Methods and devices for proteomics data complexity reduction

• US 20030139885A1
• Filed: 11/05/2002
• Published: 07/24/2003
• Est. Priority Date: 11/05/2001
• Status: Abandoned Application

• Current Assignee: IRM LLC
• Original Assignee: IRM LLC
• Inventors: Horn, David M., Peters, Eric C., Brock, Ansgar
• Application Number:
  US10/289,462
• Publication Number:
  US 20030139885A1
• Time in Patent Office: Days
• US Class Current: 702/19
• CPC Class Codes: B82Y 30/00 Nanotechnology for materials or surface science, e.g. nanocomposites
• CPC Class Codes: G01N 2035/00158 Elements containing microarrays, i.e. "biochip"
• CPC Class Codes: G01N 2458/15 Non-radioactive isotope labels, e.g. for detection by mass spectrometry
• CPC Class Codes: G01N 33/6818 Sequencing of polypeptides
• CPC Class Codes: G01N 33/6842 Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6851 Methods of protein analysis involving laser desorption ionisation mass spectrometry
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids
• CPC Class Codes: G16B 50/00 ICT programming tools or database systems specially adapted for bioinformatics
• CPC Class Codes: H01J 49/0036 Step by step routines describing the handling of the data generated during a measurement

Abstract

Provided are methods and systems for identification of proteins using high mass accuracy mass spectrometry. Not only do high mass accuracy measurements provide greater confidence in protein identification assignments, but they also enable proteins to be identified with either less sequence coverage or fewer additional tandem MS experiments. In addition, high mass measurement accuracy optionally allows protein identifications to be made on the basis of the mass of a single peptide, providing higher-throughputs in the analysis of mixtures due to the significant decrease in time spent on additional tandem MS experiments. In addition, a concomitant time saving in the cross correlation process of mass spectral data with in silico digested databases would also be achieved.

Claims

1. A method of reducing a number of peaks to further be analyzed in a mass spectrum for a sample, the method comprising:

generating a first amino acid sequence database comprising an amino acid sequence of at least one protein known to be present in the sample;

calculating a first list of theoretical masses for a first set of in silico peptides generated from one or more of the amino acid sequences in the first database; and

correlating the first list of theoretical masses with positions of the unidentified MS peaks and identifying one or more MS peaks that correspond to masses for the in silico peptides, thereby reducing the number of peaks to further be analyzed in the mass spectrum.

2. The method of claim 1, wherein all members of the first database are proteins known to be present in the sample.

3. The method of claim 1, wherein the sample comprises a plurality of proteolytic peptides generated by action of a proteolytic agent upon member proteins in the sample, and wherein calculating the first list of theoretical masses comprises generating the in silico proteolytic peptides using cleavage parameters of the proteolytic agent.

4. The method of claim 1, wherein the unidentified MS peaks are obtained using a mass spectrometer that provides a mass accuracy of 5 ppm or better.

5. The method of claim 1, wherein the unidentified MS peaks are obtained using a mass spectrometer that provides a mass accuracy of 1 ppm or better.

6. The method of claim 1, wherein generating the first database comprises providing amino acid sequences derived from protein sequencing data, nucleic acid sequencing data, tandem MS data or 2DE-MS data.

7. The method of claim 1, wherein generating the first database comprises i) selecting an unidentified MS peak and performing tandem mass spectrometry, thereby identifying a corresponding peptide sequence;

and ii) determining a parent protein sequence comprising the identified corresponding peptide sequence; and

wherein calculating the first list of theoretical masses comprises calculating masses for additional in silico peptides from the determined protein sequence.

8. The method of claim 7, wherein correlating the first list of theoretical masses with positions of the unidentified MS peaks further comprises identifying additional MS peaks that correspond to the theoretical masses of the additional it silico peptides and removing the additional MS peaks from a data set of unidentified MS peaks.

