Methods and apparatus for sequencing polymers with a statistical certainty using mass spectrometry

• US 5,869,240 A
• Filed: 05/19/1995
• Issued: 02/09/1999
• Est. Priority Date: 05/19/1995
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Perseptive Biosystems Incorporated (Ecovyst, Inc.)
• Inventors: Patterson, Dale H.
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): REES, DIANE
• Application Number:
  US08/447,175
• Time in Patent Office: 1,362 Days
• Field of Search: 435/6, 435/91.2, 435/18, 435/91.1, 435/9, 422/62, 422/50, 364/496, 364/497, 364/498, 364/499, 530/300, 536/23.1, 536/24.3
• US Class Current: 435/6.14
• CPC Class Codes: C07K 1/128 sequencing
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: H01J 49/0036 Step by step routines describing the handling of the data generated during a measurement

Abstract

Integrated methods and apparatus for sequencing or identifying polymers by mass spectrometry with a statistical certainty. The methods involve integrating data obtained by mass spectrometry analysis of a series of polymer fragments and statistically comparing said data with hypothetical data corresponding to known sequences or identities.

Claims

1. A method of obtaining sequence information about a polymer comprising a plurality of monomers of known mass, said method comprising the steps of:

a) providing a processor in communication with a mass spectrometer;

b) providing a set of polymer fragments, each polymer fragment differing from each other polymer fragment by one or more of the monomers of known mass;

c) measuring a difference x between the mass-to-charge ratio of one pair of polymer fragments with the mass spectrometer;

d) repeating step c) a number of times, n, where n is at least two, using the processor to determine a measured mean mass-to-charge ratio difference. x, between the pair of polymer fragments;

e) asserting a mean difference, μ

, between the mass-to-charge ratio of the pair of polymer fragments measured in step c) using the processor, wherein μ

corresponds to a known mass-to-charge ratio of one monomer of the plurality of monomers of known mass;

f) selecting a desired confidence level for μ

;

g) analyzing x with the processor to determine if x is statistically different from μ

at the selected confidence level;

h) determining with the processor if the asserted mean, μ

, is not statistically different from the measured mean mass-to-charge ratio difference, x, at the selected confidence level based upon the analysis in step g) and if μ

is not statistically different, uniquely assigning one of the pair of polymer fragments to a monomer of the plurality of monomers of known mass corresponding to the mean difference μ

;

i) if the asserted mean μ

, is statistically different from the measured mean mass-to-charge ratio difference, x at the selected confidence level based upon the analysis in step g) then repeating steps e) through h) for a plurality of desired values of μ

, each value of μ

corresponding to one monomer of the plurality of monomers of known mass; and

j) if after performing step i) a unique assignment of one of the pair of polymer fragments has not been made to one monomer of the plurality of monomers of known mass, then automatically repeating steps d) through i) using the processor and the mass spectrometer for a larger number of times n.

2. The method of claim 1 wherein the analysis in step g) comprises a two-tailed t-test for an experimental mean.

3. The method of claim 1 comprising automatically repeating steps b) through j) for additional sets of polymer fragments using processor and the mass spectrometer.

4. The method of claim 1 wherein the polymer is a biopolymer.

5. The method of claim 4 wherein the biopolymer is selected from the group consisting of DNAs, RNAs, PNAs, proteins, peptides, carbohydrates and modified forms thereof.

6. The method of claim 1 further comprising the step of hydrolyzing the polymer to obtain the polymer fragments in step b).

7. The method of claim 6 wherein the polymer is hydrolyzed on a reaction surface, said surface providing spatially separate differing amounts of a hydrolyzing agent which hydrolyzes said polymer thereby to break inter-monomer bonds.

8. The method of claim 7 wherein the hydrolyzing agent is an exohydrolase.

9. The method of claim 8 wherein hydrolyzing with said exohydrolase produces a series of fragments comprising a sequence-defining ladder of said polymer.

10. The method of claim 9 wherein the exohydrolase is selected from the group consisting of:

exonucleases, exoglycosidases, and exopeptidases.

