Complete Sequence Coverage Research Articles

High-mass-measurement accuracy and 100% sequence coverage of enzymatically digested bovine serum albumin from an ESI-FTICR mass spectrum.

The application of Fourier transform ion cyclotron resonance (FTICR) mass spectrometry to the analysis of polypeptide mixtures resulting from proteolytic digestion is described. A new 11.5-T FTICR mass spectrometer has been applied for the analysis of tryptic digestion mixtures of the protein bovine serum albumin (BSA). The improved cyclotron frequency stability and reduced frequency shifts observed over a wide range of trapped ion population sizes provide the ability to signal average spectra without degrading mass measurement accuracy, requiring internal calibration or advanced data processing schemes to compensate for variations in ion cyclotron signals brought about by different population sizes. A total of 100 spectra were signal-averaged leading to the observation of a total of 123 isotope distributions with a signal-to-noise ratio greater than 3:1. From those distributions, 86 can be ascribed to tryptic fragments of BSA on the basis of mass measurement errors of 10 ppm or less. Of these, 71 were within 2 ppm error limits corresponding to complete amino acid sequence coverage and an average error of 0.77 ppm. These results indicate that high-accuracy measurements are feasible for a large number of species detected simultaneously without the necessity for internal calibration and indicate the potential of such measurements, when combined with chromatographic separations, for facilitating more rapid identification of large numbers of proteins.

Phosphopeptide analysis by matrix-assisted laser desorption time-of-flight mass spectrometry.

In this paper we present methods for identifying and sequencing phosphopeptides in simple mixtures, such as HPLC fractions, at the subpicomole level by (+) ion matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-MS). Data are presented which indicate that when a reflectron time-of-flight mass spectrometer is used, MALDI can distinguish tyrosine phosphorylation from serine and threonine phosphorylation for peptides containing a single phosphate group. Phosphopeptides are identified in the (+) ion MALDI reflector spectrum by the presence of [MH-H3PO4]+ and [MH-HPO3]+ fragment ions formed by metastable decomposition. An abundant [MH-H3PO4]+ ion, accompanied by a weaker [MH-HPO3]+ ion indicates that the peptide is most likely phosphorylated on serine or threonine. In contrast, phosphotyrosine-containing peptides generally exhibit [MH-HPO3]+ fragment ions and little, if any [MH-H3PO4]+. Ambiguities do arise, most often with phosphopeptides that contain residues which readily lose water (such as unmodified serine), but these can often be resolved by recording a complete metastable fragment ion (postsource decay) spectrum. Postsource decay is shown here to be a viable technique for sequencing phosphopeptides. It can be used to distinguish between serine/ threonine and tyrosine phosphorylation and in many cases can be used to determine the exact site of phosphorylation in a peptide sequence. Nearly complete sequence coverage and phosphorylation site mapping is generally possible using approximately 300 fmol of peptide.