Code Developments to Improve the Efficiency of Automated MS/MS Spectra Interpretation

Abstract

We report the results of our work to facilitate protein identification using tandem mass spectra and protein sequence databases. We describe a parallel version of SEQUEST (SEQUEST-PVM) that is tolerant toward arithmetic exceptions. The changes we report effectively separate search processes on slave nodes from each other. Therefore, if one of the slave nodes drops out of the cluster due to an error, the rest of the cluster will carry the search process to the end. SEQUEST has been widely used for protein identifications. The modifications made to the code improve its stability and effectiveness in a high-throughput production environment. We evaluate the overhead associated with the parallelization of SEQUEST. A prior version of software to preprocess LC/MS/MS data attempted to differentiate the charge states of ions. Singly charged ions can be accurately identified, but the software was unable to reliably differentiate tandem mass spectra of +2 and +3 charge states. We have designed and implemented a computational approach to narrow charge states of precursor ions from nominal resolution ion-trap tandem mass spectra. The preprocessing code, 2to3, determines the charge state of the precursor ion using its mass-to-charge ratio (m/z) and fragment ions contained in the tandem mass spectrum. For each possible charge state the program calculates the expected fragment ions that account for precursor ion m/z values. If any one of the numbers is less than an empirically determined threshold value then the spectrum corresponding to that charge state is removed. If both numbers are higher than the threshold value then +2 and +3 copies of the spectrum are kept. We present the comparison of results from protein identification experiments with and without using 2 to 3. It is shown that by determining the charge state and eliminating poor quality spectra 2to3 decreases the number of spectral files to be searched without affecting the search results. The decrease reduces computer requirements and researcher efforts for analysis of the results.

Large-scale database searching using tandem mass spectra: Looking up the answer in the back of the book

“…On low-resolution instruments, where these methods are ineffective, MS/MS of peptides with charge states higher than 1 are typically searched twice, once calculating a molecular weight assuming that the charge state is +2 and the second time assuming the charge state is +3. Based on observations by Dancik and colleagues 33 that complementary fragment ions (N-terminal and Cterminal fragment ions) can be used to improve molecular weight calculations, our group and others used a variation of this approach to determine peptide ion charge state 34,35 . In good-quality tandem mass spectra there are numerous complimentary ions; thus, if the precursor ion is assumed to be doubly charged, the complementary ions present in the spectrum should sum to this molecular weight.…”

“…Peptide ions selected for MS/MS at very low signal levels will produce spectra with poor signal-to-noise ratios and often incomplete sequence ions. Methods have been devised to sort the good from the bad spectra 34,37,38 . Recently, Bern et al presented two different algorithms for assessing spectral quality prior to a database search: a binary classifier, which predicts whether or not the search engine will be able to make an identification, and a statistical regression, which predicts a more universal quality metric, independent of the database search program 39 .…”

Identification of 2D-gel proteins: A comparison of MALDI/TOF peptide mass mapping to μ LC-ESI tandem mass spectrometry

“…A three-min dynamic exclusion was also applied to min-imize acquisition of redundant MS/MS data. Tandem mass spectra were directly submitted to SEQUEST-PVM [36,37] searches for protein identification. Approximately eight samples were analyzed per day by manual LC-MS/MS from sample loading onto a column to column washing between sample runs by 15 min short gradient to SEQUEST-PVM database searches.…”

Development and validation of a spectral library searching method for peptide identification from MS/MS

“…This last step of sequence database searching, that is, the inference of the peptide sequence from the MS/MS spectra of fragmented peptide ions, is a challenging, error-prone, and computationally expensive exercise, and has been a subject of intense research since the early days of proteomics [7][8][9][10]. Several popular computational tools developed for that purpose have emerged over the years, each employing different algorithms and heuristics to achieve an acceptable balance of sensitivity and accuracy [11][12][13][14][15][16].…”