Methods and devices for identifying biopolymers using mass spectroscopy

• US 20040180446A1
• Filed: 03/13/2003
• Published: 09/16/2004
• Est. Priority Date: 03/13/2003
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Fischer, Steven M., Thompson, Dean R.
• Granted Patent: US 8,507,285 B2
• Time in Patent Office: Days
• US Class Current: 436/86
• CPC Class Codes: G01N 30/72 Mass spectrometers mass spectrometers per se H01J49/00
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: H01J 49/0045 characterised by the fragmentation or other specific reaction
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/24 Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Abstract

A method of identifying a biopolymer in a sample that includes one or more biopolymers. The biopolymers may be polypeptides, polynucleotides, or polysaccharides. The method makes use of mass spectral datasets. A first dataset includes measured masses of the one or more biopolymers that are in the sample. A second dataset includes measured masses of a collection of fragments of the one or more biopolymers. The method selects a mass from the first dataset and then matches masses from the second dataset with the selected mass. The matched masses represent fragments of the biopolymer with the selected mass. Once the masses in the second dataset have been matched they are compared to determine a monomer sequence for the biopolymer with the selected mass. The method may be repeated with additional masses in the first dataset.

Claims

1. A method of identifying a biopolymer in a sample that includes one or more biopolymers, the method comprising:

selecting a mass from a first dataset, wherein said first dataset includes measured masses of the one or more biopolymers;

matching masses in a second dataset with the selected mass, wherein said second dataset includes measured masses of a collection of fragments of the one or more biopolymers, and wherein the matched masses represent fragments of the biopolymer with the selected mass; and

comparing the matched masses to determine a monomer sequence for the biopolymer with the selected mass.

2. The method of claim 1, further comprising:

obtaining a first mass spectrum of the one or more biopolymers under conditions that cause the one or more biopolymers to be detected in unfragmented form, wherein said first mass spectrum is used in producing said first dataset; and

obtaining a second mass spectrum of the one or more biopolymers under conditions that cause the one or more biopolymers to be fragmented into a collection of fragments prior to detection, wherein said second mass spectrum is used in producing said second dataset.

3. The method of claim 2, wherein said first and second mass spectra are obtained consecutively using the same spectrometer and the spectrometer is a single-stage spectrometer.

4. The method of claim 2, wherein the second mass spectrum results from a summation of two different mass spectra obtained with different fragmentation energies.

5. The method of claim 2, wherein the second mass spectrum results from a summation of two different mass spectra obtained with different fragmentation mechanisms.

6. The method of claim 2, further comprising:

obtaining a third mass spectrum of the one or more biopolymers under conditions that cause the one or more biopolymers to be fragmented into a collection of fragments prior to detection, wherein said third mass spectrum is used in producing said second dataset and said second and third mass spectra are obtained using different fragmentation energies.

7. The method of claim 2, further comprising:

obtaining a third mass spectrum of the one or more biopolymers under conditions that cause the one or more biopolymers to be fragmented into a collection of fragments prior to detection, wherein said third mass spectrum is used in producing said second dataset and said second and third mass spectra are obtained using different fragmentation mechanisms.

8. The method of claim 1, wherein the masses in the first and second datasets are neutral masses.

9. The method of claim 1, wherein the masses in the first and second datasets are monoisotopic masses.

10. The method of claim 1, further comprising:

removing masses from the first dataset that are also present in the second dataset before said selecting a mass from the first dataset.

11. The method of claim 1, further comprising:

removing masses from the second dataset that are also present in the first dataset before said selecting a mass from the first dataset.

12. The method of claim 1, wherein said matching masses in the second dataset with the selected mass comprises:

subtracting a first mass from the selected mass to produce a mass delta; and

identifying a second mass in the second dataset that matches the mass delta.

13. The method of claim 12, further comprising:

performing a successful formula call on the first or second mass.

14. The method of claim 13, wherein in the step of performing a successful formula call on the first or second mass, the first or second mass is below 500 Da.

15. The method of claim 12, wherein said matching masses in the second dataset with the selected mass further comprises one or a combination of:

identifying a mass in the second dataset that differs from the first or second mass by the theoretical mass of a one or more monomer units;

identifying a mass in the second dataset that differs from the first or second mass by the theoretical mass of one or more monomer units and a biopolymer modification; and

identifying a mass in the second dataset that differs from the first or second mass by a theoretical series offset mass.

16. The method of claim 1, wherein said matching masses in the second dataset with the selected mass comprises:

subtracting a first mass in the second dataset from the selected mass to generate a mass delta; and

performing a successful formula call on the mass delta.

17. The method of claim 16, further comprising:

identifying a second mass in the second dataset that matches the mass delta.

