title: Analysis of membrane proteins from human chronic myelogenous leukemia cells: comparison of extraction methods for multidimensional LC-MS/MS.
abstract: An important strategy for "shotgun proteomics" profiling involves solution proteolysis of proteins, followed by peptide separation using multidimensional liquid chromatography and automated sequencing by mass spectrometry (LC-MS/MS). Several protocols for extracting and handling membrane proteins for shotgun proteomics experiments have been reported, but few direct comparisons of different protocols have been reported. We compare four methods for preparing membrane proteins from human cells, using acid labile surfactants (ALS), urea, and mixed organic-aqueous solvents. These methods were compared with respect to their efficiency of protein solubilization and proteolysis, peptide and protein recovery, membrane protein enrichment, and peptide coverage of transmembrane proteins. Overall, approximately 50-60% of proteins recovered were membrane-associated, identified from Gene Ontology annotations and transmembrane prediction software. Samples extracted with ALS, extracted with urea followed by dilution, or extracted with urea followed by desalting yielded comparable peptide recoveries and sequence coverage of transmembrane proteins. In contrast, suboptimal proteolysis was observed with organic solvent. Urea extraction followed by desalting may be a particularly useful approach, as it is less costly than ALS and yields satisfactory protein denaturation and proteolysis under conditions that minimize reactivity with urea-derived cyanate. Spectral counting was used to compare datasets of proteins from membrane samples with those of soluble proteins from K562 cells, and to estimate fold differences in protein abundances. Proteins most highly abundant in the membrane samples showed enrichment of integral membrane protein identifications, consistent with their isolation by differential centrifugation.
authors.affiliation: Yale University School of Medicine, New Haven, Connecticut 06510, USA
authors.forename: Mariah C
authors.initials: MC
authors.lastname: Ruth
authors.forename: William M
authors.initials: WM
authors.lastname: Old
authors.forename: Michelle A
authors.initials: MA
authors.lastname: Emrick
authors.forename: Karen
authors.initials: K
authors.lastname: Meyer-Arendt
authors.forename: Lauren D
authors.initials: LD
authors.lastname: Aveline-Wolf
authors.forename: Kevin G
authors.initials: KG
authors.lastname: Pierce
authors.forename: Alex M
authors.initials: AM
authors.lastname: Mendoza
authors.forename: Joel R
authors.initials: JR
authors.lastname: Sevinsky
authors.forename: Micah
authors.initials: M
authors.lastname: Hamady
authors.forename: Robin D
authors.initials: RD
authors.lastname: Knight
authors.forename: Katheryn A
authors.initials: KA
authors.lastname: Resing
authors.forename: Natalie G
authors.initials: NG
authors.lastname: Ahn
mesh_terms: D002457:Cell Extracts / Q000032:analysis*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D020014:K562 Cells; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis*; D009363:Neoplasm Proteins / Q000032:analysis / Q000737:chemistry*; D053719:Tandem Mass Spectrometry
chemical_list: D002457:Cell Extracts; D008565:Membrane Proteins; D009363:Neoplasm Proteins