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Innovations in Phosphopeptide Identification Using Column Technology

Learn about the advanced SAX-RP-MS/MS method to identify phosphopeptides with mass spectrometry. This technology separates peptides based on pH and offers improved phosphoproteome analysis. Explore how proteins are identified without the need for derivatization or metal chelation.

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Innovations in Phosphopeptide Identification Using Column Technology

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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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