Integral membrane proteins: bottom-up, top-down and structural proteomics

Abstract

Integral membrane proteins and lipids constitute the bilayer membranes that surround cells and sub-cellular compartments, and modulate movements of molecules and information between them. Since membrane protein drug targets represent a disproportionately large segment of the proteome, technical developments need timely review. Areas covered: Publically available resources such as Pubmed were surveyed. Bottom-up proteomics analyses now allow efficient extraction and digestion such that membrane protein coverage is essentially complete, making up around one third of the proteome. However, this coverage relies upon hydrophilic loop regions while transmembrane domains are generally poorly covered in peptide-based strategies. Top-down mass spectrometry where the intact membrane protein is fragmented in the gas phase gives good coverage in transmembrane regions, and membrane fractions are yielding to high-throughput top-down proteomics. Exciting progress in native mass spectrometry of membrane protein complexes is providing insights into subunit stoichiometry and lipid binding, and cross-linking strategies are contributing critical in-vivo information. Expert commentary: It is clear from the literature that integral membrane proteins have yielded to advanced techniques in protein chemistry and mass spectrometry, with applications limited only by the imagination of investigators. Key advances toward translation to the clinic are emphasized.

Single-cell proteomics reveals changes in expression during hair-cell development

“…Moreover, levels of cytoskeletal proteins like actin and tubulin are similar to those seen in bulk proteomics experiments (Shin et al, 2013). By contrast, as in most shotgun mass spectrometry experiments, membrane proteins are underrepresented relative to their presence in the genome (Kar et al, 2017). Membrane proteins are generally expressed at low levels, however, which also contributes to our inability to see more than a few such proteins (e.g., ATP1A1, SLC17A8, and ATP2B2).…”

Cross-Linking Mass Spectrometry: An Emerging Technology for Interactomics and Structural Biology

“…In recent years, in vitro XL-MS studies of protein complexes have produced enormous amounts of molecular details for understanding their architectures, functions and regulation 11,12,19,27,28,36,40–42,155,164,165 . However, it is more biologically relevant to perform in vivo cross-linking experiments as they can provide a more authentic map of protein-protein interactions (PPIs) occurring in native cellular environments 30 .…”

“…In recent years, significant technological advancements in XL-MS studies have dramatically propelled the field forward, enabling a wide range of applications in vitro and in vivo , not only at the level of protein complexes but also at the proteome scale. Several recent reviews have focused on specific aspects of XL-MS including reagent design 39 and applications 12,19,27,28,40–42 . This review offers an overview of recent developments in XL-MS studies especially during the last three years.…”

Characterization of Contractile Proteins from Skeletal Muscle Using Gel-Based Top-Down Proteomics

“…Since peptide-based and bottom-up bioanalytical methods can often not properly distinguish among discrete proteoforms, such as individual isoforms or post-translationally modified protein variants, biochemical approaches using top-down proteomics with a focus on the mass spectrometric characterization of intact protein species can be advantageous [49,50,51]. Importantly, specific forms of top-down proteomics can provide data for accurate protein mass determination and detailed information on the identity of specific protein isoforms and their PTMs [52,53,54], making it an ideal investigative tool of modern protein biochemistry for determining the structure and function of macromolecular protein complexes [55,56,57]. In this review, the usage of top-down proteomics in the field of skeletal muscle biochemistry is outlined with special emphasis on gel-based techniques, especially 2DGE, for studying normal, adapting and aging muscles.…”