Jun.29,2006  8  US 2006/0141632 Al  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]  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.  [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.  [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%.  [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  [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  EXAMPLE 11  PSD MALDI Sequencing Technique  Analysis Using a Mass Spectrometric Technique  EXAMPLE 10  0  H2N0  )  r-DPGYIGSR  HS  0  HO  II  o  )s~Nr  O  \  H  N - DPGYIGSR  HO  I  \  H  s  o~~  0 