation fragmentation device is preferably repeatedly switched between the first and second modes during one experimental trial or one sample analysis.

  In the first mode of operation, the parent or precursor ions are preferably directed, redirected or deflected onto the surface or target plate.

  In the second mode of operation, the parent or precursor ions are preferably not directed onto the surface or target plate, are not redirected, or deflected.

  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 plate is preferably configured in a plane that is substantially parallel to the direction of travel of the parent or precursor ions in the second mode of operation.

According to one aspect of the present invention, a method of mass spectrometry comprising:
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device;
Repeatedly switching, modifying or changing a collision, fragmentation or reaction device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the first sample At least some of which are fragmented or reacted to produce fragment, daughter, product or adduct ions, and in the second mode, substantially less parent or precursor ions are fragmented or reacted, step When,
Passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device;
Repeatedly switching, modifying or changing a collision, fragmentation or reaction device between a first mode and a second mode, wherein in the first mode the parent or precursor ions from the second sample At least some of which are fragmented or reacted to produce fragment, daughter, product or adduct ions, and in the second mode, substantially less parent or precursor ions are fragmented or reacted, step When,
Recognizing a first parent or precursor ion of interest from a first sample;
Automatically determining the intensity of a first parent or precursor ion of interest, wherein the first parent or precursor ion of interest has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
Determining a first ratio of the intensity of the first parent or precursor ion of interest to the intensity of other parent or precursor ions in the first sample;
Determining a second ratio of the intensity of second parent or precursor ions to the intensity of other parent or precursor ions in the second sample;
Comparing the first ratio to the second ratio; and
Collision, fragmentation or reaction devices are: (i) electron impact or impact dissociation fragmentation device, (ii) photo-induced dissociation (“PID”) fragmentation device, (iii) laser induced dissociation fragmentation device, (iv) infrared radiation induced dissociation Devices, (v) ultraviolet radiation induced dissociation devices, (vi) nozzle-skim interface fragmentation devices, (vii) in-source fragmentation devices, (viii) ion source collision induced dissociation fragmentation devices, (ix) thermal or temperature source fragmentation devices (X) electric field induced fragmentation device, (xi) magnetic field induced fragmentation device, (xii) enzymatic digestion or enzymatic degradation fragmentation device Chair, (xiii) ion-ion reaction fragmentation device, (xiv) ion-molecule reaction fragmentation device, (xv) ion-atom reaction fragmentation device, (xvi) ion-metastable ion reaction fragmentation device, (xvii) ion-metas Table molecular reaction fragmentation device, (xviii) ion-metastable atomic reaction fragmentation device, (xix) ion-ion reaction device for reacting ions to form adducts or product ions, (xx) reacting ions Ion-molecule reaction devices for forming adducts or product ions, (xxi) ion-atom reactions for reacting ions to form adducts or product ions Device, (xxii) Ion for reacting ions to form adduct or product ions-Metastable ion reaction device, (xxiii) Ion for reacting ions to form adduct or product ions- Selected from the group consisting of metastable molecular reaction devices, and ion-metastable atomic reaction devices for reacting (xxiv) ions to form adducts or product ions,
A method is provided.

  According to the preferred embodiment, the parent or precursor ion comprises a divalent, trivalent, tetravalent or pentavalent or higher ion.

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

  A reaction device should be understood to include a device that is reconfigured or reacted so that ions, atoms or molecules form new species of ions, atoms or molecules. An XY reaction fragmentation device should be understood to mean a device in which X and Y combine to form a product and then fragment. This is different from the fragmentation device itself where ions can be fragmented without first forming a product. An XY reaction device should be understood to mean a device in which X and Y combine to form a product, and then the product does not necessarily fragment.

  According to the present invention, ions are collided, fragmented or reacted in devices other than collision-induced dissociation fragmentation devices. According to a particularly preferred embodiment, an electron capture dissociation (“ECD”) or electron transfer dissociation (“ETD”) fragmentation device can be used to fragment analyte ions.

  A polypeptide chain consists of amino acid residues having a predetermined mass. There are three different bonds along the peptide backbone, and if one bond breaks, the charge can remain at either the N-terminal part of the structure or the C-terminal part of the structure. When a polypeptide is fragmented, six possible fragmentation series commonly referred to as a, b, c and x, y, z result.

  When using collision-induced dissociation, the most common fragmentation pathway is for fragmentation that occurs via the amide bond (II). If the charge remains at the N-terminus, the ion is called a b-series ion. If charge remains at the C-terminus, the ion is called a y-series ion.

The subscript can be used to indicate how many amino acid residues are included in the fragment. For example, b ₃ is a fragment ion obtained as a result of cleaving the amino bond (II) so that a charge remains at the N-terminus, and there are 3 amino acid residues in the fragment.

  According to one embodiment of the invention, when fragmenting ions using an electron capture dissociation (“ECD”) or electron transfer dissociation (“ETD”) fragmentation device, the polypeptide chain is formed by collision-induced dissociation of the polypeptide. When fragmented, it may be fragmented at a location different from the location where fragmentation is expected to occur. In particular, electron capture dissociation (“ECD”) or electron transfer dissociation (“ETD”) devices allow x and c series fragment ions to be primarily produced. In certain situations, it is particularly advantageous to fragment ions into x and c series fragment ions rather than b and y series fragment ions (in the case of collision induced dissociation). In some situations, more complete sequences are possible using ECD or ETD, and there is less ambiguity in identifying fragment ions. This makes the peptide sequencing process easier.

  Polypeptides can also be modified by post-translational modifications such as phosphorylation. The use of ECD or ETD fragmentation devices and the resulting fragmentation series allows easier observation of post-translational modifications such as phosphorylation. It is also possible to determine 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 is a relatively slow process and can be thought of as a process where fragmentation often occurs as a result of multiple collisions between ions and gas molecules. As a result, fragmentation is easy to average and a relatively wide range of fragmentation products is usually observed. Conversely, surface induced dissociation can be considered a relatively rapid or instantaneous process. As a result, polypeptides can be fragmented in a very specific manner. In certain situations, this is particularly useful because it can reveal certain useful information about the structure of the polypeptide.

  Thus, it can be seen that the present invention is particularly advantageous in that the parent or precursor ions are fragmented via a fragmentation pathway that is preferably different from that due to collision-induced dissociation. Furthermore, the present invention also allows post-translational modification of the peptide to be observed and the position of the modification in the peptide to be determined. The present invention is also particularly advantageous over conventional approaches for attempting to elucidate structural information about analyte ions by fragmenting analyte ions and analyzing corresponding fragment ions.

  Thus, it can be seen that the present invention is particularly advantageous in that the parent or precursor ions are fragmented via a fragmentation pathway that is preferably different from that due to collision-induced dissociation. Furthermore, the present invention also allows post-translational modification of the peptide to be observed and the position of the modification in the peptide to be determined.

  Therefore, the present invention is particularly advantageous compared to the conventional configuration.

  Also, instead of determining a first ratio of a first parent or precursor ion to another parent or precursor ion, a given fragment, product, daughter or adduct ion of the first parent or precursor ion Other configurations are conceivable in which the first ratio to can be determined. Similarly, a second ratio of a second parent or precursor ion to a given fragment, product, daughter or adduct ion can be determined and the first and second ratios can be compared.

  The method preferably involves at least 0.1, 0.2, 0.3, 0.4, 0.5, 0... Collision, fragmentation or reaction device between at least the first mode and the second mode. Automatically switching, modifying or changing once every 6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 seconds including.

  Whether other parent or precursor ions present in the first sample and / or other parent or precursor ions present in the second sample are endogenous or exogenous to the sample It can be either. Other parent or precursor ions present in the first sample and / or other parent or precursor ions present in the second sample can further be used as chromatographic retention time criteria.

  According to one embodiment, parent or precursor ions, preferably peptide ions, from two different samples are analyzed in separate experimental trials. In each experimental trial, parent or precursor ions are passed to the collision, fragmentation or reaction device. The collision, fragmentation or reaction device is preferably switched repeatedly between a fragmentation or reaction mode and a mode that does not substantially fragment or react. The ions that emerge from, or have been transported through, the collision, fragmentation or reaction device are then preferably mass analyzed. The intensity of the parent or precursor ion having a predetermined mass to charge ratio in one sample is then compared to the intensity of the parent or precursor ion having the same predetermined mass to charge ratio in the other sample. A direct comparison of parent or precursor ion expression levels can be made, or the intensity of the parent or precursor ions in the sample can be first compared to an internal standard. Thus, the ratio of the parent or precursor ion relative to the internal reference of the parent or precursor ion in one sample to the intensity of the parent or precursor ion in the other sample and preferably the parent or precursor ion related to the same internal reference of the parent or precursor ion in the other sample An indirect comparison can be made between the ratio of the intensity to the intensity. A comparison of the two ratios can then be made. While the preferred embodiment has been described with respect to comparing parent or precursor ion expression levels in two samples, it is clear that the expression levels of parent or precursor ions in more than two samples can be compared.

  Parental or precursor ions preferably have their expression levels of 1%, 10%, 50%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, It can be considered that the two samples differ significantly if they differ by more than 1000%, 5000% or 10000%.

  The collision, fragmentation or reaction device is preferably (i) 0.0001 mbar or more, (ii) 0.001 mbar or more, (iii) 0.05 mbar or more, (iv) 0.01 mbar or more, (v) 0.0001 to 100 mbar. And (vi) maintained at a pressure selected from the group consisting of 0.001-10 mbar. Preferably, the collision, fragmentation or reaction device is (i) 0.0001 mbar or higher, (ii) 0.0005 mbar or higher, (iii) 0.001 mbar or higher, (iv) 0.005 mbar or higher, (v) 0.01 mbar or higher. , (Vi) 0.05 mbar or more, (vii) 0.1 mbar or more, (viii) 0.5 mbar or more, (ix) 1 mbar or more, (x) 5 mbar or more, and (xi) 10 mbar or more. Maintained at pressure. Preferably, the collision, fragmentation or reaction device is (i) 10 mbar or less, (ii) 5 mbar or less, (iii) 1 mbar or less, (iv) 0.5 mbar or less, (v) 0.1 mbar or less, (vi) 0. Selected from the group consisting of 05 mbar or less, (vii) 0.01 mbar or less, (viii) 0.005 mbar or less, (ix) 0.001 mbar or less, (x) 0.0005 mbar or less, and (xi) 0.0001 mbar or less. Maintained at pressure.

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

  A parent ion that is considered to be the parent or precursor ion of interest is preferably identified. This may include determining the mass to charge ratio of the parent or precursor ion of interest, preferably with an accuracy of 20 ppm, 15 ppm, 10 ppm or 5 ppm or less. The determined mass-to-charge ratio of the subject's parent or precursor ions can then be compared to a database of ions and their mass-to-charge ratio so that the identity of the subject's parent or precursor ions can be established.

  According to the preferred embodiment, the step of identifying the parent or precursor ion of interest comprises one or more fragments, products, daughters or adducts that are determined to be derived from fragmentation of the parent or precursor ion of interest. Identifying the ions. Preferably, the step of identifying one or more fragments, products, daughters or adduct ions comprises a mass to charge ratio of one or more fragments, products, daughters or adduct ions of 20 ppm, 15 ppm, 10 ppm or 5 ppm. The following further includes the step of determining.

  The step of identifying the first parent or precursor ion of interest is observed in the mass spectrum obtained when the parent or precursor ion is in collision, fragmentation or reaction device in low fragmentation or reaction mode for a predetermined period of time. Whether and when the first fragment, product, daughter or adduct ion is collision, fragmentation or reaction device is in high fragmentation or reaction mode, just before a predetermined period of time, or collision, fragmentation or reaction device is high fragmentation Or it may include determining whether it is observed in a mass spectrum obtained either immediately after a predetermined period of time when in reaction mode.

  Identifying the first parent or precursor ion of interest may comprise comparing the elution time of the parent or precursor ion to the pseudoelution time of the first fragment, product, daughter or adduct ion. Fragments, products, daughters or adduct ions are said to have a pseudo-elution time. This is because fragments, products, daughters or adduct ions do not actually physically elute from the chromatography column. However, at least some of the fragment, product, daughter or adduct ions are fairly unique for a particular parent or precursor ion, and the parent or precursor ion is chromatographic column only at a particular time. The corresponding fragment, product, daughter or adduct ions can be similarly observed only at substantially the same elution time as their associated parent or precursor ions. Similarly, identifying the first parent or precursor ion of interest comprises comparing the elution profile of the parent or precursor ion to the pseudoelution profile of the first fragment, product, daughter or adduct ion. May be included. Again, although fragments, products, daughters or adduct ions do not actually physically elute from the chromatography column, they can be considered to have an effective elution profile. Because they tend to be observed only when a particular parent or precursor ion elutes from the column and as the intensity of the eluting parent or precursor ion changes over a few seconds, it is also a characteristic fragment This is because the intensity of the product, daughter or adduct ions changes as well.

