Mass spectrometry with selective ion filtration by digital thresholding

• US 20050270191A1
• Filed: 05/24/2005
• Published: 12/08/2005
• Est. Priority Date: 05/24/2004
• Status: Active Grant

• Current Assignee: Ibis Biosciences Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Hofstadler, Steven A., Drader, Jared J., Schink, Amy L.
• Granted Patent: US 7,714,275 B2
• Time in Patent Office: Days
• US Class Current: 341/50
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: H01J 49/0009 Calibration of the apparatus
• CPC Class Codes: H01J 49/0027 Methods for using particle spectrometers
• CPC Class Codes: H01J 49/0036 Step by step routines describing the handling of the data generated during a measurement
• CPC Class Codes: H01J 49/02 Details
• CPC Class Codes: H01J 49/165 Electrospray ionisation
• CPC Class Codes: H01J 49/26 Mass spectrometers or separator tubes
• CPC Class Codes: H01J 49/40 Time-of-flight spectrometers H01J49/36 takes precedence

Abstract

The methods described herein generally relate to characterization of large analytes, such as biomolecules, by molecular mass analysis. Specifically, the methods are directed to molecular mass analysis of singly- or multiply-charged ions by selective ion filtering carried out by a digital thresholding process.

Claims

1. A method of identifying a multiply-charged ion comprising:

a) specifying a digital signal threshold on a mass spectrometer, wherein the mass spectrometer comprises;

i) an ion detector;

ii) a digitizer for converting an analog signal to a digital signal;

iii) an analog signal transfer means for transferring an analog signal from the detector to the digitizer; and

iv) a digital threshold filter, for setting a digital signal threshold, in digital data communication with the digitizer, in response to a digital signal input from the digitizer, independently outputting a digital signal to a data file only if the digital signal input is greater than the specified digital signal threshold;

wherein upon a mass spectrometer measurement of the multiply-charged ion, the filtered digital signal output to the data file originates from the detection of the multiply-charged ion and excludes digital signal output from analog signals arising from singly-charged ions.

2. The method of claim 1 further comprising making a plurality of mass spectrometer measurements according to step a) and co-adding the resulting plurality of data files.

3. The method of claim 1 wherein the multiply-charged ion is a biomolecule.

4. The method of claim 3 wherein the biomolecule is a nucleic acid, peptide, protein, lipid, or carbohydrate.

5. The method of claim 3 wherein the biomolecule comprises a non-covalently-bound small molecule.

6. The method of claim 1 wherein the singly-charged ions are biomolecule stabilizer additives or matrix modifiers.

7. The method of claim 6 wherein the stabilizer additives are one or more of:

polyethylene glycol, glycerol, reducing agents, detergents or buffer salts, or any combination thereof.

8. The method of claim 6 wherein the matrix-modifying additives are one or more of:

ampholytes, detergents, or buffer salts, or any combination thereof.

9. The method of claim 1 wherein the analog signal is an analog voltage signal and the digital signal is a digital voltage signal.

10. The method of claim 1 wherein the mass spectrometer is a time-of-flight mass spectrometer, a quadrupole time-of-flight mass spectrometer, a linear quadrupole mass spectrometer, a linear trap mass spectrometer, an electric/magnetic sector mass spectrometer, or a quadrupole ion trap mass spectrometer.

11. A method for determining the molecular mass of a plurality of analytes in a mixture comprising:

a) specifying a unique digital signal threshold for at least some members of a plurality of digital threshold filters on a mass spectrometer, wherein the mass spectrometer comprises;

i) an ion detector;

ii) a digitizer for converting an analog signal to a digital signal;

iii) an analog signal transfer means for transferring an analog signal from the detector to the digitizer; and

iv) a plurality of digital threshold filters for setting a digital signal threshold in digital data communication with the digitizer, in response to a digital signal input from the digitizer independently outputting a digital signal to a corresponding data file only if the digital signal input is greater than the specified digital signal threshold;

b) making a mass spectrometer measurement of the mixture, wherein each unique digital signal threshold differentially filters digital signals arising from the plurality of analytes and produces a unique digital signal output to each corresponding data file wherein the measurement results in storage of a plurality of data files; and

c) analyzing each member of the plurality of data files wherein the molecular mass of at least one member of the plurality of analytes is contained therein.

12. The method of claim 11 further comprising making a plurality of mass spectrometer measurements according to step b) and co-adding the resulting plurality of data files obtained from the plurality of mass spectrometer measurements.

