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
dataset
posted on 2006-03-03, 00:00 authored 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. AhnAn 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