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GB2367185A - Method of analysis of a molecule using a tandem mass spectrometer - Google Patents

Method of analysis of a molecule using a tandem mass spectrometer Download PDF

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Publication number
GB2367185A
GB2367185A GB0114295A GB0114295A GB2367185A GB 2367185 A GB2367185 A GB 2367185A GB 0114295 A GB0114295 A GB 0114295A GB 0114295 A GB0114295 A GB 0114295A GB 2367185 A GB2367185 A GB 2367185A
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Prior art keywords
mass spectrometer
complex
ions
molecule
ionized
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GB2367185B (en
GB0114295D0 (en
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Steve Thompson
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Scientific Analysis Instruments Ltd
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Scientific Analysis Instruments Ltd
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    • HELECTRICITY
    • H01ELECTRIC ELEMENTS
    • H01JELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00Particle spectrometers or separator tubes
    • H01J49/26Mass spectrometers or separator tubes
    • H01J49/28Static spectrometers
    • H01J49/30Static spectrometers using magnetic analysers, e.g. Dempster spectrometer
    • H01J49/305Static spectrometers using magnetic analysers, e.g. Dempster spectrometer with several sectors in tandem
    • HELECTRICITY
    • H01ELECTRIC ELEMENTS
    • H01JELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00Particle spectrometers or separator tubes
    • H01J49/26Mass spectrometers or separator tubes
    • HELECTRICITY
    • H01ELECTRIC ELEMENTS
    • H01JELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00Particle spectrometers or separator tubes
    • H01J49/26Mass spectrometers or separator tubes
    • H01J49/34Dynamic spectrometers
    • H01J49/40Time-of-flight spectrometers

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  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

A method of analysis of a molecule, particularly a peptide molecule, including the steps of: modifying the molecule by attaching a second molecule to form a complex, the second molecule being selected to promote unimolecular dissociation; and analysing the complex using a tandem mass spectrometer comprising a first mass spectrometer tuned to select the complex when ionized and a second mass spectrometer having an energy bandwidth of sufficient size to enable it to produce a spectrum of at least 80% of the ionized complex and its dissociation products without adjustment of its electromagnetic properties. The tandem mass spectrometer used may be capable of producing a spectrum of the ionized complex of substantially all of the ionized complex and its dissociation products without adjustment. Preferably the tandem mass spectrometer comprises a pair of time-of-flight mass spectrometers, the second comprising a harmonic field mirror reflectron.