  An ion can be determined to be a parent or precursor ion by comparing two sequentially obtained mass spectra. The first mass spectrum is obtained when the collision, fragmentation or reaction device is in high fragmentation and reaction mode, and the second mass spectrum is when the collision, fragmentation or reaction device is in low fragmentation and reaction mode Is obtained. An ion is determined to be a parent or precursor ion if the peak corresponding to the ion in the second mass spectrum is higher in intensity than the peak corresponding to the ion in the first mass spectrum. Similarly, an ion is determined to be a fragment, product, daughter or adduct ion when the peak corresponding to the ion in the first mass spectrum is more intense than the peak corresponding to the ion in the second mass spectrum. Can be done. According to another embodiment, a mass filter may be provided upstream of the collision, fragmentation or reaction device. The mass filter is configured to transport ions having a mass to charge ratio in the first range, but substantially attenuate ions having a mass to charge ratio in the second range. An ion is determined to be a fragment, product, daughter or adduct ion if it is determined to have a mass to charge ratio within the second range.

  It is determined that the first parent or precursor ion and the second parent or precursor ion preferably have mass to charge ratios that differ by no more than 40 ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm, 15 ppm, 10 ppm, or 5 ppm. It can be determined that the first parent or precursor ion and the second parent or precursor ion have eluted from the chromatography column after substantially the same elution time. Also, it is determined that the first parent or precursor ion has produced one or more first fragment, product, daughter or adduct ions, and the second parent or precursor ion is one or more It can be determined that a second fragment, product, daughter or adduct ion has been generated. The one or more first fragments, product, daughter or adduct ions and the one or more second fragments, product, daughter or adduct ions have substantially the same mass to charge ratio. The mass-to-charge ratio of the one or more first fragments, product, daughter or adduct ions and one or more second fragments, product, daughter or adduct ions is 40 ppm, 35 ppm, 30 ppm, 25 ppm, 20 ppm , 15 ppm, 10 ppm, or 5 ppm or less.

  Also, it can be determined that the first parent or precursor ion gave rise to one or more first fragment, product, daughter or adduct ions, and the second parent or precursor ion is one or more. Of the second fragment, product, daughter or adduct ion. A first parent or precursor ion and a second parent or precursor ion are observed in a mass spectrum associated with data obtained in a low fragmentation or reaction mode at a given point in time, and one or more first and second Fragments, products, daughters or adduct ions, just before a given point in time when the collision, fragmentation or reaction device is in high fragmentation or reaction mode, or when the fragmentation or reaction device is in high fragmentation or reaction mode Is observed in the mass spectrum associated with the data obtained either immediately after a given point in time.

  If the first fragment, product, daughter or adduct ion has substantially the same pseudoelution time as the second fragment, product, daughter or adduct ion, then the first parent or precursor ion is 1 It may be determined that one or more first fragments, products, daughters or adduct ions have been generated, and the second parent or precursor ion is one or more second fragments, products, daughters or adducts It can be determined that ions have been generated.

  The first parent or precursor ion may be determined to have produced one or more first fragment, product, daughter or adduct ions, and the second parent or precursor ion may be one or more first It can be determined that two fragments, products, daughters or adduct ions were generated. The first parent or precursor ion is determined to have an elution profile that correlates to the pseudoelution profile of the first fragment, product, daughter or adduct ion, and the corresponding second parent or precursor ion is It is determined to have an elution profile that correlates to the pseudoelution profile of the second fragment, product, daughter or adduct ion.

  According to another embodiment, the first parent or precursor ion being compared and the second parent or precursor ion being compared can be determined to be multivalent. This may exclude many fragments, products, daughters or adduct ions that tend to be monovalent quite often. According to a more preferred embodiment, the first parent or precursor ion and the second parent or precursor ion can be determined to have the same charge state. According to another embodiment, parent or precursor ions being compared in two different samples can be determined to give rise to fragment, product, daughter or adduct ions having the same charge state.

  The first sample and / or the second sample is a portion of a plurality of different biopolymers, proteins, peptides, polypeptides, oligonucleotides, oligonucleosides, amino acids, carbohydrates, sugars, lipids, fatty acids, vitamins, hormones, DNA Or fragment, cDNA portion or fragment, RNA portion or fragment, mRNA portion or fragment, tRNA portion or fragment, polyclonal antibody, monoclonal antibody, ribonuclease, enzyme, metabolite, polysaccharide, phosphorylated peptide, phosphorylated protein , Glycopeptides, glycoproteins or steroids. Also, the first sample and / or the second sample has at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, having different identities. It may contain 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 molecules.

  The first sample can be taken from a diseased organism and the second sample can be taken from an unaffected organism. Alternatively, the first sample can be taken from a treated organism and the second sample can be taken from a non-treated organism. According to another embodiment, the first sample can be taken from a mutant organism and the second sample can be taken from a wild type organism.

  Molecules from the first and / or second sample are preferably high performance liquid chromatography (“HPLC”), anion exchange, anion exchange chromatography, cation exchange, cation exchange chromatography, ion-pair reverse phase chromatography, Chromatography, one-dimensional electrophoresis, multi-dimensional electrophoresis, size exclusion, affinity, reverse phase chromatography, cavitation-electrophoretic chromatography (“CEC”), electrophoresis, ion mobility separation, field asymmetric ion mobility separation (“FAIMS” "), Or separated from a mixture of other molecules before being ionized by capillary electrophoresis.

  According to a particularly preferred embodiment, the first and second sample ions may comprise peptide ions. Peptide ions preferably include the digestion product of one or more proteins. Attempts can be made to identify proteins that correlate with the parent peptide ion of interest. Preferably, a determination is made as to which peptide product is expected to be formed when the protein is digested, and then whether any predicted peptide product correlates to the parent or precursor ion of interest Will be judged. A determination can also be made as to whether a subject's parent or precursor ion correlates with one or more proteins.

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

  A check may be made to determine that the first and second parent or precursor ions being compared are indeed parent or precursor ions, rather than fragment, product, daughter or adduct ions. A high fragmentation mass spectrum associated with data obtained in high fragmentation or reaction mode may be compared to a low fragmentation mass spectrum associated with data obtained in low fragmentation or reaction mode. Here, both mass spectra were obtained substantially simultaneously. A first and / or second parent or precursor ion is determined not to be a fragment, product, daughter or adduct ion if it has a greater intensity in the low fragmentation mass spectrum compared to the high fragmentation mass spectrum. obtain. Similarly, a fragment, product, daughter or adduct ion may be recognized by noting an ion that has a greater intensity in the high fragmentation mass spectrum compared to the low fragmentation mass spectrum.

  The parent or precursor ions from the first sample and the parent or precursor ions from the second sample are preferably passed to the same collision, fragmentation or reaction device. However, according to less preferred embodiments, the parent or precursor ions from the first sample and the parent or precursor ions from the second sample can be passed to different collision, fragmentation or reaction devices.

According to one aspect of the invention,
An electron capture dissociation fragmentation device configured and adapted to repeatedly switch, modify or change between a first mode and a second mode in use, wherein in the first mode, at least some An electron capture dissociation fragmentation device in which the parent or precursor ions are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode substantially fewer parent or precursor ions are fragmented. When,
A mass analyzer;
A control system, wherein in use: (i) recognizing a first parent or precursor ion of interest from a first sample, the first parent or precursor ion of interest comprising a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of interest;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
And (iv) a control system that compares the intensity of the first parent or precursor ion of interest with the intensity of the second parent or precursor ion.

According to one aspect of the invention,
An electron transfer dissociation fragmentation device configured and adapted to repeatedly switch, modify or change between a first mode and a second mode in use, wherein in the first mode, at least some Electron transfer dissociation fragmentation in which parent or precursor ions are fragmented as they interact with reagent ions to produce fragment or daughter ions, and in the second mode substantially fewer parent or precursor ions are fragmented The device,
A mass analyzer;
A control system, wherein in use: (i) recognizing a first parent or precursor ion of interest from a first sample, the first parent or precursor ion of interest comprising a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of interest;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
And (iv) a control system that compares the intensity of the first parent or precursor ion of interest with the intensity of the second parent or precursor ion.

According to one aspect of the invention,
A surface induced dissociation fragmentation device configured and adapted to be repeatedly switched, modified or changed between a first mode and a second mode in use, wherein in the first mode at least some A surface-induced dissociation fragmentation device that is fragmented when parent or precursor ions strike a surface or target plate to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented When,
A mass analyzer;
A control system, wherein in use: (i) recognizing a first parent or precursor ion of interest from a first sample, the first parent or precursor ion of interest comprising a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of interest;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
And (iv) a control system that compares the intensity of the first parent or precursor ion of interest with the intensity of the second parent or precursor ion.

According to one aspect of the invention,
A collision, fragmentation or reaction device configured and adapted to be repeatedly switched, modified or changed between a first mode and a second mode in use, wherein at least some of the first mode Collisions in which the parent or precursor ions are fragmented or reacted to produce fragment, product, daughter or adduct ions, and in the second mode substantially fewer parent or precursor ions are fragmented or reacted With fragmentation or reaction devices,
A mass analyzer;
A control system, wherein in use: (i) recognizing a first parent or precursor ion of interest from a first sample, the first parent or precursor ion of interest comprising a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of interest;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
(Iv) a control system that compares the intensity of the first parent or precursor ion of interest with the intensity of the second parent or precursor ion;
Collision, fragmentation or reaction devices are: (i) electron impact or impact dissociation fragmentation device, (ii) photo-induced dissociation (“PID”) fragmentation device, (iii) laser induced dissociation fragmentation device, (iv) infrared radiation induced dissociation Devices, (v) ultraviolet radiation induced dissociation devices, (vi) nozzle-skim interface fragmentation devices, (vii) in-source fragmentation devices, (viii) ion source collision induced dissociation fragmentation devices, (ix) thermal or temperature source fragmentation devices (X) electric field induced fragmentation device, (xi) magnetic field induced fragmentation device, (xii) enzymatic digestion or enzymatic degradation fragmentation device Chair, (xiii) ion-ion reaction fragmentation device, (xiv) ion-molecule reaction fragmentation device, (xv) ion-atom reaction fragmentation device, (xvi) ion-metastable ion reaction fragmentation device, (xvii) ion-metas Table molecular reaction fragmentation device, (xviii) ion-metastable atomic reaction fragmentation device, (xix) ion-ion reaction device for reacting ions to form adducts or product ions, (xx) reacting ions Ion-molecule reaction devices for forming adducts or product ions, (xxi) ion-atom reactions for reacting ions to form adducts or product ions Device, (xxii) Ion for reacting ions to form adduct or product ions-Metastable ion reaction device, (xxiii) Ion for reacting ions to form adduct or product ions- Selected from the group consisting of metastable molecular reaction devices, and ion-metastable atomic reaction devices for reacting (xxiv) ions to form adducts or product ions,
A mass spectrometer is provided.

According to one aspect of the invention,
An electron capture dissociation fragmentation device configured and adapted to repeatedly switch, modify or change between a first mode and a second mode in use, wherein in the first mode, at least some An electron capture dissociation fragmentation device in which the parent or precursor ions are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode substantially fewer parent or precursor ions are fragmented. When,
A mass analyzer;
A control system, wherein in use: (i) recognizing a first parent or precursor ion of interest from a first sample, the first parent or precursor ion of interest comprising a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of interest;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
(Iv) determining a first ratio of the intensity of the first parent or precursor ion of interest to the intensity of other parent or precursor ions in the first sample;
(V) determining a second ratio of the intensity of the second parent or precursor ion to the intensity of other parent or precursor ions in the second sample;
(Vi) A mass spectrometer is provided that includes a control system that compares a first ratio to a second ratio.

According to one aspect of the invention,
An electron transfer dissociation fragmentation device configured and adapted to repeatedly switch, modify or change between a first mode and a second mode in use, wherein in the first mode, at least some Electron transfer dissociation fragmentation in which parent or precursor ions are fragmented as they interact with reagent ions to produce fragment or daughter ions, and in the second mode substantially fewer parent or precursor ions are fragmented The device,
A mass analyzer;
A control system, wherein in use: (i) recognizing a first parent or precursor ion of interest from a first sample, the first parent or precursor ion of interest comprising a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of interest;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
(Iv) determining a first ratio of the intensity of the first parent or precursor ion of interest to the intensity of other parent or precursor ions in the first sample;
(V) determining a second ratio of the intensity of the second parent or precursor ion to the intensity of other parent or precursor ions in the second sample;
(Vi) A mass spectrometer is provided that includes a control system that compares a first ratio to a second ratio.

