WO2001027971A1

WO2001027971A1 - Momentum acceleration orthogonal time of flight mass spectrometer

Info

Publication number: WO2001027971A1
Application number: PCT/US2000/028547
Authority: WO
Inventors: Edward M. Yin; Norman William Parker
Original assignee: Ion Diagnostics, Inc.
Priority date: 1999-10-14
Filing date: 2000-10-13
Publication date: 2001-04-19
Also published as: AU1570501A

Abstract

A method of orthogonal time of flight spectrometry is based upon applying a short, high voltage pulse to an electrode (315) to give ions (340, 350) within a pushout region (300) a constant momentum acceleration rather than a constant energy acceleration, thereby all ions (340, 350) in a beam (290) passed into the pushout region (300) are accelerated through a drift tube (325) and to a detector (330) at the same time.

Description

MOMENTUM ACCELERATION ORTHOGONAL TIME OF FLIGHT MASS

SPECTROMETER

Inventors: Edward M. Yin and N. William Parker

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/159,945 filed October 14, 1999.

BACKGROUND OF THE INVENTION

Field of the Invention

This invention relates to the field of mass spectrometry, and in particular to orthogonal time of flight mass spectrometers, suitable for high mass, high resolution mass spectrometry for use in proteomics and genomics.

Description of the Related Art

There is a growing need for compact, high-resolution mass spectrometers.

The advent of so-called soft ionization techniques, such as matrix-assisted laser

desorption ionization (MALDI) and electrospray ionization, has had a profound

effect on the utility of mass spectrometry for studying biological molecules. These

ionization techniques have rapidly extended the class of molecules that can be

analyzed, and it is now possible to use either ionization technique to generate ions

across a very large mass range with minimal interference from fragment ions. Mass spectrometry is expected to play an increasingly important role in studies of protein identification, protein expression and protein sequencing. The characterization and functional identification of proteins has been termed proteomics. Genomics is operationally defined as investigations into the structure and function of very large numbers of genes undertaken in a simultaneous fashion, including the genetic mapping, physical mapping and sequencing of entire genomes.

MALDI and electrospray ionization sources tend to produce ions over long time durations (from 10 μs to continuous), which would give poor mass resolution in a purely linear spectrometer system. Mass spectrometers that utilize MALDI and electrospray ion sources most commonly employ an orthogonal extraction (pushout) region, combined with an ion mirror (reflectron), which corrects for beam width within the pushout region. The pushout region allows for a continuous feed of ions into the spectrometer with little loss in mass resolution. These systems have achieved remarkable mass resolutions of up to 10,000 at masses of roughly 6000 Da (A.N. Krutchinsky, et. al., JASMS, vol. 9, pp 569-579 (1998)). However, reflectrons are not as effective in improving mass resolution for heavy ions (> 10,000 Da). Some applications, including proteomics and genomics, would benefit greatly from a mass spectrometer that could analyze heavy ions (> 10,000 Da) with high mass resolution. Also, reflectrons degrade instrument sensitivity because the ion beams are defocused and tend to diverge before reaching the detector region, thereby significantly lowering collection yield. Sensitivity is a very important parameter in many applications due to the small amounts of samples that are typically available. A method of correcting for beam width within the pushout region that does not reduce sensitivity is desired. FIG 1A shows a cross-sectional side view of a prior art orthogonal time of flight (o-ToF) mass spectrometer. o-ToF spectrometers allow for a compact (bench top) instrument that can accommodate ionization sources that continuously create ions. Mass spectrometers must be operated in a vacuum, with a pressure not to exceed 10^"5 Torr. From FIG 1A, an ionization source 100 ionizes the sample, and the ions are passed into an ion guide 105. The ion guide 105 typically decelerates the ions using some form of collisional process, and also collimates the ion beam using apertures. The collimated beam of low energy ions then collects in the pushout region 110, and an electronic pulse is applied to the pushout electrode 115 in order to send the ions through an extraction grid 120 and into the drift region 122. In order to provide high mass resolution, typical o-ToF spectrometers use a reflectron 125 to reflect all ions of the same mass into the detector 130 at the same time.

