Enrichment of integral membrane proteins for proteomic analysis using liquid chromatography-tandem mass spectrometry.
An increasing number of proteomic strategies rely on liquid chromatography-tandem mass spectrometry (LC-MS/MS) to detect and identify constituent peptides of enzymatically digested proteins obtained from various organisms and cell types. However, sample preparation methods for isolating membrane proteins typically involve the use of detergents and chaotropes that often interfere with chromatographic separation and/or electrospray ionization. To address this problem, a sample preparation method combining carbonate extraction, surfactant-free organic solvent-assisted solubilization, and proteolysis was developed and demonstrated to target the membrane subproteome of Deinococcus radiodurans. Out of 503 proteins identified, 135 were recognized as hydrophobic on the basis of their calculated hydropathy values (GRAVY index), corresponding to coverage of 15% of the predicted hydrophobic proteome. Using the PSORT algorithm, 53 of the proteins identified were classified as integral outer membrane proteins and 215 were classified as integral cytoplasmic membrane proteins. All identified integral cytoplasmic membrane proteins had from 1 to 16 mapped transmembrane domains (TMDs), and 65% of those containing four or more mapped TMDs were identified by at least one hydrophobic membrane spanning peptide. The extensive coverage of the membrane subproteome (24%) by identification of highly hydrophobic proteins containing multiple TMDs validates the efficacy of the described sample preparation technique to isolate and solubilize hydrophobic integral membrane proteins from complex protein mixtures.
Blonder
Josip
J
Environmental and Molecular Sciences Laboratory, Pacific Northwest National Laboratory, PO Box 999, MSIN K8-98, Richland, Washington 99352, USA.
Goshe
Michael B
MB
Moore
Ronald J
RJ
Pasa-Tolic
Ljiljana
L
Masselon
Christophe D
CD
Lipton
Mary S
MS
Smith
Richard D
RD
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D034301:Deinococcus / Q000737:chemistry / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000032:analysis*; D013696:Temperature
D001426:Bacterial Proteins; D008565:Membrane Proteins; D020543:Proteome