According to one aspect of the invention,
A surface induced dissociation fragmentation device configured and adapted to be repeatedly switched, modified or changed between a first mode and a second mode in use, wherein in the first mode at least some A surface-induced dissociation fragmentation device that is fragmented when parent or precursor ions strike a surface or target plate to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented When,
A mass analyzer;
A control system, wherein in use: (i) recognizing a first parent or precursor ion of interest from a first sample, the first parent or precursor ion of interest comprising a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of interest;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
(Iv) determining a first ratio of the intensity of the first parent or precursor ion of interest to the intensity of other parent or precursor ions in the first sample;
(V) determining a second ratio of the intensity of the second parent or precursor ion to the intensity of other parent or precursor ions in the second sample;
(Vi) A mass spectrometer is provided that includes a control system that compares a first ratio to a second ratio.

According to one aspect of the invention,
A collision, fragmentation or reaction device configured and adapted to be repeatedly switched, modified or changed between a first mode and a second mode in use, wherein at least some of the first mode Collisions in which the parent or precursor ions are fragmented or reacted to produce fragment, product, daughter or adduct ions, and in the second mode substantially fewer parent or precursor ions are fragmented or reacted With fragmentation or reaction devices,
A mass analyzer;
A control system, wherein in use: (i) recognizing a first parent or precursor ion of interest from a first sample, the first parent or precursor ion of interest comprising a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of interest;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
(Iv) determining a first ratio of the intensity of the first parent or precursor ion of interest to the intensity of other parent or precursor ions in the first sample;
(V) determining a second ratio of the intensity of the second parent or precursor ion to the intensity of other parent or precursor ions in the second sample;
(Vi) a control system that compares the first ratio to the second ratio;
Collision, fragmentation or reaction devices are: (i) electron impact or impact dissociation fragmentation device, (ii) photo-induced dissociation (“PID”) fragmentation device, (iii) laser induced dissociation fragmentation device, (iv) infrared radiation induced dissociation Devices, (v) ultraviolet radiation induced dissociation devices, (vi) nozzle-skim interface fragmentation devices, (vii) in-source fragmentation devices, (viii) ion source collision induced dissociation fragmentation devices, (ix) thermal or temperature source fragmentation devices (X) electric field induced fragmentation device, (xi) magnetic field induced fragmentation device, (xii) enzymatic digestion or enzymatic degradation fragmentation device Chair, (xiii) ion-ion reaction fragmentation device, (xiv) ion-molecule reaction fragmentation device, (xv) ion-atom reaction fragmentation device, (xvi) ion-metastable ion reaction fragmentation device, (xvii) ion-metas Table molecular reaction fragmentation device, (xviii) ion-metastable atomic reaction fragmentation device, (xix) ion-ion reaction device for reacting ions to form adducts or product ions, (xx) reacting ions Ion-molecule reaction devices for forming adducts or product ions, (xxi) ion-atom reactions for reacting ions to form adducts or product ions Device, (xxii) Ion for reacting ions to form adduct or product ions-Metastable ion reaction device, (xxiii) Ion for reacting ions to form adduct or product ions- Selected from the group consisting of metastable molecular reaction devices, and ion-metastable atomic reaction devices for reacting (xxiv) ions to form adducts or product ions,
A mass spectrometer is provided.

  The mass spectrometer preferably further includes an ion source. The ion source is preferably (i) an electrospray ionization (“ESI”) ion source, (ii) atmospheric pressure photoionization (“APPI”) ion source, (iii) atmospheric pressure chemical ionization (“APCI”) ion source, (Iv) matrix-assisted laser desorption ionization (“MALDI”) ion source, (v) laser desorption ionization (“LDI”) ion source, (vi) atmospheric pressure ionization (“API”) ion source, (vii) silicon Desorption ionization (“DIOS”) ion source, (viii) Electron impact (“EI”) ion source, (ix) Chemical ionization (“CI”) ion source, (x) Field ionization (“FI”) Ion source, (xi) field desorption (“FD”) ion source, (xii) inductively coupled plasma (“ICP”) ion source, (xiii) fast atom bombardment (“FAB”) ions (Xiv) liquid secondary ion mass spectrometry (“LSIMS”) ion source, (xv) desorption electrospray ionization (“DESI”) ion source, (xvi) nickel-63 radioactive ion source, (xvii) atmospheric pressure matrix Selected from the group consisting of a assisted laser desorption ionization ion source and (xviii) a thermal spray ion source.

  The ion source can include a pulsed or continuous ion source.

  According to particularly preferred embodiments, the ion source is electrospray, atmospheric pressure chemical ionization (“APCI”), atmospheric pressure photoionization (“APPI”), matrix-assisted laser desorption ionization (“MALDI”), laser desorption. It may include an ionization (“LDI”), inductively coupled plasma (“ICP”), fast atom bombardment (“FAB”), or liquid secondary ion mass spectrometry (“LSIMS”) ion source. Such an ion source can be provided with an eluent over a period of time, which has been separated from the mixture by liquid chromatography or capillary electrophoresis.

  Alternatively, the ion source may include an electron impact (“EI”), chemical ionization (“CI”) or field ionization (“FI”) ion source. Such an ion source can be provided with an eluent over a period of time, which has been separated from the mixture by gas chromatography.

  Mass analyzers have quadrupole mass filters, time-of-flight (“TOF”) mass analyzers (orthogonal acceleration time-of-flight mass analyzers are particularly suitable), 2D (straight) or 3D (two end cap electrodes) Two donut-shaped electrodes) may include an ion trap, a sector magnetic field analyzer or a Fourier transform ion cyclotron resonance (“FTICR”) mass analyzer.

  The mass analyzer is preferably (i) a quadrupole mass analyzer, (ii) a 2D or linear quadrupole mass analyzer, (iii) a pole or 3D quadrupole mass analyzer, (iv) Penning trap mass spectrometry. Instrument, (v) ion trap mass analyzer, (vi) sector magnetic mass analyzer, (vii) ion cyclotron resonance (“ICR”) mass analyzer, (viii) Fourier transform ion cyclotron resonance (“FTICR”) mass spectrometry (Ix) electrostatic or orbitrap mass analyzer, (x) Fourier transform electrostatic or orbitrap mass analyzer, (xi) Fourier transform mass analyzer, (xii) time-of-flight mass analyzer, (xiii) orthogonal From an accelerated time-of-flight mass analyzer, (xiv) an axial accelerated time-of-flight mass analyzer, and (xv) a quadrupole rod set mass filter or analyzer It is selected from that group.

The mass spectrometer may further include an ion trap or ion guide disposed upstream and / or downstream of the collision, fragmentation or reaction device. The ion trap or ion guide is preferably
(I) a quadrupole rod set, a hexapole rod set, an octupole rod set or a multipole rod set or segmented multipole rod set including a rod set comprising more than eight rods ion trap or ion guide;
(Ii) In an ion tunnel or ion funnel ion trap or ion guide comprising a plurality of electrodes with openings or at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 electrodes In use, ions are transported through the aperture and at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the electrodes, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% have apertures of substantially the same size or area, or sequentially in size or area An ion tunnel or ion funnel ion trap or ion guide having a larger and / or smaller aperture,
(Iii) one stack or array plane, plate or mesh electrode, wherein one stack or array plane, plate or mesh electrode is 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 planes, plates or mesh electrodes, at least 5%, 10%, 15 of the planes, plates or mesh electrodes %, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% is a plane of a stack or array, plate or mesh electrode, and (iv) an ion trap or An ion trap or ion guide comprising a plurality of groups of electrodes arranged axially along the length of the ion guide, wherein each group of electrodes comprises: (a) a first and a second electrode and ions for the ion guide Means for applying a DC voltage or potential to the first and second electrodes for confining in the first radial direction therein; and (b) second and fourth electrodes and ions in the ion guide second. Selected from the group consisting of ion traps or ion guides, including means for applying an AC or RF voltage to the third and fourth electrodes for confining them radially.

  The ion trap or ion guide preferably comprises an ion tunnel or ion funnel ion trap or ion guide, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the electrodes. , 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% are (i) ≦ 1.0 mm, (ii) ≦ 2.0 mm, (iii) ≦ 3.0 mm, (iv) ≦ 4.0 mm, (v) ≦ 5.0 mm, (vi) ≦ 6.0 mm, (vii) ≦ 7.0 mm, (viii) ≦ 8. It has an inner diameter or an inner dimension selected from the group consisting of 0 mm, (ix) ≦ 9.0 mm, (x) ≦ 10.0 mm, and (xi)> 10.0 mm.

  The ion trap or ion guide preferably applies an AC or RF voltage to at least 5%, 10%, 15% of the plurality of electrodes of the ion trap or ion guide to radially confine ions within the ion trap or ion guide. 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100 And further includes a first AC or RF voltage means configured and adapted to apply to the%.

  The first AC or RF voltage means is preferably (i) <50V peak-to-peak, (ii) 50-100V peak-to-peak, (iii) 100-150V peak-to-peak, (iv) 150-200V peak-to-peak (Vi) 200-250V peak-to-peak, (vi) 250-300V peak-to-peak, (vii) 300-350V peak-to-peak, (viii) 350-400V peak-to-peak, (ix) 400-450V peak-to-peak , (X) 450-500V peak-to-peak, and (xi)> 500V peak-to-peak, configured and adapted to apply an AC or RF voltage having an amplitude selected from the group consisting of:

  The first AC or RF voltage means is preferably (i) <100 kHz, (ii) 100-200 kHz, (iii) 200-300 kHz, (iv) 300-400 kHz, (v) 400-500 kHz, (vi) 0 .5 to 1.0 MHz, (vii) 1.0 to 1.5 MHz, (viii) 1.5 to 2.0 MHz, (ix) 2.0 to 2.5 MHz, (x) 2.5 to 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 to 5.5 MHz, (xvi) 5.5 to 6.0 MHz, (xvii) 6.0 to 6.5 MHz, (xviii) 6.5 to 7.0 MHz, (xix) 7.0 to 7.5 MHz, (Xx) 7.5-8.0 MHz, (xx ) 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 Configured and adapted to apply an AC or RF voltage having a frequency selected from the group consisting of:

  The ion trap or ion guide preferably receives a beam or group of ions and converts or splits the beam or group of ions to provide 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 ions in separate packets and / or in an ion trap or ion guide at any particular time Constructed and adapted to be isolated, the ions of each packet are confined and / or isolated separately in separate axial potential wells formed in an ion trap or ion guide.

  The mass spectrometer, in one mode of operation, removes at least some ions at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40% of the axial length of the ion trap or ion guide. %, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% through or along upstream and / or downstream Means further configured and adapted to drive the

  The mass spectrometer preferably has at least some ions at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% of the axial length of the ion trap or ion guide. 1 to drive upstream and / or downstream along%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% Further included is a first transient DC voltage means configured and adapted to apply the above transient DC voltage or potential or one or more transient DC voltages or potential waveforms to the electrodes forming the ion trap or ion guide.

  The mass spectrometer preferably has at least some ions at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% axial length of the ion trap or ion guide. 2 or more to drive upstream and / or downstream along 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% Further includes AC or RF voltage means configured and adapted to apply a phase shift AC or RF voltage to the electrode forming the ion trap or ion guide.

  The collision, fragmentation or reaction device is an ion tunnel comprising a quadrupole rod set, a hexapole rod set, an octupole or higher order rod set, or a plurality of electrodes having apertures, where the ions are open. It may include an ion tunnel that is transported through. The openings are preferably substantially the same size. The collision, fragmentation or reaction device may more generally include a plurality of electrodes connected to an AC or RF voltage source for radially confining ions within the collision, fragmentation or reaction device. An axial DC voltage gradient may or may not be applied along at least a portion of the length of the ion tunnel collision, fragmentation or reaction device. Collision, fragmentation or reaction devices can be housed in the housing, or otherwise be substantially hermetic, apart from an opening for entering and exiting ions and a port for introducing optional gas. The body may be configured to form around a collision, fragmentation or reaction device. Gases such as helium, argon, nitrogen, air or methane can be introduced into the collision, fragmentation or reaction device.

  Other configurations are also conceivable in which the collision, fragmentation or reaction device repeatedly switches between high fragmentation or reaction mode and low fragmentation or reaction mode and is not variable or changed. For example, the collision, fragmentation or reaction device may be configured to remain on invariably and fragment or react ions received within the collision, fragmentation or reaction device. An electrode or other device may be provided upstream of the collision, fragmentation or reaction device. A high fragmentation or reaction mode of operation occurs if ions or other devices allow ions to collide, pass to the fragmentation or reaction device. A low fragmentation or reaction mode of operation occurs if ions are bypassed by the electrode or other device to bypass the collision, fragmentation or reaction device and are thus prevented from being fragmented or reacted within the collision, fragmentation or reaction device.

  Also contemplated are other embodiments that may be useful when a particular parent or precursor ion cannot be readily observed to co-elute with other commonly observed peptide ions. In such a situation, the expression level of the fragment, product, daughter or adduct ion is compared between the two samples.

