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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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