title: 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.
authors.affiliation: Milwaukee School of Engineering , Milwaukee, Wisconsin 53202, United States.
authors.forename: Matthew
authors.initials: M
authors.lastname: Waas
authors.forename: Subarna
authors.initials: S
authors.lastname: Bhattacharya
authors.forename: Sandra
authors.initials: S
authors.lastname: Chuppa
authors.forename: Xiaogang
authors.initials: X
authors.lastname: Wu
authors.forename: Davin R
authors.initials: DR
authors.lastname: Jensen
authors.forename: Ulrich
authors.initials: U
authors.lastname: Omasits
authors.forename: Bernd
authors.initials: B
authors.lastname: Wollscheid
authors.forename: Brian F
authors.initials: BF
authors.lastname: Volkman
authors.forename: Kathleen R
authors.initials: KR
authors.lastname: Noon
authors.forename: Rebekah L
authors.initials: RL
authors.lastname: Gundry
mesh_terms: D000097:Acetonitriles / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D019791:Guanidine / Q000494:pharmacology*; D006860:Hydrogen Bonding; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*; D012995:Solubility; D012997:Solvents / Q000494:pharmacology*; D013379:Substrate Specificity; D013501:Surface-Active Agents / Q000494:pharmacology*; D014357:Trypsin / Q000378:metabolism
chemical_list: D000097:Acetonitriles; D008565:Membrane Proteins; D012997:Solvents; D013501:Surface-Active Agents; D014357:Trypsin; D019791:Guanidine; C032159:acetonitrile