  According to the preferred embodiment, instead of comparing the expression levels of parent or precursor ions in two different samples and considering whether the expression levels are significantly different to allow further investigation, preferably the parent of the subject Alternatively, it is first recognized that precursor ions are present in the sample.

  According to one preferred embodiment, recognizing the first parent or precursor ion of the subject comprises recognizing the first fragment, product, daughter or adduct ion of the subject.

  The first fragment, product, daughter or adduct ion of interest can be identified as needed, for example, by determining their mass to charge ratio, preferably no more than 20 ppm, 15 ppm, 10 ppm or 5 ppm. .

  If the fragment, product, daughter or adduct ion of interest is recognized and optionally identified, then it is necessary to determine which parent or precursor ion gives rise to that fragment, product, daughter or adduct ion. is there.

  The step of recognizing the first parent or precursor ion of interest is observed in the mass spectrum obtained when the parent or precursor ion has been in collision, fragmentation or reaction device in low fragmentation or reaction mode for a predetermined period Whether and when the first fragment, product, daughter or adduct ion of interest is collision, fragmentation or reaction device is in high fragmentation or reaction mode, just before a predetermined period of time, or collision, fragmentation or reaction device is Determining if observed in a mass spectrum obtained either immediately after a predetermined period of time when in high fragmentation or reaction mode.

  Recognizing the first parent or precursor ion of interest includes comparing the elution time of the parent or precursor ion to the pseudoelution time of the first fragment, product, daughter or adduct ion of interest. obtain. Recognizing the first parent or precursor ion of interest includes comparing the elution profile of the parent or precursor ion to the pseudoelution time of the first fragment, product, daughter or adduct ion of interest. obtain.

  According to another less preferred embodiment, the parent or precursor ions of interest are immediately determined by their mass-to-charge ratio without the need to recognize and identify the fragment, product, daughter or adduct ions of interest. Can be recognized. According to this embodiment, the step of recognizing the first parent or precursor ion of interest preferably comprises determining the mass to charge ratio of the parent or precursor ion, preferably no more than 20 ppm, 15 ppm, 10 ppm or 5 ppm. including. The determined mass to charge ratio of the parent or precursor ions can then be compared to a database of ions and their corresponding mass to charge ratios.

  According to another embodiment, the step of recognizing the first parent or precursor ion of interest comprises the step that the parent or precursor ion is a fragment, product, daughter or as a result of the loss of the predetermined ion or predetermined neutral molecule. Determining whether to generate adduct ions.

  The parent ion of interest can be identified in a manner similar to the preferred embodiment above.

  Other preferred features of the preferred embodiment apply equally to other configurations.

  The above embodiment relating to recognizing a parent or precursor ion of interest and comparing the expression level of the parent or precursor ion in one sample with the corresponding parent or precursor ion in another sample is the preferred embodiment. It will be apparent that the methods and apparatus can be used. Thus, also the same preferred features described for the preferred embodiment described above recognize the parent or precursor ion of interest and then the expression level of the parent or precursor ion in one sample corresponding to that in another sample. It can be used with the above embodiments relating to comparison with parent or precursor ions.

  If a parent or precursor ion having a specific mass to charge ratio is expressed differently in two different samples, the parent or precursor ion of interest is further examined according to the preferred embodiment. This further investigation may include the step of attempting to identify a parent or precursor ion of interest that is differentially expressed in two different samples. Several checks can be made to verify that the parent or precursor ions whose expression levels are being compared in two different samples are indeed the same ions.

  Much information can be obtained by measuring changes in protein abundance in complex protein mixtures. For example, changes in the abundance of proteins in cells (often referred to as protein expression levels) can be due to different cellular stresses, stimulatory effects, disease effects or drug effects. Such proteins can provide relevant targets for research, screening or clinical practice. The identification of such proteins can usually be of interest. Such proteins can be identified by the method of the preferred embodiment above.

  Thus, according to the preferred embodiment, the new criteria for finding the parent or precursor ion of interest is based on the quantification of proteins in two different samples. This requires determining the relative abundance of their peptide products in more than one sample. However, to determine relative abundance, the same peptide ion must be compared in two (or more) different samples and ensuring that this occurs is not a simple matter. It is therefore necessary to be able to recognize and preferably identify a peptide ion to the extent that it can be uniquely recognized in a sample. Such peptide ions are sufficient by measuring the mass of the parent or precursor ion and by measuring the mass-to-charge ratio of one or more fragment, product, daughter or adduct ions derived from the parent or precursor ion. Can be recognized. The specificity with which a peptide can be recognized can be increased by determining the exact mass of the parent or precursor ion and / or the exact mass of one or more fragment, product, daughter or adduct ions.

  Also, the same method for recognizing the parent or precursor ion in one sample is preferably used to recognize the same parent or precursor ion in another sample, thereby providing a parent or precursor in two different samples. It becomes possible to measure the relative abundance of ions.

  Relative abundance measurements make it possible to find proteins with significant changes or differences in expression levels. Using the above method, with the same data, several or all fragments, products, daughters or adduct ions associated with each such peptide product ion are found by their respective elution time agreement. Identification of the existing protein. Again, by accurately measuring the mass of the parent or precursor ion and the associated fragment, product, daughter or adduct ion, the specificity and confidence that a protein can be identified is substantially improved.

  Also, the specificity with which a peptide can be recognized can be increased by comparison of retention times. For example, HPLC or CE retention or elution times are measured as part of the procedure for mapping fragment, product, daughter or adduct ions to parent or precursor ions, and these elution times are 2 The above samples can be compared. The elution time can be used to reject measurement results that are not within a predetermined time difference. Alternatively, the retention time can be used to establish recognition of the same peptide if it falls within a predetermined mutual window. In general, there is some redundancy if the exact mass of the parent or precursor ion, the exact mass of one or more fragments, product, daughter or adduct ions, and the retention time are all measured and compared. obtain. In many cases, only two of these measurements are sufficient to recognize the same peptide parent or precursor ion in more than one sample. For example, it is probably sufficient to measure the exact parent or precursor ion mass to charge ratio and fragment, product, daughter or adduct ion mass to charge ratio, or the exact parent or precursor ion mass to charge ratio and retention time. is there. Nevertheless, further measurement results can be used to establish recognition of the same parent peptide ion.

  The relative expression level of matching parent peptide ions can be quantified by measuring the peak area relative to the internal standard.

  The preferred embodiment does not require interruption of data acquisition and is therefore particularly suitable for quantitative applications. According to one embodiment, one or more endogenous peptides that are common to both mixtures that do not vary with the experimental condition of the sample can be used as an internal standard or reference for measuring relative peak areas. According to another embodiment, if such an internal standard does not exist or cannot be trusted, an internal standard can be added to each sample. In addition, the internal standard can function as a chromatographic retention time standard and a mass accuracy standard, whether present naturally or added.

  Ideally, more than one peptide parent or precursor ion can be measured for each protein to be quantified. For each peptide, the same recognition means are preferably used when comparing the respective intensities of different samples. The measurement of different peptides has the function of confirming the relative abundance measurement results. Furthermore, the measurement results from several peptides provide a means to determine the average relative abundance and to determine the relative significance of the measurement results.

  According to one embodiment, all parent or precursor ions can be identified and their relative abundance can be determined by comparing their intensity with that of the same identity in one or more other samples. .

  In another embodiment, the relative abundance of all target parent or precursor ions discovered based on their relationship to a given fragment, product, daughter or adduct ion is determined by their intensity and one or more It can be determined by comparing to those of the same identity in other samples.

  In another embodiment, the relative abundance of all subject parent or precursor ions discovered based on the occurrence of a given mass loss is determined by their intensity and the same identity in one or more other samples. It can be determined by comparing things.

  In another embodiment, it may simply be sought to quantify the already identified protein. The protein can be present in a complex mixture and the same separation and recognition means as described above can be used. Here it is only necessary to recognize the relevant peptide products or products and measure their intensity in one or more samples. The basis of recognition may be the recognition of the mass or exact mass of the peptide parent or precursor ion and the recognition of the mass or exact mass of one or more fragments, products, daughters or adduct ions. Their retention times are also compared, thereby providing a means to confirm recognition of the same peptide or to reject mismatched peptides.

  The preferred embodiment can be used for proteomics research. However, the same methods of identification and quantification can be used in other areas of analysis such as metabolomics studies.

  The above method can be used for mixture analysis where different components of a mixture are first separated or partially separated by means such as chromatography, which elutes the components sequentially.

  The ion source is preferably capable of producing mainly molecular or quasi-molecular ions and relatively few (if any) fragment, product, daughter or adduct ions. Examples of such ion sources include atmospheric pressure ion sources (eg, electrospray and APCI) and matrix-assisted laser desorption ionization (MALDI).

  If the two main modes of operation of the collision, fragmentation or reaction device are set appropriately, the parent or precursor ions will be relatively higher in intensity in the mass spectrum without substantial fragmentation or reaction. Can be recognized by facts. Similarly, fragment, product, daughter or adduct ions may be recognized by the fact that they are relatively higher in intensity in the mass spectrum when using substantial fragmentation or reaction.

  The mass analyzer may include a quadrupole, time-of-flight, ion trap, sector magnetic field or FT-ICR mass analyzer. According to a preferred embodiment, the mass analyzer should be able to determine a precise or accurate mass charge value for the ions. This is to maximize selectivity for detection of characteristic fragment, product, daughter or adduct ions or mass loss and to maximize specificity for protein identification.

  The mass analyzer preferably samples or records the entire spectrum simultaneously. This ensures that the elution time observed for all masses is not altered or distorted by the mass analyzer, resulting in elution times of different masses such as parent and fragment, product, daughter or adduct ions Accurate matching can be possible. This also helps to ensure that quantitative measurements are not compromised by the need to measure the presence of transient signals.

  A mass filter (preferably a quadrupole mass filter) may be provided upstream of the collision, fragmentation or reaction device. The mass filter may have a high-pass filter characteristic and be configured to transport ions having a mass to charge ratio of, for example, 100, 150, 200, 250, 300, 350, 400, 450, or 500 or more. . Alternatively, the mass filter may have a low pass or band pass filter characteristic.

  An ion guide may be provided upstream of the collision, fragmentation or reaction device. The ion guide may include any one of a hexapole, quadrupole, octupole or higher order multipole rod set. In another embodiment, the ion guide may include an ion tunnel ion guide that includes a plurality of electrodes having openings, wherein ions are transported through the openings in use. Preferably, at least 90% of the electrodes have substantially the same size opening. Alternatively, the ion guide may include a plurality of ring electrodes (“ion funnels”) having a substantially tapered inner diameter.

  A parent ion that belongs to a specific class of parent ions and is recognizable by a characteristic fragment, product, daughter or adduct ion or characteristic neutral loss is a parent or precursor ion scan or a constant neutral loss scan. Conventionally discovered by the method. Conventional methods for recording parent or precursor ion scans or constant neutral loss scans are scans of one or both quadrupoles in a triple quadrupole mass spectrometer, or tandem quadrupole orthogonal TOF mass spectrometry. A quadrupole scan in the meter, or a scan of at least one element in other types of tandem mass spectrometers. As a result, these methods result in a low duty cycle associated with the scanning device. Further, as a result, information can be discarded and lost while the mass spectrometer is occupied by the parent or precursor ion scan or constant neutral loss scan record. Furthermore, as a result, these methods are not suitable for use when the mass spectrometer needs to analyze substances that elute directly from a gas or liquid chromatography device.

  According to one embodiment, a tandem quadrupole orthogonal time-of-flight mass spectrometer is used such that sequential low and high collision energy mass spectra are recorded. Switching back and forth, modifying or changing is preferably not interrupted. Instead, a complete set of data is acquired, which can then be processed later. Fragment, product, daughter or adduct ions can be associated with parent or precursor ions by their respective elution time match. In this way, the parent or precursor ion of interest can be determined or otherwise data acquisition is not interrupted and information is not lost.

