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Comparison of three methods for fractionation and enrichment of low molecular weight proteins for SELDI-TOF-MS differential analysis |
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| Authors: | Muriel De Bock Marie-Alice Meuwis Tran Quang Minh Jean-Paul Chapelle Michel Malaise Marianne Fillet |
| Institution: | a Clinical Chemistry, GIGA Research, University of Liège, CHU, B36, B-4000 Liège 1, Belgium b Rheumatology, GIGA Research, University of Liège, CHU, B36, B-4000 Liège 1, Belgium c Proteomic Platform, GIGA Research, University of Liège, CHU, B36, B-4000 Liège 1, Belgium d Dept. of Analytical Pharmaceutical Chemistry, Institute of Pharmacy, University of Liège, CHU, B36, B-4000 Liège 1, Belgium e Affiland s.a., rue de l’Yser 304, 4430 Ans, Belgium f Biochemistry, Institute of Pathology B23, University of Liège, Belgium g Hepato-Gastroenterology, GIGA Research, University of Liège, CHU, B36, B-4000 Liège 1, Belgium |
| Abstract: | In most diseases, the clinical need for serum/plasma markers has never been so crucial, not only for diagnosis, but also for the selection of the most efficient therapies, as well as exclusion of ineffective or toxic treatment. Due to the high sample complexity, prefractionation is essential for exploring the deep proteome and finding specific markers.In this study, three different sample preparation methods (i.e., highly abundant protein precipitation, restricted access materials (RAM) combined with IMAC chromatography and peptide ligand affinity beads) were investigated in order to select the best fractionation step for further differential proteomic experiments focusing on the LMW proteome (MW inferior to 40,000 Da). Indeed, the aim was not to cover the entire plasma/serum proteome, but to enrich potentially interesting tissue leakage proteins. These three methods were evaluated on their reproducibility, on the SELDI-TOF-MS peptide/protein peaks generated after fractionation and on the information supplied.The studied methods appeared to give complementary information and presented good reproducibility (below 20%). Peptide ligand affinity beads were found to provide efficient depletion of HMW proteins and peak enrichment in protein/peptide profiles. |
| Keywords: | CM10 weak cationic exchanger arrays HAP highly abundant proteins HMW high molecular weight IMAC-RAM restricted access materials (RAM) combined with IMAC chromatography LMW low molecular weight PF4 platelet factor 4 PRM-30 Proteomics-30® resin for molecular mass < 30 kDa RSD relative standard deviation RT room temperature SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis SELDI-TOF-MS surface-enhanced laser desorption/ionisation-time-of-flight-mass spectrometry |
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