Improved detection limits for electrospray ionization on a magnetic sector mass spectrometer by using an array detector.

Array detection was compared with point detection for solutions of hen egg-white lysozyme, equine myoglobin, and ubiquitin analyzed by electrospray ionization with a magnetic sector mass spectrometer. The detection limits for samples analyzed by using the array detector system were at least 10 times lower than could be achieved by using a point detector on the same mass spectrometer. The minimum detectable quantity of protein corresponded to a signal-to-background ratio of approximately 2∶1 for a 500 amol/µL solution of hen egg-white lysozyme. However, the ultimate practical sample concentrations appeared to be in the 10-100 fmol/µL range for the analysis of dilute solutions of relatively pure proteins or simple mixtures.

Mass spectrometric identification of modifications to human serum albumin treated with hydrogen peroxide.

Oxidized amino acid residues in human serum albumin exposed to hydrogen peroxide have been identified in tryptic peptides using liquid secondary ion mass spectrometry. Sites of oxidation identified include Cys34, Met123, Met298, Met446, and Met548. The extent of oxidation varied with location in the protein sequence, suggesting a relationship between oxidation and protein three-dimensional structure. The data presented here for human serum albumin demonstrate the utility of mass spectrometry in studying protein alterations. This type of information may be helpful in assessing the ability of proteins to act as antioxidants in biological systems which are subject to oxidant stress as in cases of inflammation and in the aging process.

Analysis of G protein gamma subunit heterogeneity using mass spectrometry.

The diversity of the gamma subunits in bovine brain G protein preparations was investigated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Analysis of these G protein mixtures revealed at least four gamma subunit masses by the following four criteria. 1) The measured masses were in the same mass range as the predicted molecular weights of gamma isoforms. 2) The masses were reproducible between the same or different preparations of G proteins. 3) The masses were independent of the matrix used for MALDI analysis. 4) The masses comigrated with the gamma subunit, as part of the heterotrimer, the beta gamma dimer, or the separated gamma subunit. These measured masses were compared with those calculated from cDNA sequences of known bovine brain gamma isoforms with the addition of plausible post-translational modifications. The mass of each spectral peak was consistent with the calculated mass for only one of four known bovine brain gamma subunit isoforms, but the data suggest modifications of the gamma subunits in addition to those already known or suspected at their carboxyl termini. Besides these four major masses, several additional, less resolved spectral peaks were observed whose measured masses did not correlate with any known gamma subunit or plausible modification. MALDI mass spectrometry promises to be a powerful technique for the analysis of the diversity of the gamma subunit in G proteins and for the characterization of their post-translational modifications.