9. The method of claim 1, wherein generating the first database comprises:

providing a mass peak list comprising the positions of the unidentified MS peaks of the sample, wherein the MS peaks represent a plurality of proteolytic peptides generated by action of a proteolytic agent upon member proteins in the sample.

providing a second list of theoretical masses for a plurality of in silico proteolytic peptides generated from a second database of protein sequences by the in silico action of the proteolytic agent upon member sequences in the second database; and

comparing the second list with the mass peak list, thereby assigning corresponding MS peaks and identifying additional member proteins of the sample for inclusion in the first database.

10. The method of claim 9, further comprising:

regenerating the first database to include sequences for the identified additional member proteins; and

repeating the calculating, correlating and regenerating steps until no additional member proteins are identified.

11. The method of claim 9, wherein the second list comprises a first set of unique masses representing unique peptide sequences and a second set of masses representing more than one peptide sequence, and wherein comparing the second list with the mass peak list comprises comparing the first set of unique masses with the mass peak list.

12. The method of claim 11, wherein comparing the first set of unique masses with the mass peak list further comprises performing tandem MS on selected members of the plurality of proteolytic peptides, thereby confirming the identity of the additional member proteins of the sample.

13. The method of claim 9, wherein the proteolytic agent comprises a proteolytic enzyme.

14. The method of claim 13, wherein the proteolytic enzyme is selected from the group consisting of trypsin, chymotrypsin, endoprotease ArgC, aspN, gluC, and lysC.

15. The method of claim 9, wherein the proteolytic reagent comprises cyanogen bromide, formic acid, or thiotrifluoroacetic acid.

16. The method of claim 9, wherein the plurality of in silico proteolytic peptides comprise peptides having up to three missed enzymatic cleavage sites and ranging in molecular mass from 500 Da to 10,000 Da.

17. The method of claim 9, wherein the second database of protein sequences are derived from amino acid sequences encoded by one or more members of an EST library, a cDNA library, or a genomic library.

18. The method of claim 9, wherein providing the mass peak list further comprises contacting the sample with a first derivatizing agent, wherein the first derivatization agent comprises at least two isotopic forms, and specifically labels a selected amino acid or a functional moiety when the selected amino acid or functional moiety is present in a protein in the sample, thereby labeling the selected amino acid in one or more member proteins.

19. The method of claim 18, wherein contacting the sample with the first derivatizing agent is performed prior to generating the plurality of proteolytic peptides by action of the proteolytic agent.

20. The method of claim 18, wherein contacting the sample with the first derivatizing agent is performed after generating the plurality of proteolytic peptides by action of the proteolytic agent.

21. The method of claim 18, wherein the derivatizing agent comprises 2-methoxy-4,5-dihydro-1H-imidazole and the selected amino acid comprises lysine.

22. The method of claim 18, wherein providing the second list of theoretical masses comprises:

determining a number of occurrences of the selected amino acid or functional moiety in the in silico proteolytic peptides, thereby determining a number of derivatizing agents that would be attached to the in silico proteolytic peptides; and

calculating a theoretical molecular masses for the in silico proteolytic peptides having the determined number of attached derivatizing agents.

23. The method of claim 18, wherein each member of the second database of protein sequences comprises at least one selected amino acid.

24. The method of claim 9, wherein providing the mass peak list further comprises:

fractionating the sample to generate fractions comprising a plurality of peptides; and

ionizing member polypeptides in one or more of the fractions and obtaining masses using a mass spectrometer that provides a mass accuracy of 5 ppm or better.

25. The method of claim 24, wherein fractionating the sample comprises performing liquid chromatography, reverse phase chromatography, size exclusion chromatography, strong cation or anion exchange chromatography, weak cation or anion exchange chromatography, immobilized metal ion affinity chromatography (IMAC), capillary electrophoresis, gel electrophoresis, isoelectric focusing, or a combination thereof.

26. The method of claim 24, wherein ionizing the polypeptide comprises performing ESI.

27. The method of claim 24, wherein ionizing the polypeptide comprises performing LDI.

28. The method of claim 27, wherein the LDI comprises MALDI, IR-MALDI, UV-MALDI, liquid-MALDI, surface-enhanced LDI (SELDI), surface enhanced neat desorption (SEND), desorption/ionization of silicon (DIOS), laser desorption/laser ionization MS, or laser desorption/two step laser ionization MS.

