Combine and Conquer: Surfactants, Solvents, and Chaotropes for Robust Mass Spectrometry Based Analyses of Membrane Proteins

Abstract

Mass spectrometry (MS) based proteomic technologies enable the identification and quantification of membrane proteins as well as their post-translational modifications. A prerequisite for their quantitative and reliable MS-based bottom-up analysis is the efficient digestion into peptides by proteases, though digestion of membrane proteins is typically challenging due to their inherent properties such as hydrophobicity. Here, we investigated the effect of eight commercially available MS-compatible surfactants, two organic solvents, and two chaotropes on the enzymatic digestion efficiency of membrane protein-enriched complex mixtures in a multiphase study using a gelfree approach. Multiple parameters, including the number of peptides and proteins identified, total protein sequence coverage, and digestion specificity were used to evaluate transmembrane protein digestion performance. A new open-source software tool was developed to allow for the specific assessment of transmembrane domain sequence coverage. Results demonstrate that while Progenta anionic surfactants outperform other surfactants when tested alone, combinations of guanidine and acetonitrile improve performance of all surfactants to near similar levels as well as enhance trypsin specificity to >90%, which has critical implications for future quantitative and qualitative proteomic studies.

Comparison of Different Sample Preparation Protocols Reveals Lysis Buffer-Specific Extraction Biases in Gram-Negative Bacteria and Human Cells

“…This observation is in line with recent studies that showed a detergent-specific increase in the recovery of peptides mapping to membrane proteins derived from membrane fractions. 32,53 We were particularly interested in finding terms related to different membrane substructures in bacteria to infer whether there are consistent or different effects between the buffer systems between the species. Compared to human cells, which have a single phospholipid bilayer for compartmentation, the bacterial cell envelope contains an outer membrane that is separated from the inner membrane by the peptidoglycan cell wall that spans through the periplasmic space.…”

Less is More: Membrane Protein Digestion Beyond Urea–Trypsin Solution for Next-level Proteomics

“…Amphi-polymers (amphipols) can support protein extraction and digestion, but most aggregate below pH 3-4 and require removal pre-HPLC by precipitation and centrifugation (45), interrupting automatable workflow. Several recent studies compared numerous detergents for digestion, but most remained confined to industrial poly-disperse and ionic detergents that deactivated proteins and required removal; other attempts combined harsh detergents, organic solvents, high urea, high pressure, and high temperature (46).…”

Sample preparation strategies for improving the identification of membrane proteins by mass spectrometry

“…However, although some of the methods provided good efficiencies for certain studies, Waas et al recently stated that the real deal would be the combination of surfactants, solvents, and chaotropes for robust MS-based identification of membrane proteins [37]. These authors tested a wide variety of organic solvents, chaotropic agents, surfactants, and their combinations.…”

“…These authors tested a wide variety of organic solvents, chaotropic agents, surfactants, and their combinations. Their data demonstrated that the inclusion of guanidine and acetonitrile in addition to surfactants maximized the overall digestion specificity, the total number of peptides, the average sequence coverage among all proteins, and the number of identified transmembrane domains [37].…”