1. Phosphopeptides identification Column technology Inc., WWW.columntechnology.com WWW.columntechnology

2. SAX for phosphopeptide separation Phosphopeptides have lower pIs then non- phosphopeptides

3. Off line pH gradient follow by reverse phase separation Peptides fractionated by pH step gradient The collected fractions were dried and reconstituted with 0.1% formic acid Followed by nano-spray reverse phase gradient and mass spectrometry. Proteins were identified by Sequest TM software

4. pH based SAX 2D-LC-MS/MS for Phosphoproteome

5. Peptide and phosphopeptide distribution in each fraction. pH22.533.544.555.568.5 Unique Phosphopeptides71166164109373135405872 Unique Nonphosphopeptides2996676121127132112021644164024263385 Phosphopeptide Hits200358319164524547557482 Peptides Hits794166317552890293123623399356463689305 Ratio of Phosphopeptide Hits v.s. All Peptide Hits 25.19 % 21.53 % 18.18 % 5.67 % 1.77 % 1.91 % 1.38 % 1.54 % 1.16 % 0.88 % Hits/Phosphopeptides 2.822.161.951.501.411.451.341.381.281.14

6. Phosphopeptide distributions

7. EEVAS*EPEEAAS*PTTPK EEVAS*EPEEAASPTTPK EEVASEPEEAASPTTPK 119 phosphorylation species Non-, Mono-, Di-phospho

8. Conclusions SAX-RP-MS/MS for phosphoproteome Mass compatible buffers Phosphopeptides were retained and separated by SAX. Low pH buffer used in the SAX is easy to switch to RP LC/MS. Both phosphopeptides and non-phosphopeptide can be identified. No derivatization and no metal chelation needed. Can be on line with 2D LC/MS/MS.