According to one embodiment, possible subject parent or precursor ions may be selected based on their relationship to a given fragment, product, daughter or adduct ion. Predetermined fragment, product, daughter or adduct ions may be, for example, immonium ions from peptides, phosphate phosphate group PO ₃ from phosphorylated peptides ^- functional group containing an ion or molecular particular molecule or a particular class, It may include a mass tag that is cleaved and subsequently identified and is therefore intended to report the presence of that particular molecule or class of molecules. Parent or precursor ions are selected as potential parent or precursor ions by generating mass chromatograms for a given fragment, product, daughter or adduct ion using high fragmentation or reaction mass spectra. Can be put on the candidate list. The center of each peak in the mass chromatogram is then determined along with the corresponding predetermined fragment, product, daughter or adduct ion elution time. Then, for each peak in a given fragment, product, daughter or adduct ion mass chromatogram, a low fragmentation or reaction mass spectrum obtained immediately before the given fragment, product, daughter or adduct ion elution time and Both low fragmentation or reaction mass spectra obtained immediately after a given fragment, product, daughter or adduct ion elution time are examined for the presence of previously recognized parent or precursor ions. The low fragmentation or reaction mass spectrum obtained immediately before the predetermined fragment, product, daughter or adduct ion elution time and the low fragmentation obtained immediately after the predetermined fragment, product, daughter or adduct ion elution time or A mass chromatogram is generated for any previously recognized parent or precursor ion found to be present in both of the reaction mass spectra, and the center of each peak in each mass chromatogram may correspond Determined with parent or precursor ion elution time. The potential subject parent or precursor ions are then ranked according to their elution times in agreement with the given fragment, product, daughter or adduct ion elution times, and the final potential The list of parent or precursor ions of interest precedes the potential parent or precursor ions of the subject by a predetermined amount more than their elution time for a given fragment, product, daughter or adduct ion. Or if it exceeds, it can be created by rejecting.

  According to another embodiment, a parent or precursor ion may be placed on the selection candidate list as a potential subject parent or precursor ion based on that it causes a predetermined mass loss. For each low fragmentation or reaction mass spectrum, a target fragment that can result from the loss of a given ion or neutral particle from each previously recognized parent or precursor ion present in the low fragmentation or reaction mass spectrum, A list of mass charge values of product, daughter or adduct ions is generated. Then, both the high fragmentation or reaction mass spectrum obtained immediately before the low fragmentation or reaction mass spectrum and the high fragmentation or reaction mass spectrum obtained immediately after the low fragmentation or reaction mass spectrum are the target fragments, products, daughters or adducts. The presence of a fragment, product, daughter or adduct ion having a mass charge value corresponding to the ionic mass charge value is examined. Then, a list of possible parent or precursor ions of interest (including their corresponding fragment, product, daughter or adduct ions, if necessary) is the target fragment, product, daughter or adduct Fragment, product, daughter or adduct ions with mass charge values corresponding to the ion mass charge value are low fragmentation or high fragmentation or reaction mass spectrum just before the reaction mass spectrum and high fragmentation just after low fragmentation or reaction mass spectrum. Or by including parent or precursor ions in the list if found to be present in both of the reaction mass spectra. A mass loss chromatogram can then be generated based on possible candidate parent or precursor ions and their corresponding fragment, product, daughter or adduct ions. The center of each peak in the mass loss chromatogram is determined along with the corresponding mass loss elution time. A mass chromatogram is then generated using low fragmentation or reaction mass spectra for each possible candidate parent or precursor ion. A corresponding fragment, product, daughter or adduct ion mass chromatogram is also generated for the corresponding fragment, product, daughter or adduct ion. The center of each peak in the potential candidate parent or precursor ion mass chromatogram and the corresponding fragment, product, daughter or adduct ion mass chromatogram is then the corresponding candidate parent or precursor ion. Determined along with body ion elution time and corresponding fragment, product, daughter or adduct ion elution time. The list of final candidate parent or precursor ions is then preceded by more than a predetermined amount from the corresponding fragment, product, daughter or adduct ion elution times of the possible candidate parent or precursor ions. Or, if so, may be created by rejecting potential candidate parent or precursor ions.

  Once the list of parent or precursor ions of interest is created (preferably including only some of the originally recognized parent or precursor ions and potential parent or precursor ions of interest) Parent or precursor ions can be identified.

  Identification of the parent or precursor ion can be made by using a combination of information. This may include the accurately determined mass or mass to charge ratio of the parent or precursor ion. It can also include the mass or mass to charge ratio of the fragment, product, daughter or adduct ions. In some cases, an accurately determined mass or mass to charge ratio of a fragment, product, daughter or adduct ion may be suitable. It is known that proteins can be identified from the mass or mass to charge ratio of the peptide product from the enzyme digested protein, preferably the exact mass or mass to charge ratio. These can be compared to those expected from a library of known proteins. It is also known that ambiguity can be resolved by analyzing the fragment for one or more of its peptides if this comparison result suggests more than one possible protein. The preferred embodiment allows a mixture of proteins digested by an enzyme to be identified by a single analysis. The mass or mass-to-charge ratio or exact mass or mass-to-charge ratio of all peptides and their related fragments, products, daughters or adduct ions can be searched against a library of known proteins. Alternatively, the peptide mass or mass to charge ratio or the exact mass or mass to charge ratio can be searched against a library of known proteins. If more than one protein is suggested, the correct protein is determined by searching for fragments, products, daughters or adduct ions that match those expected from related peptides from each candidate protein. obtain.

  Identifying the parent or precursor ion of each object preferably comprises recalling the elution time of the parent or precursor ion of the object and the low fragmentation obtained immediately before the elution time of the parent or precursor ion of the object or May contain previously recognized fragment, product, daughter or adduct ions present in both the reaction mass spectrum and the low fragmentation or reaction mass spectrum obtained immediately after the elution time of the parent or precursor ion of interest Generating a list of a fragment, product, daughter or adduct ion, generating a mass chromatogram of each possible fragment, product, daughter or adduct ion, and each possible fragment In product, daughter or adduct ion mass chromatograms And determining the center of the peak, fragments of potentially corresponding candidate, product, and determining the daughter or adduct ion elution time. The potential fragment, product, daughter or adduct ions can then be ranked according to how well their elution time matches the elution time of the subject parent or precursor ion. If the list of fragment, product, daughter or adduct ions then precedes or exceeds the elution time of the fragment, product, daughter or adduct ions by more than a predetermined amount from the elution time of the target parent or precursor ion Can be made by discarding fragments, products, daughters or adduct ions.

  The list of fragment, product, daughter or adduct ions is the closest parent or precursor ion present in the low fragmentation or reaction mass spectrum obtained closest in time to the elution time of the final candidate parent or precursor ion. Can be further refined or reduced by creating a list of A mass chromatogram of each parent or precursor ion included in the list is then generated and the center of each mass chromatogram is determined along with the corresponding adjacent parent or precursor ion elution time. Any fragment, product, daughter or adduct ion that then has a corresponding elution time closer to the parent or precursor ion elution time closer than the elution time of the parent or precursor ion of interest is also a fragment, product Can be rejected from the list of daughter or adduct ions.

  Fragments, products, daughters or adduct ions can be assigned to a parent or precursor ion according to their agreement in their elution times, and all fragments, products, daughters or ions associated with the parent or precursor ion Adduct ions can be listed.

  Also contemplated are other embodiments that involve a greater amount of data processing but are inherently simpler. Once the parent and fragment, product, daughter or adduct ions are identified, a parent or precursor ion mass chromatogram is generated for each recognized parent or precursor ion. The center of each peak in the parent or precursor ion mass chromatogram and the corresponding parent or precursor ion elution time are then determined. Similarly, a fragment, product, daughter or adduct ion mass chromatogram for each recognized fragment, product, daughter or adduct ion is generated, and then a fragment, product, daughter or adduct ion mass chromatogram. The center of each peak at and the corresponding fragment, product, daughter or adduct ion elution times are determined. Then, rather than identifying only a subset of the recognized parent or precursor ions, all (or almost all) of the recognized parent or precursor ions are identified. Fragment ions are assigned to parent or precursor ions according to their respective elution time agreement, and then all fragments, products, daughters or adduct ions associated with the parent or precursor ions are listed. obtain.

  Passing ions to a mass filter, preferably a quadrupole mass filter, before being passed to a collision, fragmentation or reaction device provides an alternative or additional method of recognizing fragment, product, daughter or adduct ions. . Fragment, product, daughter or adduct ions can be recognized by recognizing ions in high fragmentation or reaction mass spectra with mass to charge ratios that are not transported by collisions, fragmentation or reaction devices, i.e. fragments, products, Daughter or adduct ions are recognized by ions having a mass to charge ratio that are outside the transfer window of the mass filter. If the ions are not transported by a mass filter, they must be produced in a collision, fragmentation or reaction device.

  Various embodiments of the present invention will now be described by way of example only with reference to the accompanying drawings.

  FIG. 1 is a schematic diagram of a preferred mass spectrometer.

  FIG. 2 is a schematic diagram of the valve switch configuration during sample loading and desalting, and the inset shows the desorption of the sample from the analytical column.

  FIG. 3A shows a fragment or daughter ion mass spectrum and FIG. 3B is obtained when the mass filter upstream of the collision cell is configured to transfer ions with a mass to charge ratio> 350 to the collision cell. And the corresponding parent or precursor ion mass spectrum.

  4A shows the mass chromatogram of the parent or precursor ion, FIG. 4B shows the mass chromatogram of the parent or precursor ion, FIG. 4C shows the mass chromatogram of the parent or precursor ion, and FIG. Shows the mass chromatogram of the fragment or daughter ion, and FIG. 4E shows the mass chromatogram of the fragment or daughter.

  FIG. 5 shows mass chromatograms in which FIGS.

  FIG. 6 shows a mass chromatogram of asparagine immonium ions having a mass to charge ratio of 87.04.

  FIG. 7 shows the mass spectrum of ADH-derived peptide ion T5 having the sequence ANELLINVK and the molecular weight 1012.59.

  FIG. 8 shows the mass spectrum of tryptic digestion of β-casein obtained when the collision cell was in low fragmentation mode.

  FIG. 9 shows the tryptic digestion mass spectrum of β-casein obtained when the collision cell was in high fragmentation mode.

  FIG. 10 is a view obtained by processing and enlarging the mass spectrum shown in FIG.

  FIG. 11A shows a mass chromatogram of ions from the first sample with a mass to charge ratio of 880.4, FIG. 11B shows a similar mass chromatogram of the same ions from the second sample, and FIG. Shows a mass chromatogram of ions from a first sample with a mass to charge ratio of 582.3, and FIG. 11D shows a similar mass chromatogram of the same ions from a second sample.

  FIG. 12A shows the mass spectrum recorded from the first sample, and FIG. 12B shows a second spectrum similar to the first sample except that both samples contain a higher concentration of the digestion product of the protein casein common to both samples. The corresponding mass spectra recorded from the samples are shown.

  FIG. 13 shows the mass spectrum shown in FIG. 12A in more detail, and the inset shows an enlarged portion of the mass spectrum showing an isotope peak at a mass to charge ratio of 880.4.

  FIG. 14 shows the mass spectrum shown in FIG. 12B in more detail, and the inset shows an enlarged portion of the mass spectrum showing an isotope peak at a mass to charge ratio of 880.4.

  One preferred embodiment will now be described with reference to FIG. A mass spectrometer 6 is shown, which comprises an ion source 1 (preferably an electrospray ionization source), an ion guide 2 arranged downstream of the ion source 1, a quadrupole mass filter 3, collisions, fragmentation. Or a reaction device 4 and an orthogonal acceleration time-of-flight mass analyzer 5 incorporating a reflectron. The ion guide 2 and the mass filter 3 may be omitted as necessary. The mass spectrometer 6 is preferably connected to a chromatograph (not shown) such as a liquid chromatograph so that the sample entering the ion source 1 can be removed from the eluent of the liquid chromatograph.

The quadrupole rod set mass filter 3 is preferably placed in a vacuum chamber that is maintained at a relatively low pressure (eg, less than 10 ⁻⁵ mbar). The rod electrode containing the mass filter 3 is preferably connected to a power supply that generates both RF and DC potentials that determine the mass charge value transfer window of the mass filter 3.

  The collision, fragmentation or reaction device 4 preferably comprises a surface induced dissociation (“SID”) fragmentation device, an electron transfer dissociation fragmentation device or an electron capture dissociation fragmentation device.

  According to one embodiment, the collision, fragmentation or reaction device 4 may comprise an electron capture dissociation fragmentation device. According to this embodiment, the multivalent analyte ions are preferably made to interact with relatively low energy electrons. The electrons preferably have an energy of <1 eV or 1-2 eV. The electrons are preferably confined and directed by a relatively strong magnetic field so that they collide with analyte ions that are preferably confined in the RF ion guide, preferably located in the fragmentation device 4. An AC or RF voltage is preferably applied to the electrode of the RF ion guide so that a radial pseudopotential well is preferably generated. This radial pseudopotential well preferably acts to confine the ions radially within the ion guide so that the ions can interact with 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 made to interact with negatively charged reagent ions. Negatively charged reagent ions are preferably injected into an RF ion guide or ion trap located within the collision, fragmentation or reaction device 4. An AC or RF voltage is preferably applied to the electrode of the RF ion guide so that a radial pseudopotential well is preferably generated. The radial pseudopotential well preferably serves to confine the ions radially within the ion guide so that the ions can interact with negatively charged reagent ions. Alternatively, according to less preferred embodiments, negatively charged analyte ions can be made to interact with positively charged reagent ions.

