Jun.29,2006  10  US 2006/0141632 Al  that only y-ions or (y-NH3) ions are allowed fragments  because y- and (y-NH3) ions are the most prominent species  observed in the fragmentation patterns wherein the present  methods are utilized. Other fragment ion types like a-, b-,  (b+H$_{2}$O), (b-H$_{2}$O), (b-NH3) and internal cleavage ions can  be disallowed because they are not prominent in the spectra  of the peptides derivatized using the methods of the present  invention. The derivatives formed with the present invention  provide simple fragmentation patterns that often yield  greater database search specificity than can be obtained from  the spectra of the same peptides without derivatization.  [0088]  The methods of the present invention are further  illustrated in the non-limiting examples below. In these  examples, the ordinarily skilled artisan will recognize that  numbers of "candidate proteins" produced may differ from  those disclosed herein as new proteins are continually added  to databases.  [0089]  PSD MALDI tandem mass spectrometry of high› mass polypeptides is demonstrated with oxidized insulin  B-chain FVNQHLC(SO$_{3}$H)GSHLVEALYLVC(SO$_{3}$H)  GERGFFYTPKA (SEQ ID NO: 4) (MMcalc=3495.9 Da)  (commercially available from Sigma Chemical Co., St.  Louis, Mo.). The mass of this polypeptide far exceeds the  upper limit of most conventional triple quadrupole instru› ments. The tandem mass spectrum of the native polypeptide  shows a relatively intense ion series consisting substantially  of y-ions. All y-ions between y9 and y24 are readily  observed. The sequence-specific fragments defining the  N-terminus of the molecule, y25 to y29, are absent from the  spectrum.  [0090]  The spectrum of this polypeptide is improved by  derivatization using the acidic moiety reagent sulfobenzoic  acid cyclic anhydride ( commercially available from Aldrich  Chemical Co., St. Louis, Mo.). The derivatized polypeptide  shows significant enhancement of the y-ions derived from  the N-terminal region of the molecule (y25, y26, y27, y28,  and y29). A complete series ofy-ions is observed from y8 to  y29 following derivatization. No prominent b-ions are  detected.  [0091]  A polypeptide separated by two-dimensional gel  electrophoresis is in-gel digested with trypsin to produce  peptides of the polypeptide. The peptides show several  intense MH+ signals by MALDI including ions at m/z  1060.8, 1090.8, 1271.0, 1299.0, 1312.0, 1344.0, 1399.1,  1450.1, 1460.1, and 1794.4. Database searching using the  Protein Prospector Software against the entries in the NCBI  protein sequence library produces a list of forty-one candi› date proteins that match five or more of the input tryptic  masses (search parameters: MW range 1,000 to 150,000,  isoelectric point from 3.0 to 10.0, oxidized Met allowed as  a side-reaction and a conservative mass tolerance of +/-0.6  Da).  [0092]  The peptides of the polypeptide can be derivatized  with any one of several of the reagents discussed in the  previous examples. They include, but are not limited to,  2-sulfobenzoic acid cyclic anhydride, 3-sulfopropinoic  anhydride or chlorosulfonylacetyl chloride. In this example,  chlorosulfonylacetyl chloride is used as the acidic moiety  reagent. The derivatization procedure for low-level peptides  isolated from 2D gel electrophoresis is modified to improve  reproducibility and derivative yields. Typically, the peptide  extract from the 2D gel is concentrated to near dryness (5 to  10 µL) on a speed vac. The concentrates are acidified with  15 µL of  O  .1 % TF A and cleaned up using commercially  available C18 mini-colunms (ZipTip™, Millipore Corpora› tion, Bedford, Mass. 01730). The cleaned up sample is dried  on a speed vac and reconstituted in 10 µL of base  (THF:DIEA 19:1 v/v). In this example, 2 µL of a chloro› sulfonylacetyl chloride solution (2 µL  of the neat liquid in 1  mL THF) is added and the reaction proceeds for 1 to 2 min  at room temperature. The derivatized samples are again  dried on a speed vac and reconstituted in 10 µL  of0.1% TFA  prior to analysis. At this stage, the sample can be mixed with  MALDI matrix and a portion loaded onto the sample stage  for analysis, or it may be cleaned up again using a com› mercially available C18 mini-column (ZipTip™, Millipore  Corporation). The cleaned up sample can then be eluted  directly from the ZipTip onto the MALDI sample stage in a  1-2 µL  volume of acetonitrile:0.1 % TFA (1:1 v/v) containing  10 mg/mL of MALDI matrix. This latter procedure allows  loading all of the recovered derivatives onto the MALDI  sample stage for analysis thereby improving overall sensi› tivity of the method. PSD MALDI tandem mass spectrom› etry is carried out on the derivatized peptide weighing about  1572 Da. A sequence tag is obtained having y-ions at m/z  574.5, 661.7, 875.9, 1003.8, 1103.9, 1204.6, 1304.1, and the  (MH+-derivative) ion at m/z 1451.0. The spectrum is  searched against the protein sequence database, and six  candidate proteins (all mitochondrial aspartate aminotrans› ferases) are returned (search parameters: MW range 1,000 to  150,000, parent ion mass tolerance +/-0.6 Da, fragment ion  mass tolerance +/-2.5 Da, Par(mi)Frag(av), allowed number  of missed ions=!). This level of specificity is obtained  because the database search is constrained to consider y-ions  as the only allowed fragments. This search constraint is  possible because N-terminal and internal cleavage ions are  not prominent in the tandem mass spectra of these deriva› tives (due to derivatization according to the present inven› tion). Much less specificity is obtained (239 candidate  proteins returned) when this same tandem mass spectrum is  searched against the full library and all fragment ion types  (a, b, (b+H$_{2}$O), (b-NH3), (b-H$_{2}$O), internal and y) are  allowed. The peptide was identified as FVTVQTISGT› GALR (SEQ ID NO: 5).  [0093]  Confirmation of the protein identification is sought  by PSD MALDI of the derivative weighing about 1916 Da.  The spectrum contains 7 fragment ions at m/z 724.6, 1154.2,  1299.3, 1439.0, 1551.9, 1665.5, and 1779.2, in addition to  the protonated precursor ion. This spectrum is searched  against the database assuming the fragments to be y-ions.  The search does not return mitochondrial aspartate ami› notransferase or any other candidate protein. Researching  the database allowing bothy and (y-NH3) ions returns 16  candidate proteins, 10 of which are mitochondrial aspartate  aminotransferases. This latter search confirms the protein  identification. Inclusion of the (y-NH3) ions as possible  fragments is required for this particular search because the  peptide is an incomplete tryptic product (LIRPLYSNPPLN› GAR) (SEQ ID NO: 6) that contains a second arginine  residue. As stated herein above, derivatized incomplete  digestion products often exhibit (y-NH3) fragment ions in  the PSD tandem mass spectra.  EXAMPLE 14  EXAMPLE 13 