Coupling a Detergent Lysis/Cleanup Methodology with Intact Protein Fractionation for Enhanced Proteome Characterization

The expanding use of surfactants for proteome sample preparations has prompted the need to systematically optimize the application and removal of these MS-deleterious agents prior to proteome measurements. Here we compare four detergent cleanup methods (trichloroacetic acid (TCA) precipitation, chloroform/methanol/water (CMW) extraction, a commercial detergent removal spin column method (DRS) and filter-aided sample preparation (FASP)) to provide efficiency benchmarks with respect to protein, peptide, and spectral identifications in each case. Our results show that for protein-limited samples, FASP outperforms the other three cleanup methods, while at high protein amounts, all the methods are comparable. This information was used to investigate and contrast molecular weight-based fractionated with unfractionated lysates from three increasingly complex samples (Escherichia coli K-12, a five microbial isolate mixture, and a natural microbial community groundwater sample), all of which were prepared with an SDS-FASP approach. The additional fractionation step enhanced the number of protein identifications by 8% to 25% over the unfractionated approach across the three samples.

表面活性剂在蛋白质组样品制备中的应用日益广泛，这使得我们亟需在蛋白质组质谱检测前，系统优化这类有损质谱分析的试剂的使用与去除流程。本研究对比了四种去污剂净化方法：三氯乙酸（TCA）沉淀法、氯仿/甲醇/水（CMW）萃取法、商用去污剂去除离心柱法（DRS）与过滤辅助样品制备法（FASP），旨在为各类样品的蛋白质、肽段及光谱鉴定提供效率基准。研究结果表明，在蛋白限量样品中，FASP的表现优于其余三种净化方法；而当蛋白投入量较高时，四种方法的性能基本相当。基于上述结果，我们针对三个复杂度逐步提升的样品（大肠杆菌K-12、五种微生物分离株混合体系与天然微生物群落地下水样品），探究并对比了基于分子量分级与未分级的细胞裂解液的检测效果，所有样品均采用SDS-FASP法进行制备。相较于未分级的处理方式，额外增加的分级步骤可使三个样品的蛋白质鉴定数量提升8%至25%。