PatternLab V Handles Multiplex Spectra in Shotgun Proteomic Searches and Increases Identification.

Complex protein mixtures typically generate many tandem mass spectra produced by different peptides coisolated in the gas phase. Widely adopted proteomic data analysis environments usually fail to identify most of these spectra, succeeding at best in identifying only one of the multiple cofragmenting peptides. We present PatternLab V (PLV), an updated version of PatternLab that integrates the YADA 3 deconvolution algorithm to handle such cases efficiently. In general, we expect an increase of 10% in spectral identifications when dealing with complex proteomic samples. PLV is freely available at http://patternlabforproteomics.org.

Increasing confidence in proteomic spectral deconvolution through mass defect.

MOTIVATION: Confident deconvolution of proteomic spectra is critical for several applications such as de novo sequencing, cross-linking mass spectrometry and handling chimeric mass spectra. RESULTS: In general, all deconvolution algorithms may eventually report mass peaks that are not compatible with the chemical formula of any peptide. We show how to remove these artifacts by considering their mass defects. We introduce Y.A.D.A. 3.0, a fast deconvolution algorithm that can remove peaks with unacceptable mass defects. Our approach is effective for polypeptides with less than 10 kDa, and its essence can be easily incorporated into any deconvolution algorithm. AVAILABILITY AND IMPLEMENTATION: Y.A.D.A. 3.0 is freely available for academic use at http://patternlabforproteomics.org/yada3. SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.

Simple, efficient and thorough shotgun proteomic analysis with PatternLab V.

Shotgun proteomics aims to identify and quantify the thousands of proteins in complex mixtures such as cell and tissue lysates and biological fluids. This approach uses liquid chromatography coupled with tandem mass spectrometry and typically generates hundreds of thousands of mass spectra that require specialized computational environments for data analysis. PatternLab for proteomics is a unified computational environment for analyzing shotgun proteomic data. PatternLab V (PLV) is the most comprehensive and crucial update so far, the result of intensive interaction with the proteomics community over several years. All PLV modules have been optimized and its graphical user interface has been completely updated for improved user experience. Major improvements were made to all aspects of the software, ranging from boosting the number of protein identifications to faster extraction of ion chromatograms. PLV provides modules for preparing sequence databases, protein identification, statistical filtering and in-depth result browsing for both labeled and label-free quantitation. The PepExplorer module can even pinpoint de novo sequenced peptides not already present in the database. PLV is of broad applicability and therefore suitable for challenging experimental setups, such as time-course experiments and data handling from unsequenced organisms. PLV interfaces with widely adopted software and community initiatives, e.g., Comet, Skyline, PEAKS and PRIDE. It is freely available at http://www.patternlabforproteomics.org .

Characterizing protein conformers by cross-linking mass spectrometry and pattern recognition.

MOTIVATION: Chemical cross-linking coupled to mass spectrometry (XLMS) emerged as a powerful technique for studying protein structures and large-scale protein-protein interactions. Nonetheless, XLMS lacks software tailored toward dealing with multiple conformers; this scenario can lead to high-quality identifications that are mutually exclusive. This limitation hampers the applicability of XLMS in structural experiments of dynamic protein systems, where less abundant conformers of the target protein are expected in the sample. RESULTS: We present QUIN-XL, a software that uses unsupervised clustering to group cross-link identifications by their quantitative profile across multiple samples. QUIN-XL highlights regions of the protein or system presenting changes in its conformation when comparing different biological conditions. We demonstrate our software's usefulness by revisiting the HSP90 protein, comparing three of its different conformers. QUIN-XL's clusters correlate directly to known protein 3D structures of the conformers and therefore validates our software. AVAILABILITYAND IMPLEMENTATION: QUIN-XL and a user tutorial are freely available at http://patternlabforproteomics.org/quinxl for academic users. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RawVegetable - A data assessment tool for proteomics and cross-linking mass spectrometry experiments.

