High-Mass-Measurement Accuracy and 100 Sequence Coverage of Enzymatically Digested Bovine Serum Albumin from an ESI-FTICR Mass Spectrum

Abstract

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.

Automated de novo sequencing of proteins by tandem high-resolution mass spectrometry

“…Because of lower resolution, the nozzle-skimmer CAD spectra for ubiquitin and melittin could be calibrated only to Ϸ15 ppm accuracy. Sub-ppm accuracy has been demonstrated for instruments with magnetic fields higher than the 6 T used here (39,40,45); the algorithm will be demonstrated after first correcting the ubiquitin data to 1 ppm accuracy and the cytochrome c and melittin data to 15 ppm where necessary. The actual data then will be used for ubiquitin.…”

“…Here we examine the opposite problem of the de novo conversion of these mass values, despite consideration of many more unassignable values, into an accurate sequence with a fully automated computer program. The importance of high mass accuracy is shown by using data of Fourier transform MS (FTMS) (3,(37)(38)(39)(40) to predict sequences, and their reliability, for melittin (2.8 kDa), ubiquitin (8.6 kDa), and cytochrome c (12.4 kDa), all noncyclic proteins (except for the heme in cytochrome c) with known termini and no posttranslational modifications.…”

Advances in proteomics data analysis and display using an accurate mass and time tag approach

“…FTICR-MS is attractive for proteomics because it simultaneously provides high sensitivity, high MMA, and a wide dynamic range (Belov, et al, 2000b, Bruce, et al, 1999, Marshall, et al, 1998. In some regards, the high sensitivity analyses afforded by FTICR-MS are surprising since 30 to 50 charges typically must be trapped in an FTICR cell to provide a S/N >3, in contrast to conventional ion trap and time of flight mass spectrometers that can obtain a measurable signal from only a single ion.…”

Proteomics in pathology research

“…FTICR instruments are very powerful and provide exquisite sensitivity, high mass accuracy and high resolution; which are especially desirable properties in the analysis of complex protein mixtures. The FTICR instruments are especially compatible with ESI, 30 but may also be used with MALDI as an ionization source. 31 Despite these advantages, FTICR instruments require considerable expertise for successful operation, thus limiting somewhat their widespread utilization for routine proteomics studies.…”