29. The method of claim 24, wherein fractionating the sample further comprises depositing a plurality of fractions of an eluent onto a solid support suitable for laser desorption/ionization (LDI).

30. The method of claim 29, wherein the solid support comprises a surface modified for sample confinement.

31. The method of claim 24, wherein the mass spectrometer comprises a Fourier-transform ion cyclotron resonance mass spectrometer.

32. The method of claim 24, further comprising treating the sample to remove peptide modifications prior to the ionizing step.

33. The method of claim 24, wherein performing mass spectrometry further comprises providing one or more standards for comparison to the mass of the peak of interest, ionizing the one or more standards separately from the sample, thereby providing ionized standards, and mixing the ionized standards with an ionized sample in a gas phase.

34. The method of claim 1, wherein the sample comprises a proteome.

35. The method of claim 1, further comprising confirming an identification of a peak by tandem MS.

36. The method of claim 1, wherein calculating the first list of theoretical masses further comprises:

selecting a type of peptide modification; and

generating theoretical masses for the first set of in silico proteolytic peptides generated from the first database, wherein member proteins are assumed to contain one or more occurrences of the peptide modification, thereby identifying one or more peaks corresponding to modified member protein in the sample.

37. The method of claim 36, wherein the peptide modification comprises a post-translational modification as performed by a cell.

38. The method of claim 36, wherein the peptide modification comprises a chemical modification or an added chemical substituent.

39. The method of claim 36, wherein the peptide modification comprises a non-standard amino acid.

40. The method of claim 36, wherein the peptide modification comprises an amino acid substitution.

41. The method of claim 36, wherein the peptide modification comprises addition of one or more phosphate groups.

42. The method of claim 36, wherein the peptide modification comprises one or more myristoylate groups.

43. The method of claim 36, further comprising confirming an identification of a post-translationally modified protein by tandem MS of the member protein.

44. The method of claim 1, further comprising identifying member proteins corresponding to any remaining unidentified entries in the mass peak list by tandem MS.

45. A method of reducing a number of peaks to further be analyzed in a mass spectrum for a sample, the method comprising:

generating a first amino acid sequence database comprising an amino acid sequence of at least one protein present in the sample;

calculating a first list of theoretical masses for a first set of known in silico proteolytic peptides generated from the first database;

correlating a first theoretical mass with a position of an unidentified MS peak in a mass spectrum for the sample, thereby determining the presence in the sample of a first protein that comprises a peptide having a mass equal to the first theoretical mass; and

identifying one or more MS peaks that correspond to masses for the known in silico proteolytic peptides, thereby reducing the number of peaks to further be analyzed in the mass spectrum.

46. A method of identifying members of a plurality of proteins in a sample, the method comprising:

contacting a sample comprising a plurality of proteins with at least a first proteolytic agent that cleaves member proteins at defined cleavage sites to form proteolytic peptides;

contacting the sample with a first derivatizing agent comprising at least two isotopic forms, wherein the first derivatizing agent specifically labels a selected amino acid or functional moiety when the selected amino acid or functional moiety is present in a protein in the sample, thereby isotopically labeling one or more members of the plurality of proteins or proteolytic peptides;

fractionating the sample and depositing a plurality of fractions of an eluent onto a solid support suitable for LDI;

performing LDI-FT ICR mass spectrometry on the isotopically-labeled peptides in one or more of the fractions and determining masses of at least one pair of peaks of interest using a mass spectrometer that provides a mass accuracy of 5 ppm or better;

calculating a list of theoretical molecular masses for a plurality of in silico derivatized proteolytic peptides, wherein the member proteolytic peptides i) are derived from the amino acid sequences in a protein sequence database by predicted action of the proteolytic reagent upon members of the database;

ii) encompass peptides having up to three missed proteolytic cleavage sites;

iii) range in size between 1000 Da and 6000 Da; and

iv) comprise one or more derivatized amino acids; and

correlating the list of theoretical molecular masses to the mass peak list of experimental mass peaks, wherein a match between an experimental mass peak of a sample proteolytic peptide and a theoretical molecular mass for an in silico proteolytic peptide is indicative of the presence in the sample of the protein from which the in silico proteolytic peptide is derived, thereby assigning MS peaks in the mass peak list and identifying the members of the plurality of proteins.