The two ionization sources most commonly used in o-ToF spectrometers are a MALDI source, shown in FIG 1 B, and an electrospray source, shown in FIG 1C. These so-called soft ionization techniques have been found to ionize large biological molecules without significant fragmentation. In a MALDI source, the analyte 132 is mixed with and embedded in a laser-absorbing solid material called a matrix 133, forming a sample mixture 135. The sample mixture 135 sits on a sample plate 140. An IR or UV laser beam 145 (depending on the sensitivity of the matrix material) is pulsed onto the sample mixture 135. The laser beam 145 is absorbed by the matrix 133, and a plume 150 of vaporized matrix and analyte is emitted into the vacuum. A small portion of this plume 150 is ionized, and is extracted by applying voltage on the extraction grid 155. In most applications, the matrix 133 is significantly lower in molecular weight compared to the analyte 132, and thus will not interfere with mass measurements. After passing through

focusing optics 157, the ions then travel to the mass analyzer 160.

In an electrospray source, the analyte is mixed into a liquid solution, and this analyte solution is injected using a syringe pump 165 through a thin needle

170 into the vacuum region 177. When high voltage on the order of 4 kV is

applied to the needle 170 relative to the cylinder electrode 175, the induced field ionizes the analyte, which then passes through a small capillary 180. A skimmer

185 is used to aperture down the ion beam before passing to the mass analyzer 190. Heat and gas flows from inlet 182 are used to desolvate the ions existing in the sample solution. Collisional cooling may be performed after the ions pass

through the skimmer 185 in order to decelerate the ions before being injected into the mass analyzer 190.

Referring to FIG 1 A, standard o-ToF mass spectrometers all operate on the technique of imparting a constant energy to the ions. When a constant voltage is applied to the pushout electrode 115 at time t=0, the ions gain energy due to the field within the pushout region 110. This energy is exactly equal to the potential

difference between the initial position (at time t=0) of the ion within the pushout region and the potential of the extraction grid 120. The resulting time of flight of

this type of spectrometer is given by

_{ToF =} L _{= L} ! v V 2E

where ToF is the time of flight, L is the length of the drift region, v is the ion

velocity in the drift region, m is the ion mass and E is the ion energy (constant for

all ions), it can be seen that the time of flight for each ion is proportional to the square root of the ion mass. Thus, for higher masses, the average spacing between the corresponding TbFs of different ion masses becomes smaller.

The use of momentum acceleration for mass spectrometry was considered

during the initial development of time of flight spectrometers in the 1950's.

However, the requisite fast, high-voltage switches were not available at that time. There are only a few recent references in the literature for momentum

acceleration, primarily because of the limitations in high-voltage switching (see D. loanoviciu, Nuclear Instrum. and Methods in Phys. Res. A, vol. 427, pp 157-160 (1999)). For a mass spectrometer based on momentum acceleration, each ion receives a constant momentum increase in the pushout region rather than a

constant energy increase. Therefore,

ToF = = L ™ v p

where p is the ion momentum (constant for all ions), and the ToF is directly

proportional to the ion mass. This significantly increases the spacing between the ToFs at higher ion masses, and thus increases the resolution at these masses.

With the technological advances in high voltage pulsers, momentum acceleration is now possible. This type of mass spectrometer system inherently has increased

mass resolution over energy acceleration spectrometers.

Current mass spectrometers have high mass resolution for medium-heavy

ions (e.g., mass resolution of 12,000 at 2,500 Da), but much lower resolution for slightly heavier ions (e.g., 1000 at 12,000 Da). By improving mass resolution for

higher mass ions, biologists can run fewer experiments and use smaller amounts

of their samples. This generally translates directly into economic savings gained

through higher specificity of a measurement and the elimination of additional steps. This is especially true for DNA, in which fragments approaching 1000 base pairs (roughly 300,000 Da) must currently be broken up into much smaller fragments. A mass spectrometer with accuracy approaching one base pair (mass resolution of 1000 for 300,000 Da fragments) would greatly decrease analysis cost and increase sequencing speed.

SUMMARY OF THE INVENTION

This invention provides a method of high mass resolution orthogonal time of flight mass spectrometry. This method may be used in both proteomics and genomics. According to aspects of the invention, the method comprises: generating a beam of ions, which passes into the pushout region of a spectrometer; applying a voltage to an electrode such that all the ions with a specified mass have the same time of flight from the pushout region, through the drift tube and to the detector. The electrode can be the pushout electrode or the extraction grid. The drift tube can have a length in the range of 10 cm to 1 m. The ion beam can have a width up to 1 mm in the pushout region. The generating step can utilize a matrix-assisted laser desorption ion source, an electrospray ion source, or a field-ionization ion source. Further, the method may include the measurement of ion mass peaks - mass peaks from 30,000 to 300,000 Daltons can be measured with peak resolutions greater than 3, and mass peaks from 200,000 to 350,000 Daltons can be measured with mass resolutions greater than 4000.