11. The method of claim 10 wherein the exopeptidase is selected from the group consisting of carboxypeptidase Y, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, aminopeptidase 1, leucine aminopeptidase, proline aminodipeptidase and cathepsin C.

12. The method of claim 10 wherein the exoglycosidase is selected from the group consisting of

a) α

-Mannosidase I

b) α

-Mannosidase

c) β

-Hexosaminodase

d) β

-Galactosidase

e) α

-Fucosidase I and II

f) α

-Galactosidase

g) α

-Neuraminidase and

h) α

-Glucosidase I and II.

13. The method of claim 10 wherein the exonuclease is selected from the group consisting of

a) λ

-exonuclease

b) t7 Gene 1 exonuclease

c) exonuclease III

d) Exonuclease I

e) Exonuclease V

f) Exnonuclease II and

g) DNA Polymerase II.

14. The method of claim 7 wherein the agent is a hydrolyzing agent other than an enzyme.

15. The method of claim 7 wherein the reaction surface comprises a gradient of said hydrolyzing agent.

16. The method of claim 1 further comprising adding a matrix to the polymer fragments before measuring the mass-to-charge ratio in step c).

17. The method of claim 16 wherein the data is analyzed by matrix assisted laser desorption mass spectrometry.

46. The method of claim 1 wherein multiple different sets of polymer fragments obtained from the same polymer are provided in step b).

47. The method of claim 1 further comprising the step of eluting from a liquid chromatography column a sample comprising the polymer for which sequence information is to be obtained.

18. A method for obtaining sequence information about a polymer comprising a series of different monomers of known mass, said method comprising the steps of:

a) hydrolyzing a pole with a hydrolyzing agent wherein the hydrolysis is performed on a reaction surface, said surface providing spatially separate differing amounts of a hydrolyzing agent, said hydrolysis creating a set of polymer fragments, each polymer fragment differing from each other polymer fragment by one or more of the monomers of known mass;

b) measuring a difference x between the mass-to-charge ratio of one pair of polymer fragments;

c) repeating step b) a number of times, n, where n is at least two, to determine a measured mean mass-to-charge ratio difference, x, between the pair of polymer fragments;

d) asserting a mean difference, μ

, between the mass-to-charge ratio of the pair of polymer fragments measured in step b, wherein μ

corresponds to a known mass-to-charge ratio of one of the monomers of known mass;

e) selecting a desired confidence level for μ

;

f) determining the standard deviation s of the measured mean mass-to-charge ratio difference x determined in step c;

g) calculating a test statistic t_calculated with the following algorithm;

##EQU5## h) comparing the test statistic t_calculated calculated in step g to a t-distribution corresponding to the number of measurements, n, and the desired confidence level; and

i) determining if the asserted mean μ

is not substantially different from the measured mean mass-to-charge ratio difference, x, at the selected confidence level based upon the comparison in step h.

19. The method of claim 18 further comprising repeating steps b)-i) for additional pairs of fragments thereby to obtain sequence information.

20. The method of claim 18 further comprising the step of determining the number of measurements, n, based upon the comparison in step h.

21. The method of claim 18 wherein the polymer is a biopolymer.

22. The method of claim 21 wherein the biopolymer is selected from the group consisting of DNAs, RNAs, PNAs, proteins, peptides, carbohydrates and modified forms thereof.

23. The method of claim 18 wherein the hydrolyzing agent is an exohydrolase which produces a series of fragments comprising a sequence-defining ladder of said polymer.

24. The method of claim 23 wherein the exohydrolase is selected from the group consisting of:

exonucleases, exoglycosidases, exopeptidases.

25. The method of claim 24 wherein the exopeptidase is selected from the group consisting of carboxypeptidase Y, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, aminopeptidase 1, leucine aminopeptidase, proline, aminodipeptidase and cathepsin C.

26. The method of claim 24 wherein the exoglycosidase is selected from the group consisting of

a) α

-Mannosidese I

b) α

-Mannosidese

c) β

-Hexosaminidese

d) β

-Galactosidase

e) α

-Fucosidase I and II

f) α

-Galactosidase

g) α

-Neuraminidase and

h) α

-Glucosidase I and II.