  According to another embodiment, the collision, fragmentation or reaction device 4 may comprise a surface induced dissociation fragmentation device. According to this embodiment, the ions are preferably directed towards the surface or target plate with relatively low energy. The ions can be configured to have an energy of, for example, 1-10 eV. The surface or target plate may comprise stainless steel, or more preferably the surface or target plate may comprise a single layer of fluorocarbon or hydrocarbon coated metal plate. The monolayer preferably comprises a self-assembled monolayer. The surface or target plate may be placed in a plane that is substantially parallel to the direction of ion travel through the surface-induced dissociation fragmentation device in an operating mode in which ions are not fragmented. In modes of operation in which ion fragmentation is desired, the ions may be deflected onto or toward the surface or target plate such that the ions strike the surface or target plate at a relatively shallow angle with respect to the surface or target plate. . Fragment ions are preferably generated as a result of analyte ions striking the surface or target plate. The fragment ions are preferably directed away from or away from the surface or target plate at a relatively shallow angle relative to the surface or target plate. The fragment ions are then preferably configured to assume trajectories that preferably correspond to trajectories of ions that are transported through or through the surface-induced dissociation fragmentation device in an operating mode in which the ions are not substantially fragmented. The

  The collision, fragmentation or reaction device 4 may include an electron collision or impact dissociation fragmentation device that is fragmented when ions collide with relatively high energy electrons (eg,> 5 ev electrons).

  According to other embodiments, the collision, fragmentation or reaction device 4 is a photo-induced dissociation (“PID”) fragmentation device, a laser-induced dissociation fragmentation device, an infrared radiation-induced dissociation device, an ultraviolet radiation-induced dissociation device, a heat or temperature source. Fragmentation device, electric field induced fragmentation device, magnetic field induced fragmentation device, enzymatic digestion or enzymatic fragmentation device, ion-ion reaction fragmentation device, ion-molecule reaction fragmentation device, ion-atom reaction fragmentation device, ion-metastable ion reaction fragmentation device , Ion-metastable molecular reaction fragmentation device, ion-measuring Stable atomic reaction fragmentation device, ion-ion reaction device for reacting ions to form adduct or product ions, ion-molecule reaction device for reacting ions to form adduct or product ions Ion-atom reaction devices for reacting ions to form adducts or product ions, ion-metastable ion reaction devices for reacting ions to form adducts or product ions, reacting ions An ion-metastable molecular reaction device for forming an adduct or product ion, or an ion-metastable atomic reaction device for reacting an ion to form an adduct or product ion.

  According to one embodiment, the collision, fragmentation or reaction device may form part of the ion source 1. For example, the collision, fragmentation or reaction device may include a nozzle-to-skim 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 include a quadrupole or hexapole rod set. The quadrupole or hexapole rod set is a pressure between 10 ⁻⁴ and 10 ⁻¹ mbar, more preferably 10 ⁻³ mbar to 10 ⁻² mbar, such as helium, argon, nitrogen, air or methane Can be enclosed in a substantially hermetic casing (apart from small ion inlet and outlet holes) that can be introduced at a pressure of A suitable AC or RF potential for the electrodes making up the collision, fragmentation or reaction device 4 may be provided by a power source (not shown).

  The ions generated by the ion source 1 are preferably transferred by the ion guide 2 and passed into the vacuum chamber 8 through the inter-chamber hole 7. The ion guide 2 is preferably maintained at a pressure intermediate between the ion source 1 and the vacuum chamber 8. In the above embodiment, the ions can be mass filtered by the mass filter 3 before entering the collision, fragmentation or reaction device 4. However, the mass filter 3 is an optional feature of this embodiment. Preferably, ions leaving the collision, fragmentation or reaction device 4 or transported through the collision, fragmentation or reaction device 4 preferably pass into a mass analyzer including the time-of-flight mass analyzer 5. Other ion optical components (not shown and not described herein) may be provided, such as additional ion guides and / or electrostatic lenses. Such components can be used to maximize ion transfer between various parts or stages of the mass spectrometer. Various vacuum pumps (not shown) can be provided to maintain optimal vacuum conditions. A time-of-flight mass analyzer 5 incorporating a reflectron measures the transit time or flight time of ions contained in one packet of ions so that their mass-to-charge ratio can be determined in a known manner. Works with.

  Control means (not shown) preferably provide the necessary operating potential for the ion source 1, ion guide 2, quadrupole mass filter 3, collision, fragmentation or reaction device 4 and time-of-flight mass analyzer 5, respectively. Provides control signals for various power supplies (not shown). These control signals determine the operating parameters of the mass spectrometer (for example, the mass to charge ratio transferred through the mass filter 3 and the operation of the mass analyzer 5). The control means includes a computer (not shown) that can also be used to process the acquired mass spectral data. The computer can also display and store the mass spectrum generated by the mass analyzer 5 and receive instructions from the operator. The control means may be set to automatically perform various methods and make various decisions without operator intervention or may require operator input as appropriate at various stages.

  The control means is preferably configured to switch, change or modify the collision, fragmentation or reaction device 4 to go back and forth between at least two different modes. If the collision, fragmentation or reaction device 4 includes an electron capture dissociation fragmentation device, the electron source or beam can be switched ON in the first mode of operation and OFF in the second mode of operation. When the collision, fragmentation or reaction device 4 includes an electron transfer dissociation fragmentation device 4, in a first mode of operation, reagent ions are injected into an ion guide or ion trap containing analyte ions, and in a second mode of operation. It may be that substantially no reagent ions are injected into the ion guide or ion trap. If the collision, fragmentation or reaction device 4 comprises a surface induced dissociation fragmentation device, in the first mode of operation, analyte ions can be directed to strike or hit the surface or target plate, and in the second mode of operation. , Can be oriented straight so that analyte ions pass through the surface or target plate without impinging or hitting the surface or target plate.

  In one embodiment, the control means may switch, modify or change approximately every second between modes. The mass spectrometer 6 can be operated for tens of minutes when provided in conjunction with an ion source provided with an eluent separated from the mixture by liquid or gas chromatography. Over this period, hundreds of high and low fragmentation or reaction mass spectrum fragmentation or reaction mass spectra can be obtained.

  At the end of the experimental trial, the data obtained is preferably analyzed and parent or precursor ions and fragments, products, daughters or adduct ions are preferably obtained when in a mode with collision, fragmentation or reaction device 4. Is recognized based on a comparison of the relative intensity of the peaks in the resulting mass spectrum with the intensity of the same peak in the mass spectrum obtained after about 1 second in time when the collision, fragmentation or reaction device 4 is in the second mode.

  According to one embodiment, a mass chromatogram is generated for each parent and fragment, product, daughter or adduct ion, and the fragment, product, daughter or adduct ion is based on their relative elution times. Assigned to body ions.

  The advantage of this method is that all data is acquired and then processed so that every fragment, product, daughter or adduct ion is matched to the parent or precursor ion by their respective elution time match. Can be. This ensures that all parent or precursor ions, regardless of whether they are discovered by the presence of characteristic fragments, products, daughter or adduct ions or characteristic “neutral loss”, Allows identification from product, daughter or adduct ions.

  According to another embodiment, attempts can be made to reduce the number of parent or precursor ions of interest. A list of possible parental or precursor ions that are possible (ie, not yet finalized) can be obtained for a given fragment, product, daughter or adduct ion of interest (eg, immonium ions from a peptide). It can be created by looking for parent or precursor ions that may have been generated. Alternatively, a search can be made for parent and fragment, product, daughter or adduct ions. Here, the parent or precursor ion may have been fragmented into a first component and a second component including a predetermined ion or neutral particle, a fragment, product, daughter or adduct ion. Various steps may then be performed to further reduce / narrow the list of possible candidate parent or precursor ions, leaving some final candidate parent or precursor ions. Then, preferably thereafter, the final candidate parent or precursor ion is identified by comparing the elution times of the parent or precursor of interest and the fragment, product, daughter or adduct ions. As can be seen from the above, even though the two ions have similar mass-to-charge ratios, the chemical structure can be different and therefore the fragmentation is very likely different, so that the fragment, product, daughter or addition Parent or precursor ions can be identified based on product ions.

  Details of the sample introduction system are shown in FIG. The sample can be introduced into the mass spectrometer 6 by a Micromass® Modular CapLC system. For example, the sample is placed on a C18 cartridge (0.3 mm × 5 mm) and desalted with 0.1% HCOOH at a flow rate of 30 μL / min for 3 minutes. The 10-port valve was then switched to allow the peptide to elute onto the analytical column for separation (see inset in FIG. 2). The flow from the two pumps A and B was split so that the flow rate through the column was approximately 200 nL / min.

  A suitable analytical column is a PicoFrit® column packed with Waters® Symmetry C18. This is set to spray ions directly into the mass spectrometer 6. An electrospray potential (about 3 kV) can be applied into the liquid via a low dead volume stainless steel fitting. A small amount (about 5 psi (34.48 kPa)) of mist gas can be introduced around the spray tip to assist the electrospray process.

  Data can be acquired using a mass spectrometer fitted with a Z-spray® nanoflow electrospray ion source. The mass spectrometer can be operated in positive ion mode with a source temperature of 80 ° C. and a cone gas flow rate of 40 L / hr.

  The instrument can be calibrated with a multi-point calibration using, for example, selected fragments, products, daughters or adduct ions obtained from fragmentation of Glu-fibrinopeptide b. The data can be processed using the software MassLynx® suite.

  Switching between two different modes of operation of the collision induced dissociation fragmentation cell is not intended to be included within the scope of the present invention. However, the experimental results obtained according to the method will be described as they serve to illustrate aspects of the invention.

  FIGS. 3A and 3B show the tryptic digestion fragment or daughter and parent or precursor ion spectra of alcohol dehydrogenase (ADH), respectively. The fragment or daughter ion spectrum shown in FIG. 3A was obtained by maintaining the gas collision cell at a relatively high potential of about 30 V, resulting in significant fragmentation of ions through the gas collision cell. The parent or precursor ion spectrum shown in FIG. 3B was obtained with low collision energy (eg, 5 V or less). The data depicted in FIG. 3B was obtained using a mass filter 3 placed upstream of the collision cell and set to transport ions with a mass to charge ratio> 350. The mass spectrum in this particular example was obtained from a sample eluted from a liquid chromatograph. The spectra were acquired sufficiently quickly and close together in time so that they essentially correspond to the same components eluted from the liquid chromatograph.

  The mass spectrum shown in FIG. 3A was obtained by fragmenting ions by collision induced dissociation using a collision cell. Such an approach is not intended to be included within the scope of the present invention. However, the following description regarding the processing of the resulting mass spectrum and mass spectral data illustrates various aspects of the present invention.

  In FIG. 3B, there are several high intensity peaks in the parent or precursor ion spectrum (eg, peaks at 418.7724 and 568.7813). These are substantially less intense in the corresponding fragment ion spectrum shown in FIG. 3A. Thus, these peaks can be recognized as parent or precursor ions. Similarly, an ion that is more intense in the fragment or daughter ion spectrum shown in FIG. 3A than the parent or precursor ion spectrum shown in FIG. 3B can be recognized as a fragment or daughter ion. Also, as is apparent from the above, therefore, all ions with a mass charge value of less than 350 in the high fragmentation mass spectrum shown in FIG. 3A have a mass charge value of less than 350, and the mass charge value Can be easily recognized as fragment or daughter ions based on the fact that only parent or precursor ions greater than 350 have been transferred to the collision cell by the mass filter 5.

  4A-4E show mass chromatograms for three parent or precursor ions and two fragment or daughter ions, respectively. The parent or precursor ions are determined to have a mass to charge ratio of 406.2 (peak “MC1”), 418.7 (peak “MC2”) and 568.8 (peak “MC3”) The daughter ions were determined to have mass to charge ratios of 136.1 (peaks “MC4” and “MC5”) and 120.1 (peak “MC6”).

  The parent or precursor ion peak MC1 (mass-to-charge ratio 406.2) correlates well with the fragment or daughter ion peak MC5 (mass-to-charge ratio 136.1), ie, the mass-to-charge ratio is 406.2. It can be seen that the parent or precursor ions have fragmented to produce fragment or daughter ions 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 difficult to determine which parent or precursor ion corresponds to which fragment or daughter ion It is.

  FIG. 5 shows the peaks of FIGS. 4A-4E overlaid in sequence and redrawn at different scales. A careful comparison of the peaks of MC2, MC3, MC4 and MC6 actually shows that parent or precursor ion MC2 and fragment or daughter ion MC4 are well correlated, while parent or precursor ion MC3 is well correlated with fragment or daughter ion MC6 I understand that This is because the parent or precursor ion with a mass-to-charge ratio of 418.7 is fragmented to produce a fragment or daughter ion with a mass-to-charge ratio of 136.1, and the mass-to-charge ratio is 568.8. Indicates that the parent or precursor ions fragment to produce fragment or daughter ions with a mass to charge ratio of 120.1.