Description

METHOD OF ANALYSIS The present invention relates to a method of analysis of molecules, particularly but not exclusively peptide molecules, by tandem mass spectroscopy.
It is a well established method for the identification of proteins to separate them using two dimensional gel electrophoresis. Separation occurs in the first dimension under the action of an applied electric field, the proteins separating according to their iso-electric points. In the second dimension, separation occurs according to the diffusion rate of the protein, a property which relates roughly to its physical size. Proteins separated by this technique can be collected by staining the proteins and removing the region containing proteins of a particular iso-electric point and diffusion coefficient from the gel. The molecular weight of this protein or group of proteins can then be measured by mass spectrometry.
A more accurate identification of the protein can be achieved if the protein is further broken down by the action of an appropriate enzyme such as trypsin. This can result in a series of cleavages of the protein backbone where specific amino acid residues exist. The mass of the resulting group of peptides can be measured to high accuracy using mass spectrometry.
Ten parts per million (ppm) mass measurement accuracy is not unusual.
These peptides each represent a part of the chain of the original protein molecule. Where the cleavage rules for the enzymatic digestion are known
and where the protein is catalogued the peptide masses can be submitted for database searching and the original protein can be identified.
As the total number of recorded proteins becomes ever larger, protocols for unique identification become more challenging. Where two or more proteins were present during the digestion phase the resulting peptide spectrum can become very complex leading to the possibility of incorrect identification. What is required is a second technique which can confirm the identification derived from the peptide mass spectrum. This can be achieved if an individual peptide molecule is further dismantled, amino acid by amino acid, to reveal the type and sequence of amino acids present. This additional information has a enormous effect on reducing the number of possible matches from the database.
One such method, involving modifying a peptide molecule to promote fragmentation and using a time of flight mass spectrometer to study the fragment ions is described by Keough et al in T. Keough, R. S. Youngquist and M. P. Lacey in"A method for high sensitivity peptide sequencing using postsource decay matrix-assisted laser desorption ionization mass spectrometry", Proc. Natl. Acad. Sci. , USA 96,7133-7136, (1999).
However, the ion mirror reflectron used in this study has a limited energy bandwidth. The fragment ions have a large range of kinetic energies. The result is that only a small range of ions (fragments or precursor) is in time focus and available for collection for any one setting of
the spectrometer. To collect the whole spectrum, the spectrometer must be adjusted for each narrow range until the whole of the fragment spectrum is collected. The individual spectra are then stitched together to reveal the complete spectrum. Software tools have to be employed to correct for the calibration over each range and for the uneven background signal level.
This is a time consuming business with many opportunities for error.
Worse, each individual spectral acquisition comprises many shots of the laser used to initially ionize the precursor. Inevitably, the region of sample being analysed will become depleted of analyte material. Frequently, the complete"stitched spectrum"is comprised of spectra from many different regions of the sample. This is a most unsatisfactory way to perform the experiment.
It is an object of the present invention to overcome, or at least mitigate, problems associated with Keough's method of analysis.
According to the present invention there is provided a method of analysis of a molecule comprising the steps of: modifiying the molecule by attaching a second molecule to form a complex, the second molecule being selected to promote unimolecular dissociation of the complex; and analysing the complex using a tandem mass spectrometer comprising a first mass spectrometer tuned to select the complex when ionized and a second mass spectrometer having an energy bandwidth of sufficient size to
enable it to produce a spectrum of at least 80% of the ionized complex and its dissociation products without adjustment of its electromagnetic properties.
This allows the bulk of any complex ions and dissociation products to be analysed substantially at the same time, overcoming problems associated with Keough's technique.
Preferably, the tandem mass spectrometer is capable of producing a spectrum of substantially all of the ionized complex and its dissociation products without adjustment.
The energy bandwidth of the mass spectrometer is preferably at least 20 kev.
Preferably the tandem mass spectrometer comprises a pair of time of flight mass spectrometers.
The first mass spectrometer preferably comprises a means for generating ions from a sample, means for accelerating those ions and a field free drift space. The first mass spectrometer preferably also includes means for delaying the acceleration of ions after their generation, such that ions with the same mass to charge ratio are brought substantially to a time focus plane despite having differing initial velocities. This time focus plane preferably coincides with the entry plane of the second mass spectrometer.
An ion selector is preferably provided for selecting ions according to their arrival time at the end of the field free drift space.
The second mass spectrometer preferably comprises a harmonic field mirror reflectron. The potential V (r, z) near the axis of the second mass spectrometer is preferably given by the relationship : V (r, z) = Vo/a2 (z2-r2/2) for z in the range 0 < z < a, where z is the displacement along the axis of the second mass spectrometer measured from its entry plane and r is the radial distance from this axis.
In order that the invention may be more clearly understood an embodiment thereof will now be described, by way of example, with reference to the accompanying drawing, the single figure of which shows a schematic view of apparatus for use in the method.
First, a peptide molecule to be analysed is modified to promote its unimolecular dissociation by replacing the proton that terminates the"N" terminus of the peptide backbone with an acid group containing a sulphonate negative ion. This charge is balanced in the solution by a proton attached to the arginine residue at the"C"terminus. The modified molecule is then analysed using a tandem mass spectrometer using matrix assisted laser disorption (MALI) in order to produce ions from the modified molecules. During the analysis some of the modified molecules will undergo unimolecular dissociation, producing ion fragments.
To illustrate this further an example is described below for a tripeptide: Below the neutral tri-peptide molecule is shown in its unmodified
