Towards reproducible MSMS data preprocessing, quality control and quantification

The development of MSnbase aims at providing researchers dealing with labelled quantitative proteomics data with a transparent, portable, extensible and open-source collaborative framework to easily manipulate and analyse MS2-level raw tandem mass spectrometry data. The implementation in R gives users and developers a great variety of powerful tools to be used in a controlled and reproducible way. Furthermore, MSnbase has been developed following an object-oriented programming paradigm: all information that is manipulated by the user is encapsulated in ad hoc data containers to hide it's underlying complexity. We illustrate the usage and achievements of our software using a published spiked-in data set in which varying quantities of test proteins have been labelled with four different iTRAQ tags. In addition to providing raw MSMS data, MSnbase also stores meta-data and logs processing steps in the data object itself for optimal traceability. We provide graphics on how to inspect precursor data for quality control and how individual or merged MSMS spectra can subsequently be processed, plotted and extracted using a variety of methods. We also demonstrate how reporter ions (or any peaks of interest defined by the user) can easily be quantified and normalised using several build-in alternative strategies and how the effect of each transformation can be recorded, examined and reproduced. MSnbase constitutes a unique versatile working and development environment to process labelled MSMS data and provides in turn important feedback for data acquisition optimisation. We conclude by presenting future extensions of MSnbase and highlight its usage in reproducible proteomics research

Ciprofloxacin binding to GyrA causes global changes in the proteome of Pseudomonas aeruginosa.

Ciprofloxacin is one of the most widely-used antibiotics, and has proven especially effective at controlling infections associated with the opportunistic human pathogen, Pseudomonas aeruginosa. In this work, we show that sub-inhibitory concentrations of ciprofloxacin induce discrete changes in the intracellular proteome. Central metabolism and cell envelope-associated functions are particularly affected. In spite of the low magnitude of the intracellular proteomic changes, we found that sub-lethal concentrations of ciprofloxacin had substantial effects on motility and exoprotein secretion. Crucially, the proteomic and phenotypic modulations that we observed were absolutely dependent upon the presence of wild-type GyrA; an isogenic strain of P. aeruginosa carrying a ciprofloxacin-insensitive form of GyrA (a T83→I mutant) did not display ciprofloxacin-dependent changes unless complemented with wild-type gyrA in trans. These results show that the diverse effects of sub-inhibitory ciprofloxacin on the cell are routed through its primary target in the cell, DNA gyrase.This work was supported by BBSRC grants BB/C500252/1 and BB/M019411/1

The chicken B-cell line DT40 proteome, beadome and interactomes.

In developing a new quantitative AP-MS method for exploring interactomes in the chicken B-cell line DT40, we also surveyed the most abundant proteins in this organism and explored the likely contaminants that bind to a variety of affinity resins that would later be confirmed quantitatively [1]. We present the 'Top 150 abundant DT40 proteins list', the DT40 beadomes as well as protein interaction lists for the Phosphatidyl inositol 5-phosphate 4-kinase 2β and Fanconi anaemia protein complexes.We thanks Prof. R. Irvine for providing the JPR3 cell line, to Dr. E. Rajendra for providing the FANCC cell line, FANC antibodies and helpful discussions, to Dr. M. Deery and J. Howard for assistance with MS. This work was funded by the Biotechnology and Biological Sciences Research Council (UK) Grant BB/H024085/1.This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.dib.2014.12.00

Drought Stress Causes Specific Changes to the Spliceosome and Stress Granule Components.

The spliceosome processes RNAs from a pre-RNA state to a mature mRNA thereby influencing RNA availability for translation, localization, and turnover. It consists of complex structures containing RNA-binding proteins (RBPs) essential for post-transcriptional gene expression control. Here we investigate the dynamic modifications of spliceosomal RBPs under stress and in particular drought stress. We do so by mRNA interactome capture in Arabidopsis thaliana using label free quantitation. This approach identified 44 proteins associated with the spliceosome and further 32 proteins associated with stress granules. We noted a high enrichment in the motifs RDRR and RSRSRS that are characteristic of RNA interacting proteins. Identification of splicing factors reflect direct and/or indirect stress induced splicing events that have a direct effect on transcriptome and proteome changes under stress. Furthermore, detection of stress granule components is consistent with transcriptional arrest. Identification of drought induced stress granule components is critical in determining common abiotic stress-induced foci that can have biotechnological applications. This study may therefore open ways to modify plant stress responses at a systems level through the modification of key spliceosome components

