Development Of Affinity Combinational Fractionation System For Proteomics

Efficient and high resolution separation of the protein mixture prior totrypsin digestion and mass spectrometry analysis is generally used to reducethe complexity of samples, an approach that highly increases the probabilityof detecting low-copy-number proteins. In this study, an affinitycombinational fractionation sytem was employed to prefractionate the tisseextract prior to trypsin digestion and LC-MS/MS analysis. After reducingthe complexity of tissue protein sample, more protein information could beobtained from MS-based proteomics analysis. Two affinity combinationalfractionation methods, tandem affinity fractionation and cascade affinityfractionation, were studied and applied in the proteomics analysis of rat livercytosol and mouse testis.Different amino acids, peptides, or amino compounds were linked toimmobilized matrix Sepharose4B, and thousands of these synthetic affinityligands constituted an affinity ligand library in our lab. Structural differencesbetween these affinity ligands result in different non-bonded protein-ligand interactions. Thus, each ligand exhibits a specific affinity to some proteingroups. We first screened out affintiy ligands with medium absorbance abilityand large band distribution difference in protein absorbance profiles. Thus,some affinity ligands (including A1-4, A6, A7-56, A8-54, A11-70, A15,A17-56, A25-35, A29-32, and A84) were selected for components of affinitycombinational fractionation sytem.In tandem affinity fractionation, A7-56, A84, A11-70, A6, and A29-32were orderly connected. Rat liver cytosol sample with8mg protein weightwas loaded into the tandem columns, after being bound and flushed fully, fivetandem columns were divided into single column, and bound protein of eachcolumn was respectively eluted. Ultimately, rat liver cytosol sample (Fraction0) was fractionated into5elution fractions (Fraction1to5) and1flow-through (Fraction6). All the fractions collected from tandem affinityprefractionation were digested and then analyzed by LC-MS/MS, whichresulted in high confidence identification of665unique rat protein groups,1.8times as many proteins as were detected in the unfractionated sample (371protein groups). Of these,430unique proteins (64.7%) only characterized inspecific fractions, indicating that the crude tissue extract was well-distributedby tandem affinity fractionation. Of665detected proteins,6proteins werewith Mr <10kDa,56with Mr>100kDa,10with pI <4.3,4with pI>10,55 with GRAVY value>0, and41with1or more TM Helices. This approachhighlighted the sensitivity of this method to a wide variety of protein classes.Three affinity ligands (A15, A8-54, and A11-70) were orderly selectedfor three-cascade affinity fractionation. First, rat liver cytosol (F0) wasloaded into A15, after binding and elution F0was distributed into2fractions(flow-through and elution, F1-1and F1-2). Second, F1-1and F1-2wereseparately loaded into A8-54, and4new fractions (F2-1to F2-4) wereobtained. Finally, F2-1, F2-2, F2-3, and F2-4were separately loaded into A11-70,and8new fractions (F3-1to F3-8) were obtained. All fractions collected wereseparately digested and then analyzed by LC-MS/MS.391non-redundantprotein groups were identified in F0,499protein groups in F1-1and F1-2,616in F2-1-F2-4, and738in F3-1-F3-8. Ultimately, a total of859unique proteingroups were identified in all cascade fractions,2.2times as many proteins aswere detected in the unfractionated sample. Of859detected proteins, thereare75proteins with Mr <20kDa,73with Mr>100kDa,72with GRAVYvalue>0, and49with1or more TM Helices.Three-cascade affinity fractionation system (A15~A8-54~A11-70) wasalso successfully used in proteomics of mouse testis. All fractions collectedfrom cascade affinity fractionation system were separately digested and thenanalyzed by LC-MS/MS, which resulted in high confidence identification of a total of1378non-redundant mouse testis protein groups,2.6times as manyproteins as were detected in the un-fractionated sample (526). All detectedproteins were bioinformatically categorized according to theirphysicochemical characteristics (such as relative molecular mass, pI, GRAVYvalue, TM Helices), subcellular location and function annotation. Of1378detected mouse testid proteins, there are12proteins with Mr <10kDa,169with Mr>100kDa,38with pI <4.5,51with pI>10,93with GRAVY value>0,81with1or more TM Helices,310without subcellular locationinformation, and221without GO function annotation. There were16detectedproteins involving in the spermatogenesis and sperm development.Combined usage of affinity combinational fractionation system with massspectrometry-based proteomic analysis was proved to be simple, low-cost,and effective, providing the prospect of broad application in proteomics.