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Data-independent acquisition (DIA) mass spectrometry, also known as Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH), is a popular label-free proteomics strategy to comprehensively quantify peptides/proteins utilizing mass spectral libraries to decipher inherently multiplexed spectra collected linearly across a mass range. Although there are many spectral libraries produced worldwide, the quality control of these libraries is lacking. We present the DIALib-QC (DIA library quality control) software tool for the systematic evaluation of a library's characteristics, completeness and correctness across 62 parameters of compliance, and further provide the option to improve its quality. We demonstrate its utility in assessing and repairing spectral libraries for correctness, accuracy and sensitivity.

Data-independent acquisition mass spectrometry promises higher performance in terms of quantification and reproducibility compared to data-dependent acquisition mass spectrometry methods. To enable high-accuracy quantification of Staphylococcus aureus proteins, we have developed a global ion library for data-independent acquisition approaches employing high-resolution time of flight or Orbitrap instruments for this human pathogen. We applied this ion library resource to investigate the time-resolved adaptation of S. aureus to the intracellular niche in human bronchial epithelial cells and in a murine pneumonia model. In epithelial cells, abundance changes for more than 400 S. aureus proteins were quantified, revealing, e.g., the precise temporal regulation of the SigB-dependent stress response and differential regulation of translation, fermentation, and amino acid biosynthesis. Using an in vivo murine pneumonia model, our data-independent acquisition quantification analysis revealed for the first time the in vivo proteome adaptation of S. aureus. From approximately 2.15 × 10 5   S. aureus cells, 578 proteins were identified. Increased abundance of proteins required for oxidative stress response, amino acid biosynthesis, and fermentation together with decreased abundance of ribosomal proteins and nucleotide reductase NrdEF was observed in post-infection samples compared to the pre-infection state.

Quantitative proteomics employing mass spectrometry is an indispensable tool in life science research. Targeted proteomics has emerged as a powerful approach for reproducible quantification but is limited in the number of proteins quantified. SWATH-mass spectrometry consists of data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics (accuracy, sensitivity, and selectivity) of targeted proteomics at large scale. While previous SWATH-mass spectrometry studies have shown high intra-lab reproducibility, this has not been evaluated between labs. In this multi-laboratory evaluation study including 11 sites worldwide, we demonstrate that using SWATH-mass spectrometry data acquisition we can consistently detect and reproducibly quantify >4000 proteins from HEK293 cells. Using synthetic peptide dilution series, we show that the sensitivity, dynamic range and reproducibility established with SWATH-mass spectrometry are uniformly achieved. This study demonstrates that the acquisition of reproducible quantitative proteomics data by multiple labs is achievable, and broadly serves to increase confidence in SWATH-mass spectrometry data acquisition as a reproducible method for large-scale protein quantification.SWATH-mass spectrometry consists of a data-independent acquisition and a targeted data analysis strategy that aims to maintain the favorable quantitative characteristics on the scale of thousands of proteins. Here, using data generated by eleven groups worldwide, the authors show that SWATH-MS is capable of generating highly reproducible data across different laboratories.

Posttranslational modifications of proteins play an important role in biology. For example, phosphorylation is a key component in signal transduction in all three domains of life, and histones can be modified in such a variety of ways that a histone code for gene regulation has been proposed. Shotgun proteomics is commonly used to identify posttranslational modifications as well as chemical modifications from sample processing. However, it favors the detection of abundant peptides over the repertoire presented, and the data analysis usually requires advance specification of modification masses and target amino acids, their number constrained by available computational resources. Recent advances in data independent acquisition mass spectrometry technologies such as SWATH-MS enable a deeper recording of the peptide contents of samples, including peptides with modifications. Here, we present a novel approach that applies the power of SWATH-MS analysis to the automated pursuit of modified peptides. With the new SWATHProphet(PTM) functionality added to the open source SWATHProphet software, precursor ions consistent with a modification are identified along with the mass and localization of the modification in the peptide sequence in a sensitive and unrestricted manner without the need to anticipate the modifications in advance. Using this method, we demonstrate the detection of a wide assortment of modified peptides, many unanticipated, in samples containing unpurified synthetic peptides and human urine, as well as in phospho-enriched human tissue culture cell samples., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)