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minutes at room temperature prior to dilution of the resulting derivatized analyte and mass spectral analysis. The concen› tration of the acidic moiety reagent is decreased by a factor of as much as 100 when derivatizing smaller quantities. 

[0058] 

<img src='content_image/935030.jpg'>

[0059] ASHLGLAR (1 nmol, commercially available from Sigma Chemical Co., St. Louis Mo.) (SEQ ID NO: 1) is mixed with 2 µL of 0.02 M sulfoacetic acid, which is formed by mixing 2 µL of neat chlorosulfonylacetyl chloride (commercially available from Aldrich Chemical Co., Mil› waukee, Wis.) with 500 µL of water. The mixture is dried and then reconstituted in 20 µL oftetrahydrofuran:diisopro› pylethyl amine (4:1 v:v). 0.1 M chlorosulfonylacetyl chlo› ride (2 µL, commercially available from Aldrich Chemical Co., Milwaukee, Wis.) in dry tetrahydrofuran is added and the mixture is vortexed for 30 seconds. The derivatization reaction proceeds for approximately two minutes at ambient temperature. The derivatized analyte is dried, reconstituted in 20 µL water and further diluted prior to mass spectral analysis. Chlorosulfonylacetyl chloride is also a useful reagent for derivatization of 2D gel isolates, but a modified synthetic procedure provides more consistent product yields. The modified procedure is discussed in Example 14. 

[0060] 

<img src='content_image/935032.jpg'>

S-acetylmercaptosuccinic anhydride ( commercially avail› able from Aldrich Chemical Co., Milwaukee, Wis.) is pre› pared at a concentration of 0.1 M in dry tetrahydrofuran prior to use. ASHLGLAR (1 nmol, commercially available from Sigma Chemical Co., St. Louis Mo.) (SEQ ID NO: 1) 

is diluted into 20 µL of 0.05 M trimethylamine. The S-acetylmercaptosuccinic anhydride solution (5 µL) is added and the reaction mixture is vortexed for 30 seconds. The reaction proceeds for about two minutes at room temperature and is then oxidized with 10 µL of formic acid (88%):H$_{2}$ 0$_{2 }$ (30%) prepared at a ratio of 19:1 (v:v). Oxi› dation proceeds for 16 hours at room temperature, and the sample is dried prior to dilution and mass spectral analysis. The concentration of the acidic moiety reagent is decreased by a factor of as much as 100 when derivatizing smaller quantities. 

[0061] 

<img src='content_image/935031.jpg'>

[0062] To ASHLGLAR (1 nmol, commercially available from Sigma Chemical Co., St. Louis, Mo.) (SEQ ID NO: 1) in 0.1 M trimethylamine (20 µL) is added 2-iminothiolane (Traut’s reagent, commercially available from Aldrich Chemical Co., Milwaukee, Wis.) (3 µL, 0.1 Min deionized water). The reaction proceeds for 5 minutes at room tem› perature. The product is oxidized with 3 µL of formic acid (88%):H$_{2}$ 0$_{2 }$ (30%) prepared at a ratio of 19:1 (v:v). Oxi› dation proceeds for 5 minutes at room temperature, and the derivatized analyte is dried prior to mass spectral analysis. 

[0063] 

<img src='content_image/935033.jpg'>

[0064] The polypeptide CDPGYIGSR (commercially available from Sigma Chemical Co., St. Louis, Mo.) (SEQ ID NO: 2) is oxidized by mixing 1 to 5 nM of polypeptide (in 5-20 µL water) with 10 µL of formic acid (88%):H$_{2}$ 0$_{2 }$ (30%) prepared at a ratio of 19:1 (v:v). Oxidation proceeds for 30 minutes at room temperature, and the derivatized analyte is dried prior to mass spectral analysis. 