In preferred embodiments: the voltage is a voltage pulse followed by a monotonically increasing voltage tail; the detector is a multi-channel plate. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows a schematic cross-sectional side view of a prior art orthogonal time-of-flight mass spectrometer.

FIG. 1 B shows a schematic cross-sectional side view of a prior art matrix- assisted laser desorption ionization source.

FIG. 1C shows a schematic cross-sectional side view of a prior art electrospray ion source.

FIG. 2A shows a schematic cross-sectional side view of an embodiment of the present invention.

FIG. 2B shows a schematic diagram of the ion x-coordinate versus time within an embodiment of the present invention.

FIG. 2C shows a graph of the calculated Electrode Potential versus Time to be applied to the pushout electrode of an embodiment of the present invention.

FIG. 3 shows a graph of the calculated ion time-of-flight versus the ion mass (measured in units of DNA bases = 300 Da) for both the prior art constant energy o-ToF spectrometer and for an embodiment of the present invention.

FIG. 4 shows a schematic of a control system for an embodiment of the present invention.

FIG. 5A shows a simulation of the ion trajectories from the pushout region to the detector in an embodiment of the present invention, including a more detailed view of the trajectories near the pushout region. FIG. 5B shows a graph of the ion locations within the pushout and detector regions (measured from the pushout region) versus the ion mass (measured in units of DNA bases = 300 Da) for an embodiment of the present invention.

FIG. 6 shows a graph of the Mass Resolution versus Ion Mass (measured in units of DNA bases ≡ 300 Da) calculated from a simulation of an embodiment of the present invention, with and without the voltage tail.

FIG. 7A shows a graph of Mass Resolution versus Ion Mass (measured in Daltons) calculated from a simulation of an embodiment of the present invention that has been optimized for 10,000 Da ion mass.

FIG. 7B shows a graph of Mass Resolution versus Ion Mass (measured in Daltons) calculated from a simulation of an embodiment of the present invention for two different drift tube lengths.

FIG. 7C shows a graph of Mass Resolution versus Ion Mass (measured in Daltons) calculated from a simulation of an embodiment of the present invention optimized for 300,000 Da ion mass.

FIG. 8 shows a graph of Peak Resolution versus Ion Mass (measured in units of DNA bases = 300 Da) calculated from a simulation of an embodiment of the present invention as applied to the sequencing of DNA.

DETAILED DESCRIPTION

FIG 2A shows a schematic cross-sectional side view of a preferred embodiment of this invention, in which an ion beam 290, a pushout region 300, an ion source 305, a pushout electrode 315, an extraction grid 320, a drift tube 325, a detector 330, and an accelerating region 335 are shown. In FIG 2B ions located at the front 340, and back 350 of the beam are shown, as well as the time and position 347 at which these ions hit the detector. In FIG 2C, a voltage pulse 380, and a voltage tail 382 are shown. In FIG 2A, low energy ions 290 are injected into the pushout region 300 by an ion source 305. These ions 290 should have the following characteristics: ion energy 1-5 eV with less than a ± 1 eV energy spread, and beam collimation with less than 0.2° half angle beam divergence. Any ionization source that can provide these characteristics can be used with this invention. Such sources include, but are not limited to, electrospray ion sources, MALDI sources and field-ionization ion sources. A chamber pumped by vacuum pumps with a pressure less than 10^"5 Torr is required to enable all embodiments of this invention. As can be seen in FIG 2A, the system does not use a reflectron, but rather a simple linear drift tube 325. This is advantageous in terms of spectrometer sensitivity because a much larger portion of the ions are collected by the detector 330. In order to implement a momentum acceleration mode of operation, a short voltage pulse 380, as seen in FIG 2C, is applied to the pushout electrode 315 or the extraction grid 320. A preferred embodiment would have the pulse 380 applied to the pushout electrode 315 in order to keep the drift region 325 field-free. FIG 2B shows the effect of replacing the reflectron used in most mass spectrometers with a pulse shaping of the voltage applied to the pushout region as shown in FIG 2C.

In a typical o-TOF pushout system, a voltage is applied to the pushout electrode and held constant until all of the ions have left the pushout region 300. This results in transfer of a constant energy to each of the ions, depending on their location within the pushout region 300, but independent of ion mass. In the present invention, the short voltage pulse 380 is turned off before any of the ions of interest have left the region 300. Therefore, the lighter ions travel further in the electric field and gather more energy, while the heavier ions travel less distance

and gather less energy. If we examine the simple physics of the system, the force, F, on an ion in the pushout region 300 is

F = ma = ^~ (1 ) d where m is the mass of the ion, a is the acceleration during the applied electric

field, z is the ion charge, q is the Coulomb charge, V is the magnitude of the potential applied to the pushout electrode, and d is the width of the pushout region

300. From Equation (1), the ion acceleration, a, is

a = ^ = ^ (2) τ md

where τ is the duration of the pulse 380 and Av is the change in ion velocity due to

the voltage pulse 380, assuming constant acceleration. Thus,

mΔv _ τqV .„,

- — )

^ ^ Constant (4)

where Ap=mAv is the change in momentum. Therefore, if the charge z on each

ion is the same, all ions will see the same change in momentum, rather than energy.

