Phosphopeptides identification Column technology Inc.,

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

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

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

Peptide and phosphopeptide distribution in each fraction. pH Unique Phosphopeptides Unique Nonphosphopeptides Phosphopeptide Hits Peptides Hits Ratio of Phosphopeptide Hits v.s. All Peptide Hits % % % 5.67 % 1.77 % 1.91 % 1.38 % 1.54 % 1.16 % 0.88 % Hits/Phosphopeptides

Phosphopeptide distributions

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

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.