form. If this peptide is a result of a cleavage by trypsin the R1 will usually be an Arginine or Lysine amino acid :
Modification of the peptide involves removing the proton from the"N" terminus and adding a sulphonate ion. The balancing positive charge is then effectively trapped to the arginine or lysine amino acid:
In a MALT ! experiment the ion is formed by proton attachment. In the derivitised molecule the site on the arginine residue is occupied by the proton balancing the sulphonate group so the second proton which forms the positive ion is free to find a site anywhere on the backbone of the molecule just as if it were two times charged. (It is known that doubly charged ions from an electrospray ionization source of peptides where arginine is present as a terminal group undergo significantly higher rates of dissociation after low energy collisional activation than the singly charged ion. ) A typical dissociation regime is shown below.
Neutral loss"Y"series ion.
When this molecule is ionized by proton attachment the proton can take up any position on the backbone. This leads to an increased probability of fragmentation. After fragmentation the proton attaches to the amide thus terminating the remaining ionised molecule. This leaves the C-0 group carrying a net positive charge; this balances the negative charge on the sulphonate group.
The tandem mass spectrometer will now be described. Referring to the drawing the spectrometer comprises a first, time of flight, mass spectrometer A and a second, harmonic ion mirror reflectron, mass spectrometer B. A comprises an ion source 1, means for accelerating ions 2, a field free drift space 3 and an ion selector 5, all disposed in a vacuum chamber.
The ion source 1 and means for accelerating ions 2 comprises three electrodes. The first electrode comprises a sample holder 6. The second electrode 7 is displaced from the first and includes an aperture through which ions may pass. The third electrode 8 is displaced from the second electrode and also includes an aperture through which ions may pass.
The ion source 1 further comprises means 9 for directing laser light onto the sample holder 6, to enable ionisation of a sample by matrix assisted laser desorption (MALDI).
In use, the field between the first 6 and second 7 electrodes is initially kept at zero. Laser light is then directed onto a sample in the
sample holder 6. This releases matrix molecules and analyte molecules into the vacuum chamber as a rapidly expanding vapour cloud. Molecules within the expanding cloud typically take a range of velocities from 200 to 2,000 meters per second. During formation of the cloud a small fraction of the molecules are ionised; this includes the matrix molecules as well as the analyte molecules. Ionisation of analyte molecules continues during the expansion of the vapour cloud by ion-molecule reactions at the expense of the matrix ions.
A short time afterwards electric field is suddenly applied between the first 6 and second 7 electrodes to accelerate the ions in the cloud through the aperture in the second electrode 7 and a constant accelerating field is maintained between the second 7 and third 8 electrodes to accelerate the ions through the aperture in the third electrode 8 and into the field free drift space 3.
The delay between the formation of a vapour cloud by laser light and application of a field serves to increase the mass resolving power of the spectrometer, through so-called"time-lag"focussing. The principle of this method is simple : the ions of the vapour cloud are allowed to fly apart at first for a brief time in a field free region. The faster ions thereby separate further from the sample support electrode 6 than the slower ions, and from the velocity distribution of the ions, a location distribution results. Then the accelerating field is applied. As the field is applied, the faster ions,
having drifted further from the sample electrode 6, find themselves at a lower potential than the slower ions. As the faster ions leave the ion source region they have a lower ultimate velocity than the slower ions. At some point in the field free drift space the ions which were slow but are now fast will overtake the ions which were originally fast. This plane is a plane of first order time focus for ions of a given charge to mass ratio. The position of the time focus plane can be adjusted by selection of the magnitude of the accelerating field and the delay time before it is applied. With the present apparatus the time focus plane is arranged to coincide with the entrance plane of B.
If dissociation occurs in the field free drift region of a time of flight mass spectrometer the dissociation products, fragment ions and neutral submolecules will continue to drift with the same velocity. If the kinetic energy of a precursor molecular ion of mass Mo is given as Eo then the
kinetic energy Em of the fragment of mass m is given by : Em = (m/Mo) Eo Molecular ions such as peptides containing a series of amino acid residues which undergo dissociation can lead to considerable array of dissociation products with masses ranging down to a few percent of the parent ion mass. The result is a collection of dissociation products, all drifting with the same velocity as the precursor ion, but with a very large range of kinetic energies. For efficient detection of the full range of dissociation products demands that B is capable of accepting this great range of kinetic energies without loss of temporal or spatial focus.
After travelling along the field from drift space 3 ions pass through the ion selector 5. This comprises a timed ion gate selector and may take the form of a pair of electrostatic deflection plates to which positive and negative voltages may be applied. When an ion of interest approaches the ion gate these voltages are suddenly and simultaneously brought to zero.
As the ion passes through the gate region the voltages are restored to their initial values. The gate may be made inactive by holding the voltages on the plates to zero.
The ion gate selector is used to deflect ions, so that they do not enter B until a time from the issue of an ion pulse equal to the expected drift time of ions of interest has elapsed. The potential on the selector plates is then reduced to zero to allow the ions to pass into B.
In B the ions are all reflected harmonically along an axis. The potential V (r, z) near the axis of the second mass spectrometer is given by the relationship : - V (r, z) = Vo/a-r/2) for z in the range 0 < z < a, where z is the displacement along the axis of the second mass spectrometer measured from its entry plane and r is the radial distance from this axis.
B has a very large energy bandwidth making possible collection of any precursor ions and virtually all of any fragment ions, thereby obviating the need for adjustment of the electromagnetic properties of the mass spectrometer or to perform repeat experiments.
Other types of mass spectrometer may be used, providing they offer sufficient energy bandwidth.
The above embodiment is described by way of example, many variations are possible without departing from the invention.