After being pulsed out of the pushout region 300 by voltage pulse 380 and

voltage tail 382, the ions travel through the extraction grid 320 and through the drift region 325. The velocity for each ion is constant as it travels through the drift

region 325 because this region 325 is field-free. The length of drift region 325 is

typically in the range of 10 cm to 1 m, depending on the required specifications of the spectrometer. This range of drift tube lengths is acceptable for bench top applications. Because the high voltage pulse 380 is turned off before the ions reach the drift region 325, this drift region 325 can be held at low voltage (ground), unlike many standard energy acceleration mass spectrometers that require drift regions 325 at high voltage. This simplifies the electronics and electrical isolation of the vacuum chamber. The ions impinge upon a detector 330 placed at the end of the accelerating region 335. The detector 330 can be, but is not limited to, a standard multi-channel plate (MCP) electron detector. In order to detect high mass ions (> 100,000 Da), an acceleration region 335 will be required to impart further energy to the ions for detection. This acceleration region 335 will not significantly impact the mass resolution of the spectrometer. Times of flight for embodiments of the present invention will range from 10 μs to the millisecond range, with 100 μs being a typical value for the time of flight. The pushout repetition rate will be limited by this time of flight. Time of flight values for prior art constant energy spectrometers are typically at least 3 times shorter than for the present invention.

In typical o-ToF systems, a reflectron is required to account for beam width within the pushout region 300. In the present invention, the reflectron is effectively replaced by shaping the voltage tail 382 applied to the pushout electrode 315 or applied to the extraction grid 320. This is illustrated in FIG 2B. The ion at the front of the beam 340 must hit the detector 330 at the same time 347 as the ion at the back of the beam 350. Therefore, the back ion 350 must have a slightly higher velocity than the front ion 340 when it enters the drift region 325. This can be accomplished by applying a monotonically increasing voltage tail, VJAILW 382, on the pushout electrode 315. The purpose of the voltage tail 382 is to offset the mass resolution degradation due to a finite beam diameter dx within the pushout region 300. In order for all ions of the same mass across the beam diameter to reach the detector 330 at the same time 347, the back ions 350 must be accelerated a small amount more than the front ions 340. Thus, with this monotonically increasing voltage 382, the mass resolution can be optimized for any beam size for a given mass. The analytic solution for this function is derived below. A particular mass m_opt must be specified in order to calculate the required function for the voltage tail VTA_IL ). The mass resolution is optimized for ion mass m₀pt, but mass resolution is improved for all ion masses with the implementation of the voltage tail V_TAi_L.(t) 382. In FIG 2B, with an incoming ion beam width of dx, the front ion 340 and the back ion 350 represent the edges of this beam. It is required that these ions hit the detector 330 with the same time of flight, which is defined as a constant, ToF. The function t₀(x) is defined as the time when an ion originating at position x within the pushout region 300 (when the voltage pulse 380 is initiated) crosses the extraction grid 320 into the field free drift region 325. For x closer to the extraction grid 320, t₀(x) has a smaller value. The time of flight is therefore equal to the time in the pushout region 300, t₀(x), plus the time it takes for the ion to travel through the drift region 325. Since all of the ions of mass m_opt are required to hit the detector 330 with the same time of flight ToF,

ToF = t₀(x)+ (5)

where L is the length of the drift tube 325 and v_opt(fo) is the velocity of the ion of mass mop_t as it leaves the extraction grid 320. Solving for the velocity,

v_0VMx)) = ^L (6)

ToF - t₀(x) Since t₀(x) will vary depending on the initial position of the ion in the pushout

region, this equation must be satisfied for all t₀(x) to make the time of flight

independent of x. Thus, an ion velocity in the drift region 325 is required to have the following characteristic:

"^τ , <⁷⁾

This equation is valid for all t greater than the duration of the voltage pulse 380, and less than the total time of flight ToF. The duration of the voltage pulse 380 is

defined as τ so this equation should be valid for all ions of mass m_opt from τ < t <

