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5. MASS PEAK LIST FROM SPECTRA

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5. MASS PEAK LIST FROM SPECTRA. Automated & Semi-automated Software Inspect the spectra Check Mascot-Parameters. Manual PTM-validation Detect Phosphate-Fingerprints (Long & Tedious) For Large-Scale Phospho-analyses  Bioinformatics Tools are useful. Variants TiO2. SIMAC. IMAC.

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5. MASS PEAK LIST FROM SPECTRA

  • Automated & Semi-automated Software
  • Inspect the spectra
  • CheckMascot-Parameters
  • Manual PTM-validation
  • Detect Phosphate-Fingerprints
  • (Long & Tedious)
  • For Large-Scale Phospho-analyses Bioinformatics Tools are useful
  • Variants TiO2
  • SIMAC
  • IMAC
  • TiO2

1. Chemical Treatments- Sample Conditioning for Phosphoproteomic Analyses

  • According to the sample amount, characteristics & Phospho-goals
  • Optimal Phospho-Digestions (Tryp+/- Lys-C; +/-SDS; +/-Urea…)
  • Avoiding phospho-artefacts
  • Keeping phosphate-groups

6. In Summary

  • Combining different Phospho-proteomic
  • Strategies
  • Recover the highest number of
  • phosphorylated peptides/proteins

Simple samples,usually No Prefraction

Large-scale, Prefraction steps

2. Phosphoenrichments of Mono & Multiply phosphorylated peptides

3. Desalting, cleaning and concentrating phosphopeptides

  • POROS R2
  • (unbonded phospho-)
  • R3
  • &/or Graphite
  • C18
  • R3 -C18

4. Isolating and Identifying Mono &Multiply Phosphorylated Residues

Testing different

fragmentation modes

MS2, MS3, MS3 NL, MSA

(eg. Combining different ionizations via LC-ESI: CID, ECD,ETD)

Testing different Ph-elutions

DHB, Glycolic & Phtalic acids…

(eg. Check via MALDI-MS, MS/MS)

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