A comparison of sample preparation methods for the study of the human serum proteome

Abstract

This work describes our work of investigating the outcome of different sample preparation methods for label-free quantitative proteomic study of human serum proteins. All analyses were preformed on insolution trypsin digestion of human serum. MS analysis was on a Q-TOF instrument with a nanoflow split-free LC system interfaced to the ion-source (75 μ id LC columns). Data was acquired using data dependent analysis (DDA) or a high/low energy strategy. The high/low method is a “protein expression” method were a mass window of data is acquired in low energy (intact peptide) followed by a high energy acquisition on all ions in the mass window for intact peptides. This maximizes the duty cycle of the Q-TOF to yield extensive qualitative and quantitative information by concurrently analyzing the peptide ions in the mixture. Samples were prepared by a verity of methods including, top-6 or top-20 abundant protein depletion and enhancement (reproducibility) of digestion with trypsin-friendly surfactants. Fractionation of intact proteins by 1D SDS-PAGE and chromatography was investigated. In addition, peptide mixtures from digestion were separated by multidimensional chromatography. The 2D methods included SCX/RP, RP/RP, and HILIC/RP. The objective was to understand the most effective sample preparation method for studying the human serum proteome. A summary of the results will be presented.