13. The method of claim 11 wherein the analysis of step c) comprises mathematical subtraction of at least one member of the plurality of data files from at least one other member of the plurality of data files.

14. The method of claim 11 wherein the plurality of analytes comprise singly-charged ions and multiply-charged ions.

15. The method of claim 14 wherein the multiply-charged ions are biomolecules.

16. The method of claim 15 wherein the biomolecules comprise non-covalently-bound small molecules.

17. The method of claim 15 wherein the biomolecules are nucleic acids, peptides, proteins, lipids or carbohydrates.

18. The method of claim 14 wherein the singly-charged ions are biomolecule stabilizer additives or matrix modifiers.

19. The method of claim 18 wherein the stabilizer additives are one or more of:

polyethylene glycol, glycerol, reducing agents, detergents, or buffer salts, or any combination thereof.

20. The method of claim 18 wherein the matrix-modifying additives are one or more of:

ampholytes, detergents, or buffer salts, or any combination thereof.

21. The method of claim 11 wherein the analog signal is an analog voltage signal and the digital signal is a digital voltage signal.

22. The method of claim 11 wherein the mass spectrometer is a time-of-flight mass spectrometer, a quadrupole time-of-flight mass spectrometer, a linear quadrupole mass spectrometer, a linear trap mass spectrometer, an electric/magnetic sector mass spectrometer, or a quadrupole ion trap mass spectrometer.

23. A method of calibrating a mass spectrum of an analyte comprising:

a) specifying a first digital signal threshold at a first member of a plurality of digital threshold filters, on a mass spectrometer, wherein the mass spectrometer comprises;

i) an ion detector;

ii) a digitizer for converting an analog signal to a digital signal;

iii) an analog signal transfer means for transferring an analog signal from the detector to the digitizer; and

iv) a plurality of digital threshold filters for setting a digital signal threshold, in digital data communication with the digitizer, in response to a digital signal input from the digitizer, independently outputting a digital signal to a corresponding data file only if the digital signal input is greater than the specified digital signal threshold;

wherein the first digital signal threshold is specified such that digital signal output to a first data file comprises signals from the analyte and a calibrant ion;

b) specifying a second digital signal threshold at a second member of the plurality of digital threshold filters wherein the second digital signal threshold is specified such that digital signal output to a second data file comprises signals from the analyte but not from the calibrant ion;

c) subtracting the second data file from the first data file to obtain a calibration data file comprising a signal from the calibrant ion but not from the analyte; and

d) using said calibration data file to calibrate the mass spectrum of the analyte.

24. The method of claim 23 wherein the calibrant ion is a nucleic acid, peptide or small molecule.

25. The method of claim 23 wherein the analyte is a biomolecule.

26. The method of claim 25 wherein the biomolecule is a nucleic acid, peptide, protein, lipid or carbohydrate.

27. The method of claim 26 wherein the biomolecule comprises a non-covalently-bound small molecule.

28. The method of claim 23 wherein the analog signal is an analog voltage signal and the digital signal is a digital voltage signal.

29. The method of claim 23 wherein the mass spectrometer is a time-of-flight mass spectrometer, a quadrupole time-of-flight mass spectrometer, a linear quadrupole mass spectrometer, a linear trap mass spectrometer, an electric/magnetic sector mass spectrometer, or a quadrupole ion trap mass spectrometer.

30. A system comprising a mass spectrometer comprising:

an ion detector;

a digitizer for converting an analog signal to a digital signal;

an analog signal transfer means for transferring an analog signal from the detector to the digitizer;

a plurality of digital threshold filters for setting a plurality of independent digital signal thresholds, each digital threshold filter in parallel electronic communication with the digitizer, each digital threshold filter in response to a digital voltage input from the digitizer independently outputting a digital voltage only if the digital voltage input is greater than the specified digital signal threshold;

a plurality of data files; and

a plurality of parallel digital signal output transferring means, each of which is in digital data communication with one of the digital threshold filters and a corresponding data file from the plurality of data files.

31. The system of claim 30 wherein the analog signal is an analog voltage signal and the digital signal is a digital voltage signal.

32. The system of claim 30 wherein the mass spectrometer is a time-of-flight mass spectrometer, a quadrupole time-of-flight mass spectrometer, a linear quadrupole mass spectrometer, a linear trap mass spectrometer, an electric/magnetic sector mass spectrometer, or a quadrupole ion trap mass spectrometer.