We present RawVegetable, a software for mass spectrometry data assessment and quality control tailored toward shotgun proteomics and cross-linking experiments. RawVegetable provides four main modules with distinct features: (A) The charge state chromatogram that independently displays the ion current for each charge state; useful for optimizing the chromatography for highly charged ions and with lower XIC values such as those typically found in cross-linking experiments. (B) The XL-Artefact determination, which flags possible noncovalently associated peptides. (C) The TopN density estimation, for detecting retention time intervals of under or over-sampling, and (D) The chromatography reproducibility module, which provides pairwise comparisons between multiple experiments. RawVegetable, a tutorial, and the example data are freely available for academic use at: http://patternlabforproteomics.org/rawvegetable. SIGNIFICANCE: Chromatography optimization is a critical step for any shotgun proteomic or cross-linking mass spectrometry experiment. Here, we present a nifty solution with several key features, such as displaying individual charge state chromatograms, highlighting chromatographic regions of under- or over-sampling and checking for reproducibility.

Mixed-Data Acquisition: Next-Generation Quantitative Proteomics Data Acquisition.

We present the Mixed-Data Acquisition (MDA) strategy for mass spectrometry data acquisition. MDA combines Data-Dependent Acquisition (DDA) and Data-Independent Acquisition (DIA) in the same run, thus doing away with the requirements for separate DDA spectral libraries. MDA is a natural result from advances in mass spectrometry, such as high scan rates and multiple analyzers, and is tailored toward exploiting these features. We demonstrate MDA's effectiveness on a yeast proteome analysis by overcoming a common bottleneck for XIC-based label-free quantitation; namely, the coelution of precursors when m/z values cannot be distinguished. We anticipate that MDA will become the next mainstream data generation approach for proteomics. MDA can also serve as an orthogonal validation approach for DDA experiments. Specialized software for MDA data analysis is made available on the project's website.

Mixed-data acquisition: next-generation quantitative proteomics data acquisition

We present the Mixed-Data Acquisition (MDA) strategy for mass spectrometry data acquisition. MDA combines Data-Dependent Acquisition (DDA) and Data-Independent Acquisition (DIA) in the same run, thus doing away with the requirements for separate DDA spectral libraries. MDA is a natural result from advances in mass spectrometry, such as high scan rates and multiple analyzers, and is tailored toward exploiting these features. We demonstrate MDA's effectiveness on a yeast proteome analysis by overcoming a common bottleneck for XIC-based label-free quantitation; namely, the coelution of precursors when m/z values cannot be distinguished. We anticipate that MDA will become the next mainstream data generation approach for proteomics. MDA can also serve as an orthogonal validation approach for DDA experiments. Specialized software for MDA data analysis is made available on the project's website.

A quantitation module for isotope-labeled peptides integrated into PatternLab for proteomics.

We present a new module integrated into the widely adopted PatternLab for proteomics to enable analysis of isotope-labeled peptides produced using dimethyl or SILAC. The accurate quantitation of proteins lies within the heart of proteomics; dimethylation has shown to be reliable, inexpensive, and applicable to any sample type. We validate our algorithm using an M. tuberculosis dataset obtained from two biological conditions; we used three dimethyl labels, one serving as an internal control for labeling a mixture of samples from both biological conditions. This internal control certified the proper functioning of our software. Availability: http://patternlabforproteomics.org, freely available for academic use.

Top-Down Garbage Collector: a tool for selecting high-quality top-down proteomics mass spectra.

MOTIVATION: We present the first tool for unbiased quality control of top-down proteomics datasets. Our tool can select high-quality top-down proteomics spectra, serve as a gateway for building top-down spectral libraries and, ultimately, improve identification rates. RESULTS: We demonstrate that a twofold rate increase for two E. coli top-down proteomics datasets may be achievable. AVAILABILITY AND IMPLEMENTATION: http://patternlabforproteomics.org/tdgc, freely available for academic use. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

TopoLink: evaluation of structural models using chemical crosslinking distance constraints.

SUMMARY: A software was developed to evaluate structural models using chemical crosslinking experiments. The user provides the types of linkers used and their reactivity, and the observed crosslinks and dead-ends. The software computes the minimum length of a physically inspired linker that connects the reactive atoms of interest, and reports the consistency of each distance with the experimental observation. Statistics on model consistency with the links are provided. Tools to evaluate the correlation of crosslinks in ensembles of models were developed. TopoLink was used to evaluate the potential crosslinks of all structures of the CATH database. The number of crosslinks expected as a function of protein size and linker length can be used as guide for experimental design. AVAILABILITY AND IMPLEMENTATION: TopoLink is available as free software at http://m3g.iqm.unicamp.br/topolink, and distributed as source code with a user-friendly graphical interface for Windows. A web server is also provided. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.