A Bioconductor workflow for processing and analysing spatial proteomics data

Spatial proteomics is the systematic study of protein sub-cellular localisation. In this workflow, we describe the analysis of a typical quantitative mass spectrometry-based spatial proteomics experiment using the MSnbase and pRoloc Bioconductor package suite. To walk the user through the computational pipeline, we use a recently published experiment predicting protein sub-cellular localisation in pluripotent embryonic mouse stem cells. We describe the software infrastructure at hand, importing and processing data, quality control, sub-cellular marker definition, visualisation and interactive exploration. We then demonstrate the application and interpretation of statistical learning methods, including novelty detection using semi-supervised learning, classification, clustering and transfer learning and conclude the pipeline with data export. The workflow is aimed at beginners who are familiar with proteomics in general and spatial proteomics in particular.LMB and CMM are supported by a Wellcome Trust Technology Development Grant (grant number 108441/Z/15/Z). KSL is a Wellcome Trust Joint Investigator (110170/Z/15/Z). LG is supported by the BBSRC Strategic Longer and Larger grant (Award BB/L002817/1)

Effects of Traveling Wave Ion Mobility Separation on Data Independent Acquisition in Proteomics Studies

qTOF mass spectrometry and traveling wave ion mobility separation (TWIMS) hybrid instruments (q- TWIMS-TOF) have recently become commercially available. Ion mobility separation allows an additional dimension of precursor separation inside the instrument, without incurring an increase in instrument time. We comprehensively investigated the effects of TWIMS on data-independent acquisition on a Synapt G2 instrument. We observed that if fragmentation is performed post TWIMS, more accurate assignment of fragment ions to precursors is possible in data independent acquisition. This allows up to 60% higher proteome coverage and higher confidence of protein and peptide identifications. Moreover, the majority of peptides and proteins identified upon application of TWIMS span the lower intensity range of the proteome. It has also been demonstrated in several studies that employing IMS results in higher peak capacity of separation and consequently more accurate and precise quantitation of lower intensity precursor ions. We observe that employing TWIMS results in an attenuation of the detected ion current. We postulate that this effect is binary; sensitivity is reduced due to ion scattering during transfer into a high pressure “IMS zone”, sensitivity is reduced due to the saturation of detector digitizer as a result of the IMS concentration effect. This latter effect limits the useful linear range of quantitation, compromising quantitation accuracy of high intensity peptides. We demonstrate that the signal loss from detector saturation and transmission loss can be deconvoluted by investigation of the peptide isotopic envelope. We discuss the origin and extent of signal loss and suggest methods to minimize these effects on q-TWIMS-TOF instrument in the light of different experimental designs and other IMS/MS platforms described previously

SWATH-MS data of Drosophila melanogaster proteome dynamics during embryogenesis

AbstractEmbryogenesis is one of the most important processes in the life of an animal. During this dynamic process, progressive cell division and cellular differentiation are accompanied by significant changes in protein expression at the level of the proteome. However, very few studies to date have described the dynamics of the proteome during the early development of an embryo in any organism. In this dataset, we monitor changes in protein expression across a timecourse of more than 20h of Drosophila melanogaster embryonic development. Mass-spectrometry data were produced using a SWATH acquisition mode on a Sciex Triple-TOF 6600. A spectral library built in-house was used to analyse these data and more than 1950 proteins were quantified at each embryonic timepoint. The files presented here are a permanent digital map and can be reanalysed to test against new hypotheses. The data have been deposited with the ProteomeXchange Consortium with the dataset identifier PRIDE: PXD0031078

Proteomic analysis of the EhV-86 virion

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Transcriptional regulation of the genes involved in protein metabolism and processing in Saccharomyces cerevisiae.

Topological analysis of large networks, which focus on a specific biological process or on related biological processes, where functional coherence exists among the interacting members, may provide a wealth of insight into cellular functionality. This work presents an unbiased systems approach to analyze genetic, transcriