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i2MassChroQ: Software for Ion Mobility-Enabled Quantitative Proteomics in timsTOF Data Format
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International audience. IntroductionX!TandemPipeline (Langella et al. 2017) is a proteomics free and open sourceJava software program designed to filter and group peptide/proteinidentifications from MS/MS mass spectra. After a complete rewrite in C++17,X!TandemPipeline++, now named i2MassChroQ, features both native support for the timsTOF raw dataformat and peptide/protein quantification. i2MassChroQ performs peptideidentifications and area under the curve XIC-based quantifications using theBruker's native raw data format. Using a common HeLa data set published byMeier et al. (2018, PXDO1OO12), we demonstrate that i2MassChroQidentifies and quantifies significantly more proteins than competitors MaxQuantand MSFragger. It is also significantly faster.Methodsi2MassChroQ is written in portable C++17 and makes use of the Qtlibraries for the graphical user interface. Binary packages are available forLinux and MS Windows. The timsTOF native raw data reader was developed in-house withthe technical specifications provided by Bruker. The sofware wastightly optimized to ensure very fast access to the binary data. The currentversion provides real time MS/MS peptide annotation and performs extremely fastion current extractions and XIC chromatogram visualizations (typically, in lessthan five seconds for a 2 hour PASEF run).ResultsThe Bruker timsTOF line of instruments improves the identification of peptidesand proteins in complex mixtures by implementing a peculiar ion mobilitytechnology. i2MassChroQ has the distinct feature, with respect to theMaxQuant and MSFragger competitors, of natively parsing the timsTOF raw datawith original software code that puts us in total control of the dataprocessing, in particular where tight optimizations are needed for speed or incases where special data processing features need adding for increased accuracy.This is exemplified by i2MassChroQ using the renowned X!Tandem engine(Craig et al. 2004) to perform database searches right on optimized databrokered to it by our reader of native timsTOF binary data.Our software identifies roughly the same amount of peptides as when using theX!Tandem engine on MGF data provided by the Bruker Data analysis tool butperforms much faster, reducing processing time from 55 to 12 minutes on atypical quality control HeLa sample. For peptide quantifications,i2MassChroQ uses the ion mobility-enabled version of MassChroQ (Valot etal. 2011), both software pieces sharing our optimized native timsTOF datareader.Using a common HeLa data set (Meier et al. (2018), PXDO1OO12, 4 technicalreplicates), we found more quantified proteins with a minimum of 2 quantifiedions : 5783 vs 4977 (MSFragger), 3526 (MaxQuant). Correlation of quantifiedproteins between samples was 0.99. Median protein coefficient of variation (CV)was also very good : 0.059 vs 0.049 (MSFragger), 0.072 (Peaks) and 0.070(MaxQuant). Comparable results have been found using data set PXD014777(Prianichnikov et al., 2020, a mixture of proteins from three organisms, H.sapiens, S. cerevisiae, and E. coli).ConclusionOverall, while producing similar orbetter results than competing software, i2MassChroQ always performsfaster than all the other software offerings.
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