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Phosphopeptides identification

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

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Phosphopeptides identification

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

  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 SequestTM software

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

  5. Peptide and phosphopeptide distribution in each fraction.

  6. Phosphopeptide distributions

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

  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.

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