CPSA Digest 2003

Day 1

Vendor Session - Proteomics Technologies & Applications

Improved Bottom-Up Protein Identification Using A New Two-Dimensional Ion Trap Mass Spectrometer

Most current bottom-up proteomics experiments rely on high-throughput correlation analysis between uninterpreted MS/MS spectra and database entries in order to obtain highly specific, high-confidence peptide sequences to give unambiguous protein identifications. Under this scheme, the confidence level for the identification of a protein, whether pure or as part of a complex mixture, is a function of several factors including (a) the number of discrete, 'signature' peptides sequenced (i.e., protein coverage) and the size of the protein itself, (b) the quality of the spectrum-database correlations and their statistical distributions, (c) the total number of spectra analyzed, and (d) the size and redundancy of the database. We are now reporting on the significant benefits to factors (a)-(c) realized through the use of a two-dimensional ion trap mass spectrometer. Our preliminary results indicate that the two-dimensional trap produces both higher protein coverage and better quality protein identifications. The gain in coverage occurs at two levels: (a) a multifold increase in the total number of proteins identified per run and, (b) a higher number of discrete peptides sequenced per protein per run. For instance, in one of the experiments the two-dimensional trap yielded 191 high-confidence protein IDs versus 66 for the same sample using the three-dimensional trap. Furthermore, the same set of ten, top-scoring proteins were observed using both mass spectrometers; however, in each of the ten cases, protein scores were several fold higher for IDs made with the two-dimensional trap vs. the three-dimensional one. We've interpret these gains to be the direct result of faster rates of data acquisition, the enhanced quality of single MS/MS scans and improved detectability of low-level and/or poorly ionizable peptides.

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