Protein identification in biomarker discovery studies based on profiling of intact proteins: Why the challenge? Lesson gained from breast cancer tissues
| Autoři | |
|---|---|
| Rok publikování | 2012 |
| Druh | Konferenční abstrakty |
| Fakulta / Pracoviště MU | |
| Citace | |
| Popis | We showed thate the use of simple combination of protein/peptide separation technique followed by MS/MS leads to high uncertainty in protein biomarker identification in biomarker discovery studies based on profiling of intact proteins. This is given namely by the fact that database molecular weight of proteins does not reflect their posttranslational modifications. Here we propose two protocols to overcome these bottlenecks: (i) For proteins larger than 6 kDa, we have developed a method combining IMAC column preseparation, reverse phase LC, MALDI/SELDI protein profiling, tricine SDS-PAGE, protein profile matching, trypsin digestion and MS/MS identification. (ii) For identification of proteins smaller than 6 kDa, we have optimized a "top-down" procedure where tricine SDS-PAGE is replaced by LC-MS/MS of proteins on LTQ Orbitrap Velos with the use "no enzyme" option during MS/MS ion search. This protocol enabled and identification of HNRNP A2/B1 protein variant correlating with estrogen receptor expression in breast cancer. |
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