Integrated proteomics sample preparation and fractionation: Method development and applications

Mass spectrometry (MS)-based proteomics has become a powerful and routine approach for proteome profiling in the post-genome era. Standard shotgun proteomic workflow contains four parts, including sample preparation, peptide separation, MS analysis and data analysis. As compared with advanced and commercialized technology development for peptide separation, MS analysis and data analysis parts, proteomics sample preparation has lagged behind largely due to the multi-step operation and reaction. However, these less optimized steps have becoming the major bottleneck for many cutting-edge biomedical applications, especially with limited starting materials. In the past five years, we have seen exciting technology development for integrated proteomics sample preparation and peptide fractionation which significantly increase the sensitivity, robustness and throughput of the whole proteomics workflow for in vivo proteomics and clinical proteomics. In this review, we outline the recent progress in integrated sample preparation and fractionation methodology from a technical perspective. In details, integrated sample preparation based on in-solution, on-bead and immobilized-enzymatic digestion as well as peptide fractionation based on reversed-phase, ion-exchange and two-dimensional fractionation in the same device are presented. Key biomedical applications on different sample types including body fluids, tissues and cells are discussed with highlight for some of the cutting-edge applications such as high-throughput plasma proteome profiling, spatial proteomics, and single cell proteomics.