27. The method of claim 24 wherein the exonuclease is selected from the group consisting of

a) Exonuclease

b) λ

-exonuclease

c) t7 Gene 1 exonuclease

d) exonuclease III

e) Exonuclease I

f) Exonuclease V

g) Exnonuclease II and

h) DNA Polymerase II.

28. The method of claim 18 wherein the hydrolyzing agent is other than an enzyme.

29. The method of claim 28 wherein the agent comprises a combination of at least one enzyme and at least one agent other than an enzyme.

30. The method of claim 18 wherein the reaction surface comprises a continuous concentration gradient of a hydrolyzing agent.

31. The method of claim 18 further comprising adding a matrix to the polymer fragments before measuring the mass-to-charge ratio in step b).

32. A method for obtaining sequence information about a polymer having a plurality of monomers of known mass, said method comprising:

a) hydrolyzing a polymer with a hydrolyzing agent wherein the bydrolysis is performed on a reaction surface, said surface providing spatially separate differing amounts of a hydrolyzing agent, said hydrolysis creating a set of polymer fragments, each polymer fragment differing from each other polymer fragment by one or more of the monomers of known mass;

b) determining a measured mean mass-to-charge ratio difference, x between one pair of polymer fragments;

c) asserting a mean difference, μ

, between the mass-to-charge ratio of the pair of polymer fragments measured in step b, wherein μ

corresponds to a known mass-to-charge ratio of one of the monomers of known mass;

d) selecting the desired confidence level for μ

;

e) analyzing x to determine if it is statistically different from μ

at the selected confidence level;

f) repeating steps b)-e) a number of times, n, until a plurality of desired values of μ

have been asserted;

g) determining if the asserted mean μ

is not substantially different from the measured mean mass-to-charge ratio difference, x, at the selected confidence level based upon the analysis in step e; and

h) repeating steps b)-g) for additional pairs of fragments.

33. The method of claim 32 wherein the polymer is a biopolymer.

34. The method of claim 33 wherein the biopolymer is selected from the group consisting of DNAs, RNAS, PNAs, proteins, peptides, carbohydrates and modified forms thereof.

35. The method of claim 32 wherein the polymer fragments in step a) are created by concentration dependent hydrolysis of the polymer.

36. The method of claim 32 wherein the hydrolyzing agent is an exohydrolase.

37. The method of claim 36 wherein the hydrolysis caused by said exohydrolase produces a series of fragments defining a ladder of said polymer.

38. The method of claim 36 wherein the exohydrolase is selected from the group consisting of. exonucleases, exoglycosidases, and exopeptidases.

39. The method of claim 38 wherein the exoglycosidase is selected from the group consisting of

a) α

-Mannosidese I

b) α

-Mannosidese

c) β

-Hexosaminidese

d) β

-Galactosidase

e) α

-Fucosidase I and II

f) α

-Galactosidase

g) α

-Neuraminidase and

h) α

-Glucosidase I and II.

40. The method of claim 38 wherein the exonuclease is selected from the group consisting of

a) Exonuclease

b) λ

-exonuclease

c) t7 Gene 1 exonuclease

d) exonuclease III

e) Exonuclease I

f) Exonuclease V

g) Exnonuclease II and

h) DNA Polymerase II.

41. The method of claim 38 wherein the exopeptidase is selected from the group consisting of carboxypeptidase Y, carboxypeptidase A, carboxypeptidase B, carboxypeptidase P, aminopeptidase 1, leucine aminopeptidase, proline, aminodipeptidase and cathepsin C.

42. The method of claim 32 wherein said agent comprises a hydrolyzing agent other than an enzyme.

43. The method of claim 32 wherein the polymer fragments are obtained by hydrolysis with a combination of at least one enzyme and at least one hydrolyzing agent other than an enzyme.

44. The method of claim 32 wherein the reaction surface comprises a concentration gradient of said hydrolyzing agent.

45. The method of claim 32 further comprising adding a matrix to the polymer fragments before measuring the mass-to-charge ratio in step b).