A novel fractionation method prior to MS-based proteomics analysis using cascade biomimetic affinity chromatography.
This is the first report that combines cascade biomimetic affinity fractionation with MS-based proteomics analysis. Our lab has constructed an affinity ligand library composed of thousands of ligands with different protein-binding properties. Structural differences between these ligands result in different non-bonded protein-ligand interactions, thus each ligand exhibits a specific affinity to some protein groups. In this work, we first screened out three affinity ligands with large difference in protein-binding properties. Next, cascade combination of these ligands was applied to fractionate tissue sample into simple subgroups prior to trypsin digestion and LC-MS/MS analysis. In this study, 391 non-redundant protein groups were identified in unfractionated rat liver cytosol, 499 protein groups were identified in 2 fractions of the first affinity fractionation, 616 in 4 fractions of the second fractionation, and 738 in 8 fractions of the third fractionation (an 88.74% increase). Ultimately, a total of 859 unique protein groups were identified in all cascade fractions (a 119.6% increase compared with unfractionated sample). The proteins detected in each fraction were bioinformatically categorized according to their physicochemical characteristics (relative molecular mass, pI, GRAVY value and TM helices). This approach highlighted the sensitivity of this method to a wide variety of protein classes.
Tan
Qingqiao
Q
MOE Key Laboratory of Microbial Metabolism, College of Life Science and Biotechnology, Shanghai Jiao Tong University, 800 Dong-chuan Road, Shanghai 200241, China.
Dong
Dexian
D
Li
Rongxiu
R
D000818:Animals; D005591:Chemical Fractionation / Q000379:methods*; D002846:Chromatography, Affinity / Q000379:methods*; D008024:Ligands; D008099:Liver / Q000737:chemistry; D008297:Male; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics; D051381:Rats; D017207:Rats, Sprague-Dawley
D008024:Ligands; D011506:Proteins