Proteome analysis of intact proteins in complex mixtures.
Analysis of intact protein mixtures by electrospray ionization mass spectrometry requires the resolution of a complex, overlapping set of multiply charged envelopes. To ascertain the ability of a moderate resolution mass spectrometer to resolve such mixtures, we have analyzed the soluble proteins of adult chick skeletal muscle. This is a highly specialized tissue showing a marked bias in expression of glycolytic enzymes in the soluble fraction. SDS-PAGE-resolved proteins were first identified by a combination of matrix-assisted laser desorption ionization time-of-flight (TOF) and electrospray ionization tandem mass spectrometry. Then the mixture of intact proteins was introduced into the electrospray source of a Q-TOF mass spectrometer either by direct infusion or via a C4 desalting trap. In both instances, the complex pattern of peaks could be resolved into true masses, and these masses could in many instances be reconciled with the masses predicted from the known protein sequences when qualified by expected co- and post-translational modifications. These included loss of the N-terminal initiator methionine residue and N-terminal acetylation. The ability to resolve such a complex mixture of proteins with a routine instrument is of considerable value in analyses of protein expression and in the confirmation of post-translational changes in mature proteins.
Hayter
Julia R
JR
Department of Veterinary Preclinical Sciences, University of Liverpool, Crown Street Liverpool L69 7ZJ, United Kingdom.
Robertson
Duncan H L
DH
Gaskell
Simon J
SJ
Beynon
Robert J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D017124:Conserved Sequence; D024981:Glycogen Phosphorylase / Q000737:chemistry; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000032:analysis* / Q000737:chemistry; D018482:Muscle, Skeletal / Q000737:chemistry; D010736:Phosphoglycerate Mutase / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D021241:Spectrometry, Mass, Electrospray Ionization
D009124:Muscle Proteins; D020543:Proteome; D024981:Glycogen Phosphorylase; D010736:Phosphoglycerate Mutase