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 |
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| 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 |
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| 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 |
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| Combo package*
(Method 200)
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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 |
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| 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 |
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