pr050313z_si_002.xls (2.64 MB)

Analysis of Membrane Proteins from Human Chronic Myelogenous Leukemia Cells:  Comparison of Extraction Methods for Multidimensional LC−MS/MS

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posted on 03.03.2006, 00:00 by Mariah C. Ruth, William M. Old, Michelle A. Emrick, Karen Meyer-Arendt, Lauren D. Aveline-Wolf, Kevin G. Pierce, Alex M. Mendoza, Joel R. Sevinsky, Micah Hamady, Robin D. Knight, Katheryn A. Resing, Natalie G. Ahn
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, ∼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. Keywords: mass spectrometry • leukemia • membrane proteins • urea • acid labile surfactant