47. The method of claim 46, further comprising:

removing the assigned peaks from the mass peak list;

incorporating the identified members of the plurality of proteins into a database of identified proteins; and

repeating the calculation and correlating steps using in silico derivatized proteolytic peptides generated from the database of identified proteins, thereby assigning additional MS peaks in the mass peak list and identifying additional members of the plurality of proteins.

48. The method of claim 46, further comprising:

providing one or more additional databases of proteolytic peptide sequences, wherein the member proteolytic peptides i) are derived in silico by predicted action of one or more additional proteolytic reagents upon members sequences in the second database of protein sequences;

ii) encompass peptide sequences having up to three missed enzymatic cleavage sites;

iii) range in size between 1000 Da and 4000 Da; and

iii) comprise one or more derivatized amino acids; and

repeating the generating and correlating step using the one or more additional databases, thereby identifying additional members of the plurality of proteins.

49. A method for identifying two or more members of a plurality of proteins in a sample, the method comprising:

a) providing a sample comprising a plurality of proteolytic polypeptides;

b) ionizing member polypeptides by LDI and obtaining a mass of at least a first polypeptide using a mass spectrometer that provides a mass accuracy of 5 ppm or better;

c) comparing the mass of the first polypeptide to members of a database of theoretical molecular masses for a plurality of in silico proteolytic peptides, wherein each member in silico peptide has a unique theoretical mass, and wherein a match between the mass obtained for the first polypeptide and the unique theoretical mass for an in silico proteolytic peptide indicates that a parent protein comprising the in silico polypeptide is present in the sample, thereby identifying a first protein in the sample; and

d) repeating the comparing step for one or more masses obtained for additional sample polypeptides, thereby identifying additional proteins in the sample.

50. The method of claim 49, wherein the plurality of proteins comprises a proteome or a sub-proteome.

51. The method of claim 50, wherein the proteome comprises a human proteome.

52. The method of claim 50, wherein the sub-proteome comprises a preparation of ribosomes, protein complexes, or organelles and comprises at least 50 proteins.

53. The method of claim 49, wherein the plurality of proteins comprises at least 1,000 proteins.

54. The method of claim 53, wherein the plurality of proteins comprises at least 25,000 proteins.

55. The method of claim 49, wherein the method identifies at least 50 percent of the proteins in the sample.

56. The method of claim 49, wherein providing the sample further comprises contacting the plurality of proteins with a first derivatizing agent, wherein the first derivatization agent comprises at least two isotopic forms and specifically labels a selected amino acid or functional moiety when the selected amino acid or functional moiety is present in a member protein.

57. The method of claim 56, wherein the derivatizing agent comprises 2-methoxy-4,5-dihydro-1H-imidazole.

58. The method of claim 59, wherein the derivatizing agent comprises a maleimide, a haloacetyl, an iodoacetamide, or a vinylpyridine.

59. The method of claim 56, wherein the selected amino acid comprises cysteine.

60. The method of claim 56, wherein the selected amino acid comprises lysine and wherein the derivatizing agent reacts with less than 10% of N-terminal amino groups.

61. The method of claim 56, wherein the selected amino acid comprises lysine and wherein the derivatizing agent reacts with less than 1% of N-terminal amino groups.

62. The method of claim 56, wherein the selected amino acid comprises an acidic amino acid, and wherein the derivatizing agent comprises acidic methanol.