  This mass chromatogram cross-correlation may be performed by automated peak comparison means such as a suitable peak comparison software program running on a suitable computer.

FIG. 6 shows a mass chromatogram for a fragment or daughter ion having a mass to charge ratio of 87.04 extracted from HPLC separation and mass spectrometry obtained using mass spectrometer 6. It is known that the immonium ion for asparagine, which is an amino acid, has a mass charge value of 87.04. The chromatogram was extracted from all high energy spectra recorded on the mass spectrometer. FIG. 7 shows the total mass spectrum corresponding to scan number 604. This is a low energy mass spectrum recorded on the mass spectrometer 6 and a low energy spectrum next to the high energy spectrum in scan 605 corresponding to the largest peak in the mass chromatogram with a mass to charge ratio of 87.04. is there. This indicates that the parent or precursor ion for the asparagine immonium ion with a mass to charge ratio of 87.04 has a mass of 1012.54. This is because it shows a monovalent (M + H) ⁺ ion having a mass to charge ratio of 1013.54 and a divalent (M + 2H) ⁺⁺ ion having a mass to charge ratio of 507.27.

FIG. 8 shows the mass spectrum from the low energy spectrum recorded on the mass spectrometer 6 for trypsin digestion of the protein β-casein. Protein digestion products were separated by HPLC and mass analyzed. Mass spectra were recorded on a mass spectrometer 6 operating in MS mode, alternating low and high collision energies in a gas collision fragmentation cell for continuous spectra. FIG. 9 shows a mass spectrum from a high energy spectrum recorded substantially simultaneously with the low energy mass spectrum in FIG. FIG. 10 shows an enlarged view of the processed mass spectrum shown in FIG. For this spectrum, continuous data was processed to identify the peaks, display them with a line having a height proportional to the peak area, and annotate the mass corresponding to their center of mass. The peak with a mass to charge ratio of 1031.4395 is the divalent (M + 2H) ⁺⁺ ion of the peptide, and the peak with a mass to charge ratio of 982.4515 is a divalent fragment or daughter ion. It must be a fragment or daughter ion. Because it does not exist in the low energy spectrum. The mass difference between these ions is 48.9880. The theoretical mass for H ₃ PO ₄ is 97.9769, the mass to charge value for divalent H ₃ PO ₄ ⁺⁺ ions is 48.9984, and the difference from the observed value is only 8 ppm. Thus, the peak with a mass-to-charge ratio of 982.4515 relates to a fragment or daughter ion obtained from a peptide ion with a mass-to-charge ratio of 1031.4395 that has lost H ₃ PO ₄ ⁺⁺ ions.

  Presented here are some experimental data illustrating the ability of the preferred embodiment to quantify the relative abundance of two proteins contained in two different samples containing a mixture of proteins.

  The first sample contained the trypsin digestion products of the three proteins BSA, glycogen phosphorylase B and casein. These three proteins were present at a 1: 1: 1 ratio in the inset. Each of the three proteins had a concentration of 330 fmol / μl. The second sample contained the trypsin digestion products of the same three proteins, BSA, glycogen phosphorylase B, and casein. However, the protein was initially present in a 1: 1: X ratio. X was indeterminate, but was considered to be in the range of 2-3. The concentrations of protein BSA and glycogen phosphorylase B in the second sample mixture were the same as in the first sample (ie 330 fmol / μl).

  The experimental protocol followed was to load a 1 μl sample onto the HPLC column at a flow rate of 4 μl / min. The liquid flow was then split so that the flow rate to the nanoelectrospray ionization source was approximately 200 nl / min.

  Mass spectra were recorded on the mass spectrometer 6. Mass spectra were recorded with alternating low and high collision energies using a nitrogen collision gas. The low collision energy mass spectrum was recorded at a collision voltage of 10V, and the high collision energy mass spectrum was recorded at a collision voltage of 33V. The mass spectrometer was equipped with a nanolock spray device that delivered an independent liquid flow to the ionization source. The ionization source can sometimes be sampled to provide a reference mass. From the reference mass, mass calibration can be periodically verified. This ensured that the accuracy of the mass measurement was within 5 ppm RMS accuracy. Data was recorded and processed using the MassLynx® data system.

  First, the first sample was analyzed and the data was used as a reference. The first sample was then analyzed two more times. The second sample was analyzed twice. Using data from these analyses, an attempt was made to quantify the (unknown) relative abundance of casein in the second sample.

  All data files are automatically processed to generate a list of ions and their associated areas and a high collision energy spectrum for each experiment. This list is then searched against the Swiss-Prot protein database using the ProteinLynx® search engine. The chromatographic peak area was obtained using the Waters® Apex Peak Tracking algorithm. The chromatograms for each charge state found to exist were summed and then integrated.

  The relative expression levels determined by experiments with various peptide ions for the two samples, normalized to the reference data, are given in the table below.

  In the table above, peptides whose sequence was determined by high collision energy data are underlined. Confirmation means that the probability of this peptide is greater than any other peptide in the database given the current current fragmentation or reaction model given its exact mass and corresponding high collision energy data. Means. The remaining peptides are considered accurate based on their retention time and mass compared to those for the established peptides. It was expected that there could be some experimental error in the results due to injection volume error and other effects.

  Using BSA as an internal standard, the relative abundance of glycogen phosphorylase B in the first sample was determined to be 0.925 (first analysis) and 1.119 (second analysis), and the average was 1.0. The relative abundance of glycogen phosphorylase B in the second sample was determined to be 1.244 (first analysis) and 1.292 (second analysis), with an average of 1.3. These results are not inferior to the expected value of 1.

  Similarly, the relative abundance of casein in the first sample was determined to be 0.980 (first analysis) and 1.111 (second analysis), with 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), with an average of 2.7. These results are comparable to the expected values 1 and 2-3.

  The following data relates to chromatograms and mass spectra obtained from the first and second samples. A peptide derived from casein having the sequence HQGLPQEVLNNENLR elutes almost exactly simultaneously with a peptide derived from BSA having the sequence LVNELTEFAK. Although this occurs rarely, it provides an opportunity to compare the abundance of casein in two different samples.

11A-11D show four mass chromatograms. Two relate to the first sample and two relate to the second sample. FIG. 11A shows the mass chromatogram for the first sample for ions having the sequence HQGLPQEVLNNENLR and having a mass-to-charge ratio of 880.4, corresponding to casein-derived peptide ion (M + 2H) ⁺⁺ . FIG. 11B shows a mass chromatogram for a second sample corresponding to the same peptide ion with the sequence HQGLPQEVLNNENLR from casein.

FIG. 11C shows the mass chromatogram for the first sample for ions having the sequence LVNELTEFAK and corresponding to peptide ions (M + 2H) ⁺⁺ derived from BSA with a mass to charge ratio of 582.3. FIG. 11D shows a mass chromatogram for a second sample having the sequence LVNELTEFAK and corresponding to the same peptide ion from BSA. The mass chromatogram shows the intensity of peptide ions derived from BSA with a mass-to-charge ratio of 582.3 in both samples, while the peptide ions derived from casein with a mass-to-charge ratio of 880.4. Indicates that there is a difference of about 100%.

  FIG. 12A shows the parent or precursor ion mass spectrum recorded about 20 minutes after the first sample, and FIG. 12B shows the parent or precursor recorded after substantially the same time from the second sample. An ion mass spectrum is shown. The mass spectrum has an ion with a mass-to-charge ratio of 582.3 (derived from BSA) that is approximately the same in both mass spectra, while the ion with a mass-to-charge ratio of 880.4 for peptide ions from casein is It shows that the intensity in the second sample is approximately double compared to the one sample. This is consistent with expectations.

  FIG. 13 shows in more detail the parent or precursor ion mass spectrum shown in FIG. 12A. A peak corresponding to a BSA peptide ion with a mass charge of 582.3 and a peak corresponding to a casein peptide ion with a mass to charge ratio of 880.4 can be clearly seen. The inset shows the enlarged portion of the spectrum showing the isotope peak of the peptide ion with a mass to charge ratio of 880.4. Similarly, FIG. 14 shows in more detail the parent or precursor ion mass spectrum shown in FIG. 12B. Again, a peak corresponding to a BSA peptide ion with a mass to charge ratio of 582.3 and a peak corresponding to a casein peptide ion with a mass to charge ratio of 880.4 can be clearly seen. The inset shows the enlarged portion of the spectrum showing the isotope peak of the peptide ion with a mass to charge ratio of 880.4. As is apparent from comparison of FIGS. 12 to 14 and the insets of FIGS. 13 and 14, the abundance of peptide ions derived from casein and having a mass spectrum peak at a mass-to-charge ratio of 880.4 is compared to that of the first sample. Thus, the abundance in the second sample is approximately double.

  Although the present invention has been described with reference to preferred embodiments, workers skilled in the art will recognize that various changes can be made in form and detail without departing from the scope of the invention as set forth in the appended claims. To be understood.

FIG. 1 is a schematic diagram of a preferred mass spectrometer. FIG. 2 is a schematic diagram of the valve switch configuration during sample loading and desalting, and the inset shows the desorption of the sample from the analytical column. FIG. 3A shows a fragment or daughter ion mass spectrum and FIG. 3B is obtained when the mass filter upstream of the collision cell is configured to transfer ions with a mass to charge ratio> 350 to the collision cell. And the corresponding parent or precursor ion mass spectrum. 4A shows the mass chromatogram of the parent or precursor ion, FIG. 4B shows the mass chromatogram of the parent or precursor ion, FIG. 4C shows the mass chromatogram of the parent or precursor ion, and FIG. Shows the mass chromatogram of the fragment or daughter ion, and FIG. 4E shows the mass chromatogram of the fragment or daughter. FIG. 5 shows mass chromatograms in which FIGS. FIG. 6 shows a mass chromatogram of asparagine immonium ions having a mass to charge ratio of 87.04. FIG. 7 shows the mass spectrum of ADH-derived peptide ion T5 having the sequence ANELLINVK and the molecular weight 1012.59. FIG. 8 shows the mass spectrum of tryptic digestion of β-casein obtained when the collision cell was in low fragmentation mode. FIG. 9 shows the tryptic digestion mass spectrum of β-casein obtained when the collision cell was in high fragmentation mode. FIG. 10 is a view obtained by processing and enlarging the mass spectrum shown in FIG. FIG. 11A shows a mass chromatogram of ions from the first sample with a mass to charge ratio of 880.4, FIG. 11B shows a similar mass chromatogram of the same ions from the second sample, and FIG. Shows a mass chromatogram of ions from a first sample with a mass to charge ratio of 582.3, and FIG. 11D shows a similar mass chromatogram of the same ions from a second sample. FIG. 12A shows the mass spectrum recorded from the first sample, and FIG. 12B shows a second spectrum similar to the first sample except that both samples contain a higher concentration of the digestion product of the protein casein common to both samples. The corresponding mass spectra recorded from the samples are shown. FIG. 13 shows the mass spectrum shown in FIG. 12A in more detail, and the inset shows an enlarged portion of the mass spectrum showing an isotope peak at a mass to charge ratio of 880.4. FIG. 14 shows the mass spectrum shown in FIG. 12B in more detail, and the inset shows an enlarged portion of the mass spectrum showing an isotope peak at a mass to charge ratio of 880.4.