Claims (11)

  1. CLAIMS 1. A method of analysis of a molecule comprising the steps of : modifying the molecule by attaching a second molecule to form a complex, the second molecule being selected to promote unimolecular dissociation of the complex ; and analysing the complex using a tandem mass spectrometer comprising a first mass spectrometer tuned to select the complex when ionized and a second mass spectrometer having an energy bandwidth of sufficient size to enable it to produce a spectrum of at least 80% of the ionized complex and its dissociation products without adjustment of its electromagnetic properties.
  2. 2. A method as claimed in claim 1, wherein the tandem mass spectrometer is capable of producing a spectrum of substantially all of the ionized complex and its dissociation products without adjustment.
  3. 3. A method as claimed in either claim 1 or 2, wherein the energy bandwidth of the second mass spectrometer is equal to the full energy of the acceleration of the ions in an ion source associated with the mass spectrometer.
  4. 4. A method as claimed in any preceding claim, wherein the energy bandwidth of the mass spectrometer is at least 20 kev.
  5. 5. A method as claimed in any preceding claim, wherein the tandem
    mass spectrometer comprises a pair of time of flight mass spectrometers.
  6. 6. A method as claimed in any preceding claim, wherein the first mass spectrometer comprises a means for generating ions from a sample, means for accelerating those ions, a field free drift space and means for delaying the acceleration of ions after their generation, such that ions with the same mass to charge ratio are brought substantially to a time focus plane despite having differing initial velocities.
  7. 7. A method as claimed in claim 6, wherein the time focus plane coincides with an entry plane of the second mass spectrometer and an ion selector is provided for selecting ions according to their arrival time at the end of the field free drift space.
  8. 8. A method as claimed in any preceding claim, wherein the second mass spectrometer comprises a harmonic field mirror reflection.
  9. 9. A method as claimed in claim 8, wherein the potential V (r, z) near an axis of the second mass spectrometer is equal to Vo/a2 (z2-r2/2) for z in the range 0 < z < a, where z is the displacement along the axis measured from its entry plane and r is the radial distance from the axis.
  10. 10. A method of analysis of peptide molecules comprising a method as claimed in any preceding claim.
  11. 11. A method of analysis of peptide molecules substantially as herein
    described with reference to the accompanying drawings. descrlDeu w.."w
GB0114295A 2000-06-13 2001-06-13 Method of analysis Expired - Fee Related GB2367185B (en)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5538897A (en) * 1994-03-14 1996-07-23 University Of Washington Use of mass spectrometry fragmentation patterns of peptides to identify amino acid sequences in databases
US5952653A (en) * 1989-05-19 1999-09-14 Mds Health Group Limited Protein sequencing by mass spectrometry
WO2000020870A1 (en) * 1998-10-01 2000-04-13 Brax Group Limited Characterising polypeptides through cleavage and mass spectrometry
WO2000045168A1 (en) * 1999-01-28 2000-08-03 Universite De Geneve Method and kit for identifying or characterising polypeptides
EP1047107A2 (en) * 1999-04-06 2000-10-25 Micromass Limited Improved methods of identifying peptides and protein by mass spectrometry

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5952653A (en) * 1989-05-19 1999-09-14 Mds Health Group Limited Protein sequencing by mass spectrometry
US5538897A (en) * 1994-03-14 1996-07-23 University Of Washington Use of mass spectrometry fragmentation patterns of peptides to identify amino acid sequences in databases
WO2000020870A1 (en) * 1998-10-01 2000-04-13 Brax Group Limited Characterising polypeptides through cleavage and mass spectrometry
WO2000045168A1 (en) * 1999-01-28 2000-08-03 Universite De Geneve Method and kit for identifying or characterising polypeptides
EP1047107A2 (en) * 1999-04-06 2000-10-25 Micromass Limited Improved methods of identifying peptides and protein by mass spectrometry

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GB0114295D0 (en) 2001-08-01
GB0014342D0 (en) 2000-08-02

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Effective date: 20110613