63. The method of claim 56, wherein at least one isotopic form of the derivatizing agent is selected from the group consisting of deuterium, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ³⁵Cl, ³⁷Cl, ⁷⁹Br and ⁸¹Br labeled agents.

64. The method of claim 56, wherein the theoretical molecular masses are obtained by:

i) determining a number of occurrences of the selected amino acid in the in silico proteolytic peptides, thereby determining a number of derivatizing agents that would be attached to the in silico proteolytic peptides; and

ii) calculating a theoretical molecular masses for the in silico proteolytic peptides having the determined number of attached derivatizing agents.

65. The method of claim 49, wherein providing the sample further comprises fractionating the sample.

66. The method of claim 65, wherein fractionating the sample comprises performing liquid chromatography, reverse phase chromatography, size exclusion chromatography, strong cation or anion exchange chromatography, weak cation or anion exchange chromatography, immobilized metal ion affinity chromatography (IMAC), capillary electrophoresis, gel electrophoresis, isoelectric focusing, or a combination thereof.

67. The method of claim 49, wherein fractionating the sample further comprises depositing a plurality of fractions of an eluent onto a solid support suitable for LDI.

68. The method of claim 67, wherein the solid support comprises a surface modified for sample confinement.

69. The method of claim 67, wherein the solid support comprises a hydrophobic/hydrophilic MALDI plate.

70. The method of claim 49, wherein ionizing member polypeptides by LDI comprises performing MALDI, IR-MALDI, UV-MALDI, liquid-MALDI, surface-enhanced LDI (SELDI), surface enhanced neat desorption (SEND), desorption/ionization of silicon (DIOS), laser desorption/laser ionization MS, or laser desorption/two step laser ionization MS.

71. The method of claim 49, wherein the mass spectrometer comprises a Fourier-transform ion cyclotron resonance mass spectrometer.

72. The method of claim 49, further comprising identifying predicted cleavage sites for a first proteolytic reagent in amino acid sequences of one or more proteins and determining amino acid sequences of one or more in silico proteolytic peptides that would be obtained by cleavage of the protein at one or more of the predicted cleavage sites.

73. The method of claim 49, further comprising:

e) calculating theoretical molecular masses for additional in silico peptides derived from the parent protein; and

f) repeating the comparing step for a mass obtained for a second peptide and disregarding mass spectral data for the second peptide if the mass spectral data for the second peptide matches that which would be obtained for one or more of the additional in silico peptides from the previously identified protein.

74. The method of claim 73, wherein the mass spectral data for the second peptide is disregarded if a mass obtained for the second peptide is within 5 ppm of the theoretical molecular mass of the additional in silico peptide derived from the previously identified protein;

and if one or both of the following conditions apply;

an expression ratio determined for the second peptide corresponds to an expression ratio for the first peptide; and

/or

a number of derivatized amino acids of the second peptide corresponds to a number of theoretical derivatized amino acids for the second in silico peptide.

75. The method of claim 49, wherein the in silico proteolytic peptides comprise peptides having up to three missed enzymatic cleavage sites and range in molecular mass from 500 Da to 10,000 Da.

76. The method of claim 75, wherein the in silico proteolytic peptides range in molecular mass from 1000 Da to 6000 Da.

77. The method of claim 49, wherein the in silico proteolytic peptides are derived from amino acid sequences encoded by one or more members of an EST library, a cDNA library, or a genomic library.

78. The method of claim 49, wherein the in silico proteolytic peptides are derived from amino acid sequences present in, or encoded by, one or more members of a human sequence library.

79. The method of claim 49, wherein the in silico proteolytic peptides are derived from amino acid sequences present in, or encoded by, one or more members of a yeast sequence library.

80. The method of claim 49, wherein the method further comprises:

identifying one or more fractions that contain a proteolytic peptide for which no unambiguous match was observed among the in silico proteolytic peptides; and

subjecting that fraction to further analysis to identify the proteolytic peptide that is present in the fraction.