18. The method of claim 17, wherein the mass delta is below 500 Da.

19. The method of claim 17, wherein said matching masses in the second dataset with the selected mass further comprises one or a combination of:

identifying a mass in the second dataset that differs from the first or second mass by the theoretical mass of one or more monomer units;

identifying a mass in the second dataset that differs from the first or second mass by the theoretical mass one or more monomer units and a biopolymer modification; and

identifying a mass in the second dataset that differs from the first or second mass by a theoretical series offset mass.

20. The method of claim 19, wherein said identifying a mass in the second dataset that differs from the first or second mass by the theoretical mass of one or more monomer units comprises:

generating a resultant mass by adding or subtracting the sum of the theoretical masses of one or more monomer units to or from the first or second mass; and

identifying a third mass in the second dataset that matches the resultant mass.

21. The method of claim 19, wherein said identifying a mass in the second dataset that differs from the first or second mass by the theoretical mass of one or more monomer units comprises:

generating a mass delta by subtracting a mass in the second dataset from the first or second mass; and

performing a successful formula call on the mass delta.

22. The method of claim 21, wherein the mass delta is below 500 Da.

23. The method of claim 19, wherein said identifying a mass in the second dataset that differs from the first or second mass by the theoretical mass of one or more monomer units and a biopolymer modification comprises:

generating a first mass delta by subtracting a mass in the second dataset from the first or second mass;

subtracting the theoretical mass of a biopolymer modification from the first mass delta to produce a second mass delta; and

performing a successful formula call on the second mass delta.

24. The method of claim 19, wherein said identifying a mass in the second dataset that differs from the first or second mass by a theoretical series offset mass comprises:

generating a resultant mass by adding or subtracting a theoretical series offset mass to or from the first or second mass; and

identifying a third mass in the second dataset that matches the resultant mass.

25. The method of claim 1, wherein said comparing the matched masses to determine a monomer sequence for the biopolymer with the selected mass comprises:

grouping the matched masses according to the series type of their corresponding fragments;

calculating mass deltas between pairs of matched masses that correspond to fragments at adjacent positions in a represented series;

performing a formula call on the mass deltas;

assigning monomers associated with the mass deltas based on the formula call; and

determining a monomer sequence for the biopolymer with the selected mass based on said assigning.

26. The method of claim 25, further comprising:

calculating mass deltas between pairs of matched masses that correspond to fragments at non-adjacent positions in a represented series;

performing a formula call on the mass deltas; and

assigning combinations of monomers associated with the mass deltas based on the formula call.

27. The method of claim 25, further comprising:

calculating mass deltas between pairs of matched masses that correspond to fragments at adjacent positions on different represented series;

performing a formula call on the mass deltas; and

assigning monomers associated with the mass deltas based on the formula call.

28. The method of claim 25, further comprising:

calculating mass deltas between pairs of matched masses that correspond to fragments at non-adjacent positions on different represented series;

performing a formula call on the mass deltas; and

assigning combinations of monomers associated with the mass deltas based on the formula call.

29. The method of claim 25, wherein the monomer sequence includes a single possible monomer at each position in the sequence.

30. The method of claim 25, wherein the monomer sequence includes more than one possible monomer at a position in the sequence.

31. The method of claim 25, wherein the monomer sequence includes an unknown monomer at a position in the sequence.

32. The method of claim 25, wherein the monomer sequence includes a biopolymer modification at a position in the sequence.

33. The method of claim 1, wherein the biopolymer with the selected mass is a polypeptide and the monomer sequence is an amino acid sequence.

34. The method of claim 1, wherein the biopolymer with the selected mass is a polynucleotide and the monomer sequence is a nucleotide sequence.

35. The method of claim 1, wherein the biopolymer with the selected mass is a polysaccharide and the monomer sequence is a sugar sequence.

36. The method of claim 1, wherein the polysaccharide is linear.

37. The method of claim 1, wherein the polysaccharide is branched.

38. The method of claim 1, wherein:

the sample includes a plurality of biopolymers;

the first dataset includes measured masses of the plurality of biopolymers; and

the second dataset includes measured masses of a collection of fragments of the plurality of biopolymers.

39. A computer program product for identifying a biopolymer in a sample that includes one or more biopolymers, comprising a computer readable medium configured with a set of machine executable instructions, the instructions comprising:

instructions for selecting a mass from a first dataset, wherein said first dataset includes measured masses of the one or more biopolymers;

instructions for matching masses in a second dataset with the selected mass, wherein said second dataset includes measured masses of a collection of fragments of the one or more biopolymers, and wherein the matched masses represent fragments of the biopolymer with the selected mass; and

instructions for comparing the matched masses to determine a monomer sequence for the biopolymer with the selected mass.