Fractionation of peptides in proteomics with the use of pI-based approach and ZipTip pipette tips

Abstract

The aim of the work was to explore the utility of the in-solution isoelectric focusing (sIEF) fractionation method. That method was proved to be the alternative separation method of mixtures of protein tryptic digests in proteomics. Analysis of the identification of peptides was performed with the use of matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF-MS). For that research, previously designed the miniaturized multi-chamber fractionation sIEF device (75μl volume for each chamber) based on polyacrylamide gel membranes with immobilines technology was utilized. To evaluate the efficiency and accuracy of sIEF fractionation combined with MS/MS peptides identification, bovine serum albumin (BSA) digest and mixture of five proteins digest were used.First, fractionation of bovine serum albumin digest sample was performed using sIEF method. Studies performed for that simple mixture of peptides proved the ability of the sIEF device to focus peptides mostly in one chamber. Additionally performed the correlation analysis between pIcalc and pIexp values for identified peptides proved the possibility to obtain experimentally useful high correlation. That information was found to have a potential value for construction of additional constraint during false positives evaluation process among identified proteins. Next, studies on the sIEF fractionation were combined with the evaluation of practical use of ZipTip pipette tips to fractionate peptides in the case of simple mixture of proteins. For this, five proteins digest samples were used. The analysis without prior any fractionation enabled to identify very limited number of proteins. The significant improvement was obtained when one used sIEF alone or with combination with ZipTips fractionation prior to MS analysis. The proposed approach based on in-solution isoelectric focusing proved to be an efficient and accurate alternative fractionation method of protein digests and can be considered as the first useful dimension in two-dimensional proteomics separations. Moreover, analytical information from that pI-based fractionation method can be considered as the additional source of database matching constraint. It can also be a valuable tool for analytical and bioinformatic studies of peptides fractionation in proteomics.