Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching.
2871 - 2882.
We describe the impact of advances in mass measurement accuracy, +/- 10 ppm (internally calibrated), on protein identification experiments. This capability was brought about by delayed extraction techniques used in conjunction with matrix-assisted laser desorption ionization (MALDI) on a reflectron time-of-flight (TOF) mass spectrometer. This work explores the advantage of using accurate mass measurement (and thus constraint on the possible elemental composition of components in a protein digest) in strategies for searching protein, gene, and EST databases that employ (a) mass values alone, (b) fragment-ion tagging derived from MS/MS spectra, and (c) de novo interpretation of MS/MS spectra. Significant improvement in the discriminating power of database searches has been found using only molecular weight values (i.e., measured mass) of > 10 peptide masses. When MALDI-TOF instruments are able to achieve the +/- 0.5-5 ppm mass accuracy necessary to distinguish peptide elemental compositions, it is possible to match homologous proteins having > 70% sequence identity to the protein being analyzed. The combination of a +/-10 ppm measured parent mass of a single tryptic peptide and the near-complete amino acid (AA) composition information from immonium ions generated by MS/MS is capable of tagging a peptide in a database because only a few sequence permutations > 11 AA's in length for an AA composition can ever be found in a proteome. De novo interpretation of peptide MS/MS spectra may be accomplished by altering our MS-Tag program to replace an entire database with calculation of only the sequence permutations possible from the accurate parent mass and immonium ion limited AA compositions. A hybrid strategy is employed using de novo MS/MS interpretation followed by text-based sequence similarity searching of a database
|Title:||Role of accurate mass measurement (+/- 10 ppm) in protein identification strategies employing MS or MS/MS and database searching|
|Additional information:||UI - 99352973 LA - Eng RN - EC 22.214.171.124 (Protein Disulfide-Isomerase) RN - 0 (Apolipoprotein A-I) PT - JOURNAL ARTICLE ID -RR01614/RR/NCRR ID - RR08282/RR/NCRR ID - HD30367/HD/NICHD ID - + DA - 19991008 IS - 0003-2700 CY - UNITED STATES JC - 4NR AA -Author EM - 199912|
|Keywords:||Proteins, Disulfides, United States, amino acid, Animal, Apolipoprotein A I, Apolipoprotein A-I, California, Cattle, chemistry, database, Databases, Factual, GENE, Methods, Molecular Weight, Protein Disulfide Isomerase, Protein Disulfide-Isomerase, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Support, Non-U.S.Gov't, Support, U.S.Gov't, P.H.S., peptides, Acids, ANS|
|UCL classification:||UCL > School of Life and Medical Sciences > Faculty of Life Sciences > Biosciences (Division of)|
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