Mass Spectrometry Applied to Bottom-Up Proteomics: Entering the High-Throughput Era for Hypothesis Testing

Abstract

Proteins constitute a key class of molecular components that perform essential biochemical reactions in living cells. Whether the aim is to extensively characterize a given protein or to perform high-throughput qualitative and quantitative analysis of the proteome content of a sample, liquid chromatography coupled to tandem mass spectrometry has become the technology of choice. In this review, we summarize the current state of mass spectrometry applied to bottom-up proteomics, the approach that focuses on analyzing peptides obtained from proteolytic digestion of proteins. With the recent advances in instrumentation and methodology, we show that the field is moving away from providing qualitative identification of long lists of proteins to delivering highly consistent and accurate quantification values for large numbers of proteins across large numbers of samples. We believe that this shift will have a profound impact for the field of proteomics and life science research in general.

Absolute Quantification of Major Photosynthetic Protein Complexes in Chlamydomonas reinhardtii Using Quantification Concatamers (QconCATs)

“…Mass spectrometry-based shotgun or discovery proteomics aims at identifying a large number of cellular proteins and allows to quantify changes in the abundance of a subset of these proteins e.g., upon changing environmental conditions (Gillet et al, 2016). However, because standards are missing, this approach does not allow determining the absolute abundance of a protein within a cell.…”

Systems proteomics approaches to study bacterial pathogens: application to Mycobacterium tuberculosis

“…Bottom-up proteomics, currently the dominant paradigm for most proteomics applications, relies on the protein inference logic where peptides originating from proteins following digestion by a protease are identified and then mapped back to proteins for both identification and quantification (Fig. 1a) [30]. After initially determining the mass to charge ratio of molecular ions of peptides (MS1), they are fragmented in a collision cell of a mass spectrometer and the masses and intensities of fragment ions (MS2) are determined.…”

Precise Temporal Profiling of Signaling Complexes in Primary Cells Using SWATH Mass Spectrometry

“…(Upper right) Overview of MS analysis. Affinity-purified samples were acquired in both DIA/SWATH and DDA/shotgun modes (for a detailed description, see Aebersold and Mann, 2016, Gillet et al., 2016). Data were used to build a high-confidence GRB2 interactor-specific assay library.…”

“…Given the central role played by GRB2 in signal initiation and diversification, knowledge of the composition and dynamics of the signalosomes that form around it in distinct signaling cascades and cell types is, thus, key to understanding the scope of its actual functions. We, therefore, selected GRB2 as a model for evaluating whether SWATH (sequential window acquisition of all theoretical fragment ion spectra)-MS (Gillet et al., 2012, Gillet et al., 2016) could enable rapid, reliable, and accurate quantitative analysis of protein interaction dynamics in two types of primary cells that were extemporaneously isolated from mouse (Figure 1). …”