Post-Translational Modifications (Phosphorylation & Methylation)

Method Number

60 70 80 90
Method Name MALDI-TOF/TOF LC-MALDI LC-ESI-MS/MS
Instrument AutoFlexIII RP-HPLC-
-robot-spotter-AutoFlex
LTQ, LCQ
Cost (per sample) 200 300 300 250
Sample requirements Single band or pure protein solution in
1 - 5 µg quantities
> 10 pmole levels
pure and in-solution is preferred
> 10 pmole levels
pure and in-solution is preferred
Analysis Includes 1) searches for peptides modified with phosphate or methyl groups
2) maps modified amino acid position, using MS/MS fragmentation, if modified peptide is found
Expected results expect > 20% coverage of whole protein
*see Combo package
expect > 40% coverage of whole protein
*see Combo package
expect > 40% coverage of protein
*see Combo package
Potential Problems and Issues 1)protein is low in concentration or impure, so coverage is low and no PTMs are detected
2)level of phosphorylation is low
3)phospho-peptide does not “fly” well in mass spectrometer
Solutions for Common Problems 1)customer must produce a higher yield and purity protein preparation
2)enrich for phosphopeptides, using affinity chromatography (below)
3)digest sample with a different protease, like chymotrypsin instead of trypsin
Combo package*

(Method 200)

 

1)coverage is maximized up to 90/100% by combining data from:
2)multiple runs on different instruments/technologies
3)Cost = $500 for conducting analysis, using two of the three methods above
Turnaround time      
Phospho peptide purification
(Method 210)
This method enriches phosphopeptides on titanium dioxide resin
$120 for each enrichment
requires >5 µg of purified protein digest or from 1 pmole to 1 nmole peptides