Identification of membrane proteins by tandem mass spectrometry of protein ions.
The most common way of identifying proteins in proteomic analyses is to use short segments of sequence ("tags") determined by mass spectrometric analysis of proteolytic fragments. The approach is effective with globular proteins and with membrane proteins with significant polar segments between membrane-spanning alpha-helices, but it is ineffective with other hydrophobic proteins where protease cleavage sites are either infrequent or absent. By developing methods to purify hydrophobic proteins in organic solvents and by fragmenting ions of these proteins by collision induced dissociation with argon, we have shown that partial sequences of many membrane proteins can be deduced easily by manual inspection. The spectra from small proteolipids (1-4 transmembrane alpha-helices) are dominated usually by fragment ions arising from internal amide cleavages, from which internal sequences can be obtained, whereas the spectra from larger membrane proteins (5-18 transmembrane alpha-helices) often contain fragment ions from N- and/or C-terminal parts yielding sequences in those regions. With these techniques, we have, for example, identified an abundant protein of unknown function from inner membranes of mitochondria that to our knowledge has escaped detection in proteomic studies, and we have produced sequences from 10 of 13 proteins encoded in mitochondrial DNA. They include the ND6 subunit of complex I, the last of its 45 subunits to be analyzed. The procedures have the potential to be developed further, for example by using newly introduced methods for protein ion dissociation to induce fragmentation of internal regions of large membrane proteins, which may remain partially folded in the gas phase.
Carroll
Joe
J
Medical Research Council Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 0XY, United Kingdom.
Altman
Matthew C
MC
Fearnley
Ian M
IM
Walker
John E
JE
D000595:Amino Acid Sequence; D000818:Animals; D004285:Dogs; D004926:Escherichia coli; D007477:Ions / Q000737:chemistry; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D053719:Tandem Mass Spectrometry
D007477:Ions; D008565:Membrane Proteins