81. The method of claim 80, wherein the further analysis comprises tandem MS.

82. The method of claim 49, further comprising:

e) contacting the sample with at least a first proteolytic reagent that cleaves proteins at defined cleavage sites to form sample proteolytic polypeptides.

83. The method of claim 82, wherein contacting the sample with the proteolytic agent is performed prior to contacting the sample with a first derivatizing agent.

84. The method of claim 82, wherein contacting the sample with the proteolytic agent is performed after contacting the sample with a first derivatizing agent.

85. The method of claim 82, wherein the proteolytic reagent comprises a proteolytic enzyme.

86. The method of claim 82, wherein the proteolytic enzyme is selected from the group consisting of trypsin, chymotrypsin, endoprotease ArgC, aspN, gluC, and lysC.

87. The method of claim 82, wherein the proteolytic reagent comprises cyanogen bromide, formic acid, or thiotrifluoroacetic acid.

88. The method of claim 82, further comprising treating the sample to remove post-translational modifications prior to subjecting the proteolytic peptides to mass spectrometry.

89. The method of claim 82, further comprising selecting a subset of proteolytic peptides comprise peptides having greater than 5 amino acids.

90. The method of claim 82, further comprising selecting a subset of proteolytic peptides comprise peptides having greater than 10 amino acids.

91. The method of claim 82, further comprising selecting a subset of proteolytic peptides comprise peptides having greater than 25 amino acids.

92. A method for identifying two or more proteins in a sample, the method comprising:

a) contacting a sample that comprises a plurality of proteins with at least a first proteolytic reagent that cleaves proteins at defined cleavage sites to form sample proteolytic peptides;

b) subjecting at least a first proteolytic peptide to mass spectrometry to determine a mass of the first proteolytic peptide;

c) comparing the mass determined for the first proteolytic peptide to theoretical molecular masses for a plurality of in silico proteolytic peptides that are derived from amino acid sequences for a plurality of proteins, wherein a match between the mass determined for the first proteolytic peptide and the theoretical molecular mass for an in silico proteolytic peptide is indicative of the presence in the sample of the protein from which the in silico proteolytic peptide is derived;

d) calculating theoretical molecular masses for additional in silico proteolytic peptides derived from the protein identified in the comparison of the mass determined for the first proteolytic peptide to the theoretical molecular masses; and

e) repeating the comparing step for a mass obtained for a second proteolytic peptide, and disregarding mass spectral data for the second proteolytic peptide if the mass spectral data is within 5 ppm of that which would be obtained for one or more of the additional in silico proteolytic peptides from the previously identified protein.

93. The method of claim 92, wherein the mass spectrometry is performed using a mass spectrometer that provides a mass accuracy of 5 ppm or better.

94. The method of claim 92, wherein the mass spectrometry comprises FT-ICR MS.

95. An integrated system for identifying a plurality of member proteins in a sample, the system comprising:

an ionization source and a mass spectrometer that provides a mass accuracy of 5 ppm or better;

an interface for receiving mass spectral data from the mass spectrometer, wherein the mass spectral data comprises mass peaks representing masses of a plurality of proteolytic peptides generated by treating the sample with at least a first proteolytic reagent;

a database of theoretical molecular masses of in silico-generated proteolytic peptides, wherein the peptides are derived by predicted action of the proteolytic reagent upon members of a database of protein sequences; and

a computer or computer-readable medium in communication with the interface and the database, the computer or computer-readable medium comprising instructions for determining a mass of a member proteolytic peptide from the mass spectral data and comparing the determined mass to members of the database of theoretical molecular masses, wherein a match between the mass determined for the proteolytic peptide and a theoretical molecular mass for an in silico proteolytic peptide is indicative of the presence in the sample of the protein from which the in silico proteolytic peptide is derived.

96. The system of claim 95, wherein the mass spectral data comprises mass peaks obtained from a sample that was contacted with at least a first amino acid derivatizing agent, and the system comprises instructions for adjusting the molecular mass determined for the in silico proteolytic peptide by adding to a calculated molecular mass the molecular mass of the derivatizing agent multiplied by the number of occurrences of a derivatized amino acid in the proteolytic peptide.

