ABSTRACT
The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC-MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation (source code is available from http://homepages.uc.edu/~wang2x7/Research.htm ). From these assessments, we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61 to 93% of the time. When comparing across different instruments and quantitative technologies, using multiple replicates, differential genes were reproduced by other data sets from 67 to 99% of the time. Projecting gene differences to biological pathways and networks increased the degree of similarity. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation.
Subject(s)
Heterografts/chemistry , Proteomics/methods , Proteomics/standards , Breast Neoplasms/chemistry , Breast Neoplasms/metabolism , Chromatography, Liquid , Data Interpretation, Statistical , Female , Gene Expression Profiling/methods , Humans , Metabolic Networks and Pathways , Observer Variation , Proteome , Proteomics/instrumentation , Quality Control , Reproducibility of Results , Tandem Mass Spectrometry/standardsABSTRACT
After raw data have been captured by mass spectrometers in biological LC-MS/MS experiments, they must be converted from vendor-specific binary files to open-format files for manipulation by most software. This protocol details the use of ProteoWizard software for this conversion, taking format features, coding options, and vendor particularities into account. This protocol will aid researchers in preparing their data for analysis by database search engines and other bioinformatics tools.
Subject(s)
Databases, Protein , Software , Tandem Mass Spectrometry , User-Computer InterfaceABSTRACT
Frequently, proteomic LC-MS/MS data may contain sets of modifications that evade identification during standard database search. For many laboratories, the standard technique to seek posttranslational modifications (PTMs) adds a short list of specified mass shifts to database search configuration. This technique provides information for only the specified PTMs, takes substantial time to run, and drives false discoveries upward through an exponential expansion of search space. This protocol describes a more structured approach to blind PTM discovery through reducing protein lists, targeting attention to a data-driven list of mass shifts, and seeking the resulting short list of modifications through targeted search.
Subject(s)
Databases, Protein , Protein Processing, Post-Translational , Proteins/analysis , Proteomics/methods , Algorithms , Amino Acid Sequence , Chromatography, Liquid , Humans , Mass Spectrometry , Proteins/chemistry , SoftwareABSTRACT
In shotgun proteomics, database search algorithms rely on fragmentation models to predict fragment ions that should be observed for a given peptide sequence. The most widely used strategy (Naive model) is oversimplified, cleaving all peptide bonds with equal probability to produce fragments of all charges below that of the precursor ion. More accurate models, based on fragmentation simulation, are too computationally intensive for on-the-fly use in database search algorithms. We have created an ordinal-regression-based model called Basophile that takes fragment size and basic residue distribution into account when determining the charge retention during CID/higher-energy collision induced dissociation (HCD) of charged peptides. This model improves the accuracy of predictions by reducing the number of unnecessary fragments that are routinely predicted for highly-charged precursors. Basophile increased the identification rates by 26% (on average) over the Naive model, when analyzing triply-charged precursors from ion trap data. Basophile achieves simplicity and speed by solving the prediction problem with an ordinal regression equation, which can be incorporated into any database search software for shotgun proteomic identification.
Subject(s)
Algorithms , Databases, Protein , Models, Chemical , Peptide Fragments/chemistry , Peptides/analysis , Amino Acid Sequence , Animals , Electrochemistry , Humans , Information Storage and Retrieval/methods , Peptides/chemistry , Protein Precursors/chemistry , Software , Tandem Mass Spectrometry/methodsABSTRACT
The identification of peptides and proteins by LC-MS/MS requires the use of bioinformatics. Tools developed in the Tabb Laboratory contribute significant flexibility and discrimination to this process. The Bumbershoot tools (MyriMatch, DirecTag, TagRecon, and Pepitome) enable the identification of peptides represented by MS/MS scans. All of these tools can work directly from instrument capture files of multiple vendors, such as Thermo RAW format, or from standard XML-based formats, such as mzML or mzXML. Peptide identifications are written to mzIdentML or pepXML format. Protein assembly is handled by the IDPicker algorithm. Raw identifications are filtered to a confident set by use of the target-decoy strategy. IDPicker arranges large sets of input files into a hierarchy for reporting, and the software applies a parsimony algorithm to report the smallest possible number of proteins to explain the observed peptides. This protocol details the use of these tools for new users.