Proteomics on full-length membrane proteins using mass spectrometry.
A general technique has been developed that allows rapid mass spectrometric analysis of full-length membrane proteins [Whitelegge, J. P., le Coutre, J., et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 10695-10698]. Using in-line HPLC electrospray ionization mass spectrometry (LC-MS), different native and recombinant bacterial membrane proteins of up to 61 kDa are characterized. Mass spectrometric data of four entirely different membrane proteins from three bacterial organisms, two transporters, a channel, and a porin protein are presented. In addition to determination of the molecular mass with an accuracy of +/-0.01%, the technique monitors alkylation or oxidation of single Cys residues and errors in deduced amino acid sequences. Finally, using in-line LC-MS, unknown proteins can be identified from solubilized Escherichia coli membranes without prior purification.
le Coutre
J
J
Howard Hughes Medical Institute, Departments of Physiology and of Microbiology and Molecular Genetics, Molecular Biology Institute, University of California, Los Angeles 90095, USA. lecoutre@hhmi.ucla.edu
Whitelegge
J P
JP
Gross
A
A
Turk
E
E
Wright
E M
EM
Kaback
H R
HR
Faull
K F
KF
D000478:Alkylation; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000032:analysis / Q000737:chemistry; D001426:Bacterial Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry / Q000166:cytology; D029968:Escherichia coli Proteins; D008024:Ligands; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D008970:Molecular Weight; D009004:Monosaccharide Transport Proteins / Q000032:analysis / Q000737:chemistry; D009154:Mutation / Q000235:genetics; D015221:Potassium Channels / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020543:Proteome; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D012680:Sensitivity and Specificity; D051247:Sodium-Glucose Transport Proteins; D013050:Spectrometry, Fluorescence; D013113:Spin Labels; D013302:Streptomyces / Q000737:chemistry; D027981:Symporters; D014736:Vibrio parahaemolyticus / Q000737:chemistry
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008024:Ligands; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D015221:Potassium Channels; D020543:Proteome; D011993:Recombinant Fusion Proteins; D051247:Sodium-Glucose Transport Proteins; D013113:Spin Labels; D027981:Symporters; C107790:prokaryotic potassium channel; C099811:sodium glucose symporter, Vibrio parahaemolyticus; C078167:OMPA outer membrane proteins; C021499:lactose permease; D003545:Cysteine