title: Comparative study of workflows optimized for in-gel, in-solution, and on-filter proteolysis in the analysis of plasma membrane proteins.
abstract: Proteomic studies of plasma membrane proteins are challenged by the limited solubility of these proteins and the limited activity of proteolytic enzymes in solubilizing agents such as SDS. In this work, we have evaluated three bottom-up workflows to obtain tryptic peptides from plasma membrane proteins solubilized with 2% SDS. The workflows are in-gel digestion, in-solution digestion, and on-filter digestion. The efficiencies of these strategies, optimized to employ different matrices for trypsin cleavage, were compared using a plasma membrane sample enriched from multiple myeloma cells using a nanoparticle pellicle. On the basis of the number of proteins identified, number of transmembrane proteins identified, hydrophobicity, and spectral count per protein, the workflow that uses in-gel digestion is the most advantageous approach for analysis of plasma membrane proteins.
authors.affiliation: Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, USA.
authors.forename: Waeowalee
authors.initials: W
authors.lastname: Choksawangkarn
authors.forename: Nathan
authors.initials: N
authors.lastname: Edwards
authors.forename: Yan
authors.initials: Y
authors.lastname: Wang
authors.forename: Peter
authors.initials: P
authors.lastname: Gutierrez
authors.forename: Catherine
authors.initials: C
authors.lastname: Fenselau
mesh_terms: D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000737:chemistry*; D019295:Computational Biology; D003902:Detergents / Q000737:chemistry; D005782:Gels / Q000737:chemistry; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry; D008970:Molecular Weight; D009101:Multiple Myeloma / Q000737:chemistry; D053758:Nanoparticles / Q000737:chemistry; D059748:Proteolysis; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry; D012995:Solubility; D012996:Solutions / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry
chemical_list: D003902:Detergents; D005782:Gels; D008565:Membrane Proteins; D020543:Proteome; D012996:Solutions; D012967:Sodium Dodecyl Sulfate; D014357:Trypsin