Abstract
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.
Highlights
Analysis of intact protein mixtures by electrospray ionization mass spectrometry requires the resolution of a complex, overlapping set of multiply charged envelopes
Proteolysis by trypsin, for example, cleaves the protein into limit peptides typically of ϳ10 –20 amino acids long. The masses of such peptides are readily matched by the high mass accuracy obtained by the mass analyzers in common use for peptide mass fingerprinting, notably a time-of-flight (TOF)1 mass analyzer, which can achieve a mass accuracy of 10 ppm and a resolution in excess of 10,000 full-width half-maximum
It has been proposed that the mass of each protein in a proteome can, at an appropriate level of mass accuracy and resolution, be considered to be virtually unique in the data set, which raises the possibility of direct analysis of an intact protein (6)
Summary
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. The generation of the set of limit peptides introduces additional steps that can result in sample losses and that diminishes throughput To circumvent these stages, it has been proposed that the mass of each protein in a proteome can, at an appropriate level of mass accuracy and resolution, be considered to be virtually unique in the data set, which raises the possibility of direct analysis of an intact protein (6). We wished to assess the extent to which direct mass measurement of the proteins in this sample could be of value in their identification using a readily available instrument (QTOF-micro, Micromass, Manchester, UK)
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