97. The system of claim 95, wherein the mass spectral data comprises mass peaks obtained from a sample that was contacted with at least a first amino acid derivatizing agent, and the system comprises instructions for adjusting the molecular mass determined for a proteolytic peptide by subtracting from the observed molecular mass for the proteolytic peptide the molecular mass of the derivatizing agent multiplied by the number of occurrences of a derivatized amino acid in the proteolytic peptide.

98. The system of claim 97, wherein the system comprises:

a) instructions for generating a subset of in silico proteolytic peptides that comprise a selected amino acid to which the derivatizing agent can attach;

b) instructions for calculating molecular masses for the subset of in silico proteolytic peptides having an attached derivatizing agent; and

c) instructions for comparing the molecular masses for the derivatized in silico proteolytic peptides to the mass peaks for the sample proteolytic peptides.

99. The system of claim 95, wherein the mass spectrometer is an FT-ICR mass spectrometer.

100. The system of claim 95, wherein the plurality of proteins comprises a proteome or a sub-proteome.

101. The system of claim 100, wherein the proteome comprises a human or yeast proteome.

102. The system of claim 95, wherein the in silico proteolytic peptides encompass peptides having up to three missed enzymatic cleavage sites and range in size from 500 Da to 10,000 Da.

103. The system of claim 102, wherein the in silico proteolytic peptides range in molecular mass from 1000 Da to 6000 Da.

104. The system of claim 95, wherein the in silico proteolytic peptides each comprise at least 5 amino acids.

105. The system of claim 95, wherein the in silico proteolytic peptides each comprise at least 10 amino acids.

106. The system of claim 95, wherein the in silico proteolytic peptides each comprise at least 25 amino acids.

107. The system of claim 95, further comprising one or more additional databases of in silico proteolytic peptides, wherein the member in silico proteolytic peptides of the additional databases i) are derived in silico from the database of protein sequences by action of one or more additional proteolytic enzyme upon members of the database;

ii) encompass peptide sequences having up to three missed enzymatic cleavage sites; and

iii) range in size between 1000 Da and 4000 Da.

108. The system of claim 95, wherein the interface further comprises software for controlling generation and processing of the mass spectral data by the mass spectrometer.

109. The system of claim 95, further comprising a liquid chromatography system fluidically coupled to an automated sample collection system that comprises an eluent collection plate, wherein the mass spectrometer is configured to analyze ions generated from sample fractions present on the collection plate.

110. The system of claim 109, wherein the liquid chromatography system comprises a HPLC system.

111. The system of claim 109, wherein the eluent collection plate comprises a hydrophobic coating and one or more hydrophilic regions.

112. The system of claim 109, further comprising a sample source and a source of one or more proteolytic reagents, wherein the sample source and the source of proteolytic reagents are fluidically coupled to one another through a mixing region, and wherein the mixing region is fluidically coupled to the liquid chromatography system.

113. The system of claim 112, wherein one or more of the sample source, the source of proteolytic reagents, and the mixing region comprise microtiter plate wells.

114. The system of claim 112, wherein one or more of the sample source, the source of proteolytic reagents, the mixing region, and the liquid chromatography system are incorporated into a microfluidic device.

115. The system of claim 95, wherein the system comprises instructions for:

calculating theoretical molecular masses for additional in silico proteolytic peptides derived from the protein identified in the comparison of the mass obtained for the first proteolytic peptide to the theoretical molecular masses; and

disregarding mass spectral data for a second proteolytic peptide if a determined mass for the second proteolytic peptide matches a theoretical molecular mass for an additional in silico proteolytic peptides derived from the previously identified protein.

116. The system of claim 95, wherein the computer or computer readable medium sequentially compares two or more sample masses to the theoretical molecular masses for the in silico proteolytic peptides.

117. The system of claim 95, wherein the computer or computer readable medium simultaneously compares two or more sample masses to the theoretical molecular masses for the in silico proteolytic peptides.