Chapter 4: Quantitative Shotgun Proteomics with Data-Independent Acquisition and Traveling Wave Ion Mobility Spectrometry: A Versatile Tool in the Life Sciences

摘要

Data-independent acquisition (DIA) implemented in a method called MS~E can be performed in a massively parallel, time-based schedule rather than by sampling masses sequentially in shotgun proteomics. In MS~E alternating low and high energy spectra are collected across the full mass range. This approach has been very successful and stimulated the development of variants modeled after the MS~E protocol, but over narrower mass ranges. The massively parallel MS~E and other DIA methodologies have enabled effective label-free quantitation methods that have been applied to a wide variety of samples including affinity pulldowns and studies of cells, tissues, and clinical samples. Another complementary technology matches accurate mass and retention times of precursor ions across multiple chromato-graphic runs. This further enhances the impact of MS~E in counteracting the stochastic nature of mass spectrometry as applied in proteomics. Otherwise significant amounts of data in typical large-scale protein profiling experiments are missing. A variety of software packages perform this function similar in concept to matching of accurate mass tags. Another enhancement of this method involves a variation of MS~E coupled with traveling wave ion mobility spectrometry to provide separations of peptides based on cross-sectional area and shape in addition to mass/charge (m/z) ratio. Such a two-dimensional separation in the gas phase considerably increases protein coverage as well as typically a doubling of the number of proteins detected.