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 

## EXAMPLE 13 

## EXAMPLE 14 

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. 