ToF, independent of their starting position x. Equation (7) can be differentiated to obtain the acceleration of the ion of mass m_opt required to achieve this drift velocity:

a_op,TML(t) = _(ToF _ _t)2 (8)

The subscript "_opt" is used to indicate that this is acceleration only of the ion of optimized mass m_opt. The subscript "_TAIL" is used to indicate that the acceleration is during the application of the voltage tail, V_JM\_( ) 382, and not during the initial

pulse 380 (see Fig 2C). Using Equation (1 ),

Solving for VTAIL(Γ),

ΪYI dL V_ΎML( = " _{- 2} for f > τ (10) zq(ToF - 1)

The duration of the voltage pulse 380, τ, is an important parameter that is related

to the minimum detectable ion mass of the spectrometer tn_min. The term tT7_min is defined as the mass of the ion that just reaches the edge of the extraction grid when the voltage pulse 380 is turned off and the voltage tail 382 begins.

Typically, τ must be kept long and will be limited by the maximum voltage

obtainable from the electronic pulser unit (not shown). This value of τ can be

calculated in terms of the spectrometer design parameters. In the present

invention, there are several design parameters: optimal mass (m_opt), time of flight

for the optimal mass (ToF), minimum detectable mass {m_mm), drift region 325 length (L), pushout region 300 width (d), and the magnitude Vp_Uι_se and duration

(τ) of the initial pulse. In order to calculate these last two values, the boundary

conditions of the time-dependent system must be examined. It is required that at

time τ, which is the time when the lightest ion of interest passes through the

extraction grid 320, the following is true for the optimized mass m_opt (from Equation (7)):

v_opl(τ) = — ±— (11 )

ToF - τ

The acceleration required to achieve this velocity is a result of the initial voltage pulse 380. Assuming the potential during the pulse 380 is constant, then the acceleration is also constant during the pulse 380, and can be expressed as t_.PULSE (0 = ^^ = ,- _r ^L , = constant for 0 < t < τ (12) τ (ToF - τ)τ

Thus, from Equation (1), the voltage pulse 380 has the following form:

V_PULSE( =

, for θ < f < τ (13) zq(ToF - τ)τ

and V_PULSE(t) = 0 for f > τ The required duration, τ, of the voltage pulse 380 can also be derived in terms of the operating parameters. Equation (12) applies to ions of mass m_opt, therefore for ions of mass t77_mιn, the acceleration, a_mιn, PULSE, due to the pulse is given by the ratio of the masses:

m ]_, m tfm.n.pu SE = ,,pu SE — — = ^~ : — = constant (14) m_m,_n (ToF - τ)τ m_mn

Thus, the time it takes the lightest ion to travel across the pushout region 300 (d) is given by the standard equation of motion for a body initially at rest,

" ^{= α}nun,PU SE^{τ = ~}Z 7XX 7 V ' *³)

^{2 2} _m,„ (ToF - τ)

Solving for τ,

τ = ^{2m dToF} (16)

As can be seen, τ is a strong function of m_mιn, and therefore the minimum detectable mass is a very important parameter of the system. Typically, τ must be as long as possible in order to meet the requirements on the voltage pulse 380. The voltages on the pushout electrode 315 are now fully defined in Equations (10), (13), and (16). For ion masses less than m_mιn, the system behaves similarly to a standard energy acceleration mass spectrometer because each mass is imparted a constant energy. However, the mass resolution at these lower ion masses may be poor due to the lack of a reflectron.

Another parameter that has not yet been considered is the beam divergence within the pushout region 300. The above calculations have assumed a perfectly parallel beam within the pushout region 300. If this is the case, then essentially infinite mass resolution can be achieved at the optimized mass m_op using the analytic solution in Equations (10) and (13). However, an assumption of a perfectly parallel beam is not realistic. The software package called SIMION 3D, version 6, which is a ray tracing program developed at the Idaho National Engineering and Environmental Laboratory and commonly used for modelling of mass spectrometer optics, has been used to examine the effect of beam divergence (angular spread) within the pushout region 300. It is found that the mass resolution is quite sensitive to this parameter. The beam divergence introduces an energy (or velocity) component in the direction of the pushout pulse 380, which will affect the time that the ions reach the detector 330, and thus affects mass resolution. For a mass resolution approaching 10,000, the beam divergence of the ion beam within the pushout region 300 must typically be less than approximately 0.2° half angle. A beam diameter of 1 mm has been assumed.

The final parameter that should be taken into account is the required collection area of the detector 330 at the end of the drift space 325 and accelerating region 335. Because the ions have some initial energy in the pushout region 300, typically about 1 -5 eV, they will maintain a velocity component in the Y-direction throughout the time of flight. Therefore, the two key components that determine the area of the detector 330 that is required to capture all the ions are (1) ion energy as they enter the pushout region 300, and (2) time of flight. The higher the ion energy and the longer the time of flight, the larger the required collection area of the detector 330. MicroChannel plate detectors come with a variety of collection areas, up to more than 100 mm in diameter. These should be more than sufficient to capture all of the ions that are pulsed from the pushout region 300. However, detectors with smaller collection areas are preferred on the basis of lower cost and higher detection bandwidth.

Table 1 below shows the consequences of varying the following parameters: time of flight, minimum mass, optimized mass, pushout width, drift tube 325 length, and beam divergence in the pushout region 300. The table is read as follows: by increasing the value of just a single parameter (in Column 1 ), the effect on mass resolution, pulse height and duration, and required detector size are given in the corresponding box. The effects on only these 6 parameters have been included because these are the limiting factors in making a high quality spectrometer. For example, if the ToF is increased, the magnitude of the voltage pulse 380, V_puι_se, can be lowered, and the duration, τ, of the voltage pulse 380 can be increased; but the mass resolution decreases and the detector size increases. Ideally, the mass resolution should be maximized, the voltage pulse 380 height should be minimized, the voltage pulse 380 duration should be maximized, and the detector 330 collection area should be minimized. When the length of the drift tube 325 increases, the mass resolution curve changes somewhat. This will be described below.

Table 1. Effect on some design specifications when the values of the parameters in Column 1 are increased.

The Drift Length, Pushout Region Width, and Beam Divergence will be fixed by the manufacturer. However, the Time of Flight, Minimum Mass and Optimized Mass can be changed from experiment to experiment by the operator of the spectrometer. The instrument will calculate the required voltage pulse height and duration and determine if that selection of parameters is capable of being performed by the instrument, enabling optimal mass resolution to be obtained for different ion masses from experiment to experiment.

FIG 3 shows a plot of calculated time of flight versus ion mass for energy acceleration 395 and momentum acceleration 397 mass spectrometers. Simulations of various embodiments of the present invention have been carried out on SIMION 3D, version 6. In the present invention, the operation of the spectrometer is based on imparting a constant momentum to the ions within the pushout region 300 (from FIG 2A). This produces a the time-of-flight, ToF, that is directly proportional to the ion mass, as evidenced by the linear curve 397. A lineary proportionality significantly increases the spacing between the ToFs at higher ion masses, and thus increases the resolution at these masses. This difference can be seen in FIG 3. Curve 395 shows the ToF versus ion mass for a prior art constant energy spectrometer, whereas curve 397 indicates the ToF for the present invention. It can be seen that the mass resolution with curve 397 will be significantly higher than for curve 395 since the ion arrival times at the detector for successive ion masses are more widely spaced for curve 397 than for curve 395.

FIG 4 is a schematic of the mass spectrometer control system, showing a laser 400, a triggering circuit 405, a pulse generator 410, a pushout region 330, a pushout electrode 315, an extraction grid 320, a drift tube 325, a detector 330, a digital oscilloscope 415, and a pre-amplifier 425. This embodiment shows a MALDI or other matrix-assisted laser desorption (MALD) type of source, as evidenced by the incorporation of an IR laser 400. A triggering circuit 405 is used to send synchronized signals to the laser 400, the pulse generator 410, and the oscilloscope 415, which detects the current coming from the MCP detector 330 through a pre-amplifier 425. The pre-amplifier 425 is required to increase the output signal due to the low ion count. The triggering circuit 405 first sends a signal to the IR laser 400 in order to irradiate the sample 426 with a pulsed laser beam 428 and create ions. These ions are decelerated and collimated into a parallel beam (not shown in FIG 4) before passing into the pushout region 300. After a delay 427 to allow the ions to fill the pushout region 300, a signal is sent to the pulse generator 410 to implement the high voltage pulse 380 and the voltage tail 382. In the configuration shown in this embodiment, both the voltage pulse 380 and voltage tail 382 are applied to the back pushout electrode 315. The sequential forces arising from the voltage pulse 380 followed by the voltage tail 382 accelerate the ions 435 through the extraction grid 320 and into the drift region 325, before hitting the detector 330. Repetitive voltage pulses 380 and voltage tails 382 can be sent from the triggering circuit to increase the summed signal on the digital oscilloscope 415. This repetition rate cannot exceed the time of flight of the heaviest ions created by the ionization source. Typical repetition rates can be in the hundreds to thousands of Hertz range.

In FIG 5A, plots of simulated ion trajectories are plotted showing a pushout region 330, an extraction grid 320, a drift tube 325, light ion 500 and heavy ion 510 trajectories, a detector 330, and the near side 505 and far side 515 of the detector 330 (with respect to the source). The ion trajectories have been modeled extensively from the source down to the detector. SIMION 3D, version 6.0, was programmed to launch ions from the ionization source and to implement the voltage pulse 380 and voltage tail 382. Trajectories of ions for varying masses are shown in FIGS 5A. As can be seen, the lighter ions 500 travel significantly farther into the pushout region 300 than the heavier ions 510. When the voltage pulse 380 is initiated, the lighter ions 500 gather significant energy in the orthogonal direction and are accelerated through the extraction grid 320. The detailed view in FIG 5A shows the trajectories of the lighter ions 500 and heavier ions 510 as they pass through the drift region 325 and reach the detector 330. It can be seen that although the lighter ions 500 travel further into the pushout region 300, they actually hit the detector 330 on the side 505 nearer the source. Ion mass affects the location on the detector where the ions hit because ions of higher mass 510 will gain a smaller energy during the pushout pulse 380 and voltage tail 382. Therefore, the heavier ions 510 will travel down the drift tube at a larger angle with respect to the pushout axis, and they will hit the detector 330 on the side 515 further from the source. These simulated ion trajectories indicate the positioning of the pushout region 300 and detector 330 within the vacuum chamber, as well as the required size of each of these components. This has been summarized in FIG 5B.

In FIG 5B, the curves 520 and 522 show the calculated locations of the ions within the pushout region 300, as measured from the edge of the entrance of the pushout region 300. Curves 520 and 522 show the positions of the first and last ions, respectively, to enter the pushout region 300. The top curves 525 and 527 show the location of the first and last ions, respectively, as they hit the detector 330 after flying through the drift tube 325. For the ion masses of interest in this simulation, the pushout region 300 length has been determined to be roughly 60 mm, as can be seen from curves 520 and 522, which have a maximum value of approximately 60 mm and a minimum value of 0 mm. The required detector size is roughly 60 mm in length, as can be seen from curves 525 and 527, which range from a minimum of roughly 70 mm to a maximum of roughly 130 mm, giving a range of 60 mm. This range determines the necessary collection area of detector 330. Although the detector 330 is required to be fairly long in the Y direction (see FIG 5A), the width of the detector (in a direction perpendicular to the plane of FIG 2A) is not so critical because the ions will have very little energy in this direction and therefore, the ion beam spread in this direction at the plane of the detector 330 will be minimal. Thus, a preferred embodiment employs a long, rectangular detector.

FIG 6 shows a graph of the mass resolution calculated using SIMION 3D, version 6.0, of an embodiment of the present invention with and without the voltage tail 382. Both curves 600 and 605 utilize the voltage pulse 380 in order to impart a constant momentum pulse to the ions. The top curve 600 shows the mass resolutions attainable in mass spectrometers embodying the present invention in which both the voltage pulse 380 and the voltage tail 382 are employed to correct for a finite beam width. The bottom curve 605 shows the mass resolutions attainable if only the voltage pulse 380 is employed. Clearly there is a significant improvement in mass resolution when the voltage tail 382 is implemented.

In FIGS 7A, 7B and 7C, plots of the Mass Resolution versus Ion Mass are shown for various embodiments of the present invention. FIG 7A shows a calculated Mass Resolution versus Ion Mass curve under typical operating conditions of an embodiment of the present invention. The operating conditions are given in Table 2 below. The operating conditions are designed to detect ions with masses in the 1-20 kDa range, with an optimization at 10 kDa. A significant aspect of the present invention is that the shape of pulse 380 is not critical, only the pulse energy ( ). This is because the change in ion velocity is equal to

the integral of the acceleration, which is proportional to the integral of the applied potential. The important figure of merit is the energy of the pulse, which must vary less than 1% from pulse-to-pulse.