Claims (15)

A method of mass spectrometry,
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device comprising an electron capture dissociation fragmentation device;
Repeatedly switching, modifying or changing the electron capture dissociation fragmentation device between a first mode and a second mode, wherein in the first mode, the parent or from the first sample; At least some of the precursor ions are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented; Steps,
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, modifying or changing the electron capture dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or At least some of the precursor ions are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented; Steps,
Recognizing a first parent or precursor ion of interest from the first sample;
Automatically determining the intensity of the first parent or precursor ion of the object, wherein the first parent or precursor ion of the object has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from the second sample having the same first mass to charge ratio;
Comparing the intensity of the first parent or precursor ion of the object with the intensity of the second parent or precursor ion. A method of mass spectrometry,
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device comprising an electron capture dissociation fragmentation device;
Repeatedly switching, modifying or changing the electron capture dissociation fragmentation device between a first mode and a second mode, wherein in the first mode, the parent or from the first sample; At least some of the precursor ions are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented; Steps,
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, modifying or changing the electron capture dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or At least some of the precursor ions are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented; Steps,
Recognizing a first parent or precursor ion of interest from the first sample;
Automatically determining the intensity of the first parent or precursor ion of the object, wherein the first parent or precursor ion of the object has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from the second sample having the same first mass to charge ratio;
Determining a first ratio of the intensity of the first parent or precursor ion of interest 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 ion to the intensity of other parent or precursor ions in the second sample;
Comparing the first ratio to the second ratio. In the first operation mode,
(I) The electron has 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) 4-5 eV. Have
And / or (II) the electrons are confined by a magnetic field;
And / or the method further comprises providing an electron source, wherein in the first mode of operation, the electron source generates a plurality of electrons configured to interact with the parent or precursor ions. And / or in the second mode of operation, the electron source is switched off. A method of mass spectrometry,
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device comprising an electron transfer dissociation fragmentation device;
Repeatedly switching, modifying or changing the electron transfer dissociation fragmentation device between a first mode and a second mode, wherein in the first mode, the parent or from the first sample; At least some of the precursor ions are fragmented as they interact with the reagent ions to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented. , Step and
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, modifying or changing the electron transfer dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or At least some of the precursor ions are fragmented as they interact with the reagent ions to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented. , Step and
Recognizing a first parent or precursor ion of interest from the first sample;
Automatically determining the intensity of the first parent or precursor ion of the object, wherein the first parent or precursor ion of the object has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from the second sample having the same first mass to charge ratio;
Comparing the intensity of the first parent or precursor ion of the object with the intensity of the second parent or precursor ion. A method of mass spectrometry,
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device comprising an electron transfer dissociation fragmentation device;
Repeatedly switching, modifying or changing the electron transfer dissociation fragmentation device between a first mode and a second mode, wherein in the first mode, the parent or from the first sample; At least some of the precursor ions are fragmented as they interact with the reagent ions to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented. , Step and
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, modifying or changing the electron transfer dissociation fragmentation device between a first mode and a second mode, wherein in the first mode the parent or At least some of the precursor ions are fragmented as they interact with the reagent ions to produce fragment or daughter ions, and in the second mode, substantially fewer parent or precursor ions are fragmented. , Step and
Recognizing a first parent or precursor ion of interest from the first sample;
Automatically determining the intensity of the first parent or precursor ion of the object, wherein the first parent or precursor ion of the object has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from the second sample having the same first mass to charge ratio;
Determining a first ratio of the intensity of the first parent or precursor ion of interest 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 ion to the intensity of other parent or precursor ions in the second sample;
Comparing the first ratio to the second ratio.   At least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0 at least the collision, fragmentation or reaction device between the first mode and the second mode. Automatically switching, modifying or changing once every 7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 seconds; The method according to claim 1. An electron capture dissociation fragmentation device configured and adapted to be repeatedly switched, modified or changed between a first mode and a second mode in use, wherein at least some Electron capture, in which the parent or precursor ions are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode substantially fewer parent or precursor ions are fragmented A dissociation fragmentation device;
A mass analyzer;
A control system, in use, wherein: (i) recognizing a first parent or precursor ion of an object from a first sample, wherein the first parent or precursor ion of the object is a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of the subject;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
(Iv) a mass spectrometer comprising: a control system that compares the intensity of the first parent or precursor ion of the object with the intensity of the second parent or precursor ion. An electron transfer dissociation fragmentation device configured and adapted to be repeatedly switched, modified or changed between a first mode and a second mode in use, wherein at least some An electron that is fragmented as the parent or precursor ion interacts with the reagent ion to produce a fragment or daughter ion, and in the second mode, substantially fewer parent or precursor ions are fragmented. A moving dissociation fragmentation device;
A mass analyzer;
A control system, in use, wherein: (i) recognizing a first parent or precursor ion of an object from a first sample, wherein the first parent or precursor ion of the object is a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of the subject;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
(Iv) a mass spectrometer comprising: a control system that compares the intensity of the first parent or precursor ion of the object with the intensity of the second parent or precursor ion. An electron capture dissociation fragmentation device configured and adapted to be repeatedly switched, modified or changed between a first mode and a second mode in use, wherein at least some Electron capture, in which the parent or precursor ions are fragmented as they interact with electrons to produce fragment or daughter ions, and in the second mode substantially fewer parent or precursor ions are fragmented A dissociation fragmentation device;
A mass analyzer;
A control system, in use, wherein: (i) recognizing a first parent or precursor ion of an object from a first sample, wherein the first parent or precursor ion of the object is a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of the subject;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
(Iv) determining a first ratio of the intensity of the first parent or precursor ion of the subject to the intensity of other parent or precursor ions in the first sample;
(V) determining a second ratio of the intensity of the second parent or precursor ion to the intensity of other parent or precursor ions in the second sample;
(Vi) a mass spectrometer comprising: a control system that compares the first ratio to the second ratio. An electron transfer dissociation fragmentation device configured and adapted to be repeatedly switched, modified or changed between a first mode and a second mode in use, wherein at least some An electron that is fragmented as the parent or precursor ion interacts with the reagent ion to produce a fragment or daughter ion, and in the second mode, substantially fewer parent or precursor ions are fragmented. A moving dissociation fragmentation device;
A mass analyzer;
A control system, in use, wherein: (i) recognizing a first parent or precursor ion of an object from a first sample, wherein the first parent or precursor ion of the object is a first mass to charge ratio; Have
(Ii) determining the intensity of the first parent or precursor ion of the subject;
(Iii) determining the intensity of a second parent or precursor ion from a second sample having the same first mass to charge ratio;
(Iv) determining a first ratio of the intensity of the first parent or precursor ion of the subject to the intensity of other parent or precursor ions in the first sample;
(V) determining a second ratio of the intensity of the second parent or precursor ion to the intensity of other parent or precursor ions in the second sample;
(Vi) a mass spectrometer comprising: a control system that compares the first ratio to the second ratio. (A) an ion source further comprising: (i) an electrospray ionization (“ESI”) ion source, (ii) atmospheric pressure photoionization (“APPI”) ion source, (iii) atmospheric pressure chemical ionization ("APCI") ion source, (iv) matrix-assisted laser desorption ionization ("MALDI") ion source, (v) laser desorption ionization ("LDI") ion source, (vi) atmospheric pressure ionization ("API") ) Ion source, (vii) desorption ionization (“DIOS”) ion source using silicon, (viii) electron impact (“EI”) ion source, (ix) chemical ionization (“CI”) ion source, (x ) Field ionization (“FI”) ion source, (xi) field desorption (“FD”) ion source, (xii) inductively coupled plasma (“ICP”) ion source, (xiii) fast atom bombardment ("FAB") ion source, (xiv) liquid secondary ion mass spectrometry ("LSIMS") ion source, (xv) desorption electrospray ionization ("DESI") ion source, (xvi) nickel-63 radioactive ion source Selected from the group consisting of: (xvii) an atmospheric pressure matrix assisted laser desorption ionization ion source, and (xviii) a thermal spray ion source; and / or
(B) further comprising an ion trap or ion guide disposed upstream and / or downstream of the fragmentation device , the ion trap or ion guide comprising:
(I) a quadrupole rod set, a hexapole rod set, an octupole rod set or a multipole rod set or segmented multipole rod set including a rod set comprising more than eight rods ion trap or ion guide;
(Ii) In an ion tunnel or ion funnel ion trap or ion guide comprising a plurality of electrodes with openings or at least 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 electrodes In use, ions are transported through the opening and at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% of the electrodes. %, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% have openings of substantially the same size or area, or size or area An ion tunnel or ion funnel ion trap or ion guide, with openings that are sequentially larger and / or smaller
(Iii) a stack or array plane, plate or mesh electrode, wherein the one stack or array plane, plate or mesh electrode is 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 planes, plates or mesh electrodes, at least 5%, 10% of said planes, plates or mesh electrodes 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95 % Or 100% is a plane of a stack or array, a plate or mesh electrode, and (iv) an ion trap, generally placed in the plane in which ions travel in use. Is an ion trap or ion guide comprising a plurality of groups of electrodes arranged axially along the length of the ion guide, each group of electrodes comprising: (a) a first and second electrode and ions Means for applying a DC voltage or potential to the first and second electrodes for confining in a first radial direction within the ion guide; and (b) third and fourth electrodes and ions for the ions. An ion trap or ion guide comprising means for applying an AC or RF voltage to the third and fourth electrodes for confining the guide in a second radial direction;
Selected from the group consisting of: and / or
(C) further comprising a mass filter disposed upstream and / or downstream of the fragmentation device ;
The mass spectrometer in any one of Claims 7-10.   The mass analyzer is (i) a quadrupole mass analyzer, (ii) a 2D or linear quadrupole mass analyzer, (iii) a pole or 3D quadrupole mass analyzer, (iv) a Penning trap mass analyzer. (Vi) an ion trap mass analyzer, (vi) a sector magnetic mass analyzer, (vii) an ion cyclotron resonance ("ICR") mass analyzer, (viii) a Fourier transform ion cyclotron resonance ("FTICR") mass analyzer (Ix) electrostatic or orbitrap mass analyzer, (x) Fourier transform electrostatic or orbitrap mass analyzer, (xi) Fourier transform mass analyzer, (xii) time-of-flight mass analyzer, (xiii) orthogonal acceleration The group of time-of-flight mass analyzers, (xiv) axial acceleration time-of-flight mass analyzers, and (xv) quadrupole rod set mass filters or analyzers Are al selected, the mass spectrometer according to any of claims 7 11. The fragmentation device comprises (i) a quadrupole rod set, (ii) a hexapole rod set, (iii) an octupole rod set or higher order rod set, and (iv) an ion comprising a plurality of electrodes having openings. A tunnel, wherein ions are transported through the aperture, or (v) a plurality of electrodes connected to an AC or RF voltage source for radially confining ions in the fragmentation device The mass spectrometer according to any one of claims 7 to 12. A method of mass spectrometry,
Passing parent or precursor ions from the first sample to a collision, fragmentation or reaction device;
Repeatedly switching, modifying or changing the collision, fragmentation or reaction device between a first mode and a second mode, wherein in the first mode the parent from the first sample Or at least some of the precursor ions are fragmented or reacted to produce fragment, daughter, product or adduct ions, and in the second mode, substantially less parent or precursor ions are fragmented or Reacting, steps,
Passing parent or precursor ions from a second sample to a collision, fragmentation or reaction device;
Repeatedly switching, modifying or changing the collision, fragmentation or reaction device between a first mode and a second mode, wherein in the first mode the parent from the second sample Or at least some of the precursor ions are fragmented or reacted to produce fragment, daughter, product or adduct ions, and in the second mode, substantially less parent or precursor ions are fragmented or Reacting, steps,
Recognizing a first parent or precursor ion of interest from the first sample;
Automatically determining the intensity of the first parent or precursor ion of the object, wherein the first parent or precursor ion of the object has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from the second sample having the same first mass to charge ratio;
Comparing the intensity of the first parent or precursor ion of the subject with the intensity of the second parent or precursor ion;
The collision, fragmentation or reaction device comprises: (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) infrared radiation induced Dissociation device, (v) ultraviolet radiation induced dissociation device, (vi) nozzle-skim interface fragmentation device, (vii) in-source fragmentation device, (viii) ion source collision induced dissociation fragmentation device, (ix) thermal or temperature source fragmentation Device, (x) electric field induced fragmentation device, (xi) magnetic field induced fragmentation device, (xii) enzymatic digestion or enzymatic degradation fragmentation Device, (xiii) ion-ion reaction fragmentation device, (xiv) ion-molecule reaction fragmentation device, (xv) ion-atom reaction fragmentation device, (xvi) ion-metastable ion reaction fragmentation device, (xvii) ion-metas Table molecular reaction fragmentation device, (xviii) ion-metastable atomic reaction fragmentation device, (xix) ion-ion reaction device for reacting ions to form adducts or product ions, (xx) reacting ions Ion-molecule reaction device for forming adducts or product ions, (xxi) ion-source for reacting ions to form adducts or product ions Reaction device, (xxii) ion-metastable ion reaction device for reacting ions to form adduct or product ions, (xxiii) ion for reacting ions to form adduct or product ions A metastable molecular reaction device and selected from the group consisting of an ion-metastable atomic reaction device for reacting (xxiv) ions to form adducts or product ions,
Method. A method of mass spectrometry,
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, modifying or changing the surface-induced dissociation fragmentation device between a first mode and a second mode, wherein in the first mode, the parent or from the first sample; When at least some of the precursor ions hit the surface or target plate to generate fragment or daughter ions, in the second mode substantially less parent or precursor ions are fragmented; Steps,
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, modifying or changing the surface-induced dissociation fragmentation device between a first mode and a second mode, wherein in the first mode, the parent or the second sample from the second sample When at least some of the precursor ions hit the surface or target plate to generate fragment or daughter ions, in the second mode substantially less parent or precursor ions are fragmented; Steps,
Recognizing a first parent or precursor ion of interest from the first sample;
Automatically determining the intensity of the first parent or precursor ion of the object, wherein the first parent or precursor ion of the object has a first mass to charge ratio;
Automatically determining the intensity of a second parent or precursor ion from the second sample having the same first mass to charge ratio;
Comparing the intensity of the first parent or precursor ion of the object with the intensity of the second parent or precursor ion.

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