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ASHLGLAR (1 nmol) (SEQ ID NO: 1) is diluted into 20 µL of 0.05 M trimethylamine. The tetrafluorosuccinic anhy› dride solution (2 µL) is added and the reaction mixture is vortexed for 30 seconds. The reaction proceeds approxi› mately two minutes at room temperature prior to dilution and mass spectral analysis. The concentration of the cou› pling reagent is decreased by a factor of approximately 100 when derivatizing smaller quantities. 

[0075] 

<img src='content_image/925291.jpg'>

[0076] The polypeptide CDPGYIGSR (commercially available from Sigma Chemical Company, St. Louis, Mo.)(SEQ ID NO: 2) is mixed with 2 µL of 0.02M sul› foacetic acid, which is formed by mixing 2 µL of neat chlorosulfonylacetyl chloride ( commercially available from Aldrich Chemical Co., Milwaukee, Wis.) with 500 µL of water. The mixture is dried and reconstituted in 20 µL of tetrahydrofuran:diisopropylethyl amine (4:1 v/v). 0.1 M chlorosulfonylacetyl chloride (2 µL) in dry tetrahydrofuran is added and the mixture is vortexed for 30 sec. The derivatization reaction proceeds for about 2 min. at ambient temperature. The derivatized analyte is dried and reconsti› tuted in 10 µL of water. To that solution is added 10 µL of formic acid (88% ):H$_{2}$Oi30%) prepared at a ratio of 19: 1 (v:v). Oxidation proceeds for 5 min. at room temperature, producing the derivatized peptide having two sulfonic acid groups near the N-terminus. 

## Analysis Using a Mass Spectrometric Technique 

[0077] Upon derivatization, the polypeptide or one or more peptides of the polypeptide are analyzed using a mass spectrometric technique. While the technique utilized is not limited, the preferred techniques are post-source decay (PSD) matrix-assisted laser desorption ionization (MALDI) and electrospray ionization tandem mass spectrometry. See, e.g., Spengler et al., "Peptide Sequencing by Matrix-assisted Laser-desorption Mass Spectrometry", Rapid Communica› tions in Mass Spectrometry, Vol. 6, pp. 105-108 (1992); Spengler et al., "Fundamental Aspects of Postsource Decay in Matrix-Assisted Laser Desorption Mass Spectrometry", Journal of Physical Chemistry, Vol. 96, pp. 9678-9684 (1992); Kaufmann et al., "Mass Spectrometric Sequencing of Linear Peptides by Product-ion Analysis in a Reflectron Time-of-flight Mass Spectrometer Using Matrix-assisted Laser Desorption Ionization", Rapid Communications in 

Mass Spectrometry, Vol. 7, pp. 902-910 (1993); Kaufmann et al., "Sequencing of Peptides in a Time-of-flight Mass Spectrometer: Evaluation of Postsource Decay Following Matrix-assisted Laser Desorption Ionization (MALDI), International Journal of Mass Spectrometry and Ion Pro› cesses, Vol. 131, pp. 355-385 (1994); Kaufmann et al., "Post-source Decay and Delayed Extraction in Matrix› assisted Laser Desorption/Ionization-Reflectron Time-of› Flight Mass Spectrometry", Rapid Communications in Mass Spectrometry, Vol. 10, pp. 1199-1208 (1996); and Spengler, "Post-source Decay Analysis in Matrix-assisted Laser Des› orption/Ionization Mass Spectrometry of Biomolecules", Journal of Mass Spectrometry, Vol. 32, pp. 1019-1036 (1997); Carr et al., "Integration of Mass Spectrometry in Analytical Biotechnology", Analytical Chemistry, Vol. 63, pp. 2802-2824, (1991); Yates III et al., "Mining Genomes With MS", Analytical Chemistry, Vol. 68, pp. 534A-540A, (1996); Morris et al., "High Sensitivity Collisionally Acti› vated Decomposition Tandem Mass Spectrometry on a Novel Quadrupole/Orthogonal Acceleration Time-of-Flight Mass Spectrometer", Rapid Communications in Mass Spec› trometry, Vol. 10, 889-896, (1996). Most preferably, the techniques utilized are positive ion mode PSD MALDI and electrospray ionization tandem mass spectrometry. For con› venience, Examples 11 and 12 below illustrate mass spec› trometric techniques which may be utilized to analyze the polypeptide or peptides thereof. 

[0078] As the present inventors have surprisingly discov› ered, appropriately derivatized peptides or peptides of the polypeptide provide MSMS spectra predominantly charac› terized by y-ions. As is well-known in the art, y-ions indicate ionized fragments containing the original C-terminus of the polypeptide or peptide. As used herein, the term "y-ion" also includes (y-NH3) ions; to illustrate, incomplete digestion products containing a second basic (for example) residue often yield abundant (y-NH3) ions. Preferably, the spectra produced by this method are substantially free of a-ions and b-ions. A-ions and b-ions are formed by cleavages on either side of backbone carbonyl groups. Importantly, charge is retained with the N-terminal fragment with a-ions and b-ions. As used herein, the term "substantially free of’ with reference to a-ions and/orb-ions means that compared to the dominant y-ion series, a-ions and b-ions have a collective relative abundance of less than about 20%, preferably less than about 10%, and most preferably less than about 5%. 

[0079] According to the method of the present invention, it is not necessary to analyze and/or identify all digestion products to identify the polypeptide thereof, particularly if the sequence of the polypeptide resides in a sequence database. 

[0080] In this example, the mass spectrometric technique is carried out on a Voyager DE-RP or Voyager DE-STR (PerSeptive Biosystems Inc., Framingham, Mass.) (or a suitable equivalent) equipped with a N2 laser (337 nm, 3 nsec pulse width, 20 Hz repetition rate). The mass spectra are acquired in the reflectron mode with delayed extraction. External mass calibration is performed with a low-mass peptide standard, and mass measurement accuracy is typi› cally –0.3 Da. The derivatized polypeptide or peptides are 

## PSD MALDI Sequencing Technique 

## EXAMPLE 11 