Table 2. Typical operating conditions of a particular embodiment of the present invention

It can be seen in FIG 7A that there is a large peak 702 at the optimized mass of 10 kDa, and a gradual decrease of mass resolution on either side 704 and 706. FIG 7B shows a plot of the calculated mass resolution versus ion mass for the present invention with two different drift tube 325 lengths. The solid line represents the calculated mass resolution for a shorter drift tube 325 length of 10 cm, while the dashed line represents the calculated mass resolution for a longer drift tube 325 length of 1 m. As can be seen, the peak 720 is higher for a shorter (10 cm) drift tube 325, but the tails 722 and 724 drop off much faster. For the longer (1 m) drift tube 325, the peak 730 is lower, but the tails 732 and 734 are higher. There may be applications in which a sharper peak is desirable. A final plot in FIG 7C shows the calculated mass resolution for a system at much higher masses. In this case, the optimized mass is 300 kDa, corresponding to the peak 740 with tails 742 and 744. Comparison of the height of peak 720 with the height of peak 740 shows that the mass resolution does not drop off significantly for higher mass ions. This is important for many applications that would benefit from the analysis of very heavy ions. A specific example is DNA sequencing, where the larger the DNA fragment that can be analyzed (with high resolution), the faster a strand can be sequenced. All of the calculated mass resolutions in FIGS 7A, 7B and 7C were derived from simulations using SIMION 3D, version 6.0.

Genomics, and specifically the sequencing of DNA, is an application for various embodiments of the present invention. In FIG 8, the concept of peak resolution for mass spectrometric sequencing of DNA is introduced for low mass 800, middle mass 802 and high mass 804 ions. Peak resolution is defined as the time separation of neighboring mass peaks (separated by one DNA base unit = 300 Da) divided by the full-width-half-maximum (FWHM) time peak corresponding to an individual ion mass. FIG 8 shows simulations of the peak width and spacing as derived from calculations for a system optimized for DNA sequencing. These simulations are at low mass 800 (100 bases = 30,000 Da), middle mass 802 (500 bases = 15,000 Da) and high mass 804 (1000 bases = 300,000 Da). It can be seen that peak resolution is greater than 3 for all ion masses, allowing simple separation ion mass peaks. In genomics, it is important to be able to resolve the number of bases in each DNA ion up to very heavy masses. For example, 1000 bases represents approximately 300,000 Da. As can be seen from FIG 8, each of the ions 800, 802 and 804 can easily be separated from a neighboring DNA ion containing one less or one more DNA base.

Claims

WHAT IS CLAIMED IS:

1. A method of orthogonal time of flight spectrometry comprising: generating a beam of ions, which passes into a pushout region of a spectrometer; and applying a voltage to an electrode such that all the ions with a specified mass have a same Time of Flight, ToF, from the pushout region, through a drift tube and to a detector.

2. A method as in claim 1 wherein the voltage is a voltage pulse followed by a monotonically increasing voltage tail.

3. A method as in claim 1 wherein the electrode is a pushout electrode.

4. A method as in claim 1 wherein the electrode is an extraction grid.

5. A method as in claim 1 wherein the generating step utilizes a matrix- assisted laser desorption ion source.

6. A method as in claim 1 wherein the generating step utilizes an electrospray ion source.

7. A method as in claim 1 wherein the generating step utilizes a field- ionization ion source.

8. A method as in claim 1 wherein the detector is a multi-channel plate.

9. A method as in claim 1 wherein the drift tube has a length between 10 cm and 100 cm.

10. A method as in claim 1 wherein the beam of ions has a width of less than 1 mm in the pushout region.

11. A method as in claim 1 further including the step of measuring a range of ion mass peaks from 30,000 to 300,000 Daltons with peak resolutions greater than 3.

12. A method of orthogonal time of flight spectrometry comprising: generating a beam of ions, which passes into a pushout region of a spectrometer; and accelerating the ions out of the pushout region into a drift tube by applying a voltage to an electrode such that all the ions with a specified mass have a same Time of Flight, ToF, from the pushout region to a detector.

13. A method of orthogonal time of flight spectrometry comprising: generating a beam of ions, which passes into a pushout region of a spectrometer; and applying a voltage pulse followed by a monotonically increasing voltage tail to a pushout electrode such that all the ions with a specified mass have a same Time of Flight, ToF, from the pushout region, through a drift tube and to a multichannel plate detector.

14. A method as in claim 13 wherein the generating step utilizes a matrix- assisted laser desorption ion source.

15. A method as in claim 13 wherein the generating step utilizes an electrospray ion source.

16. A method as in claim 13 wherein the generating step utilizes a field- ionization ion source.

17. A method as in claim 13 wherein the drift tube has a length between 10 cm and 100 cm.

18. A method as in claim 13 wherein the beam of ions has a width of less than 1 mm in the pushout region.

19. A method as in claim 13 further including the step of measuring a range of ion mass peaks from 30,000 to 300,000 Daltons with peak resolutions greater than 3.

20. A method as in claim 13 further including the step of measuring a range of ion mass peaks from 200,000 to 350,000 Daltons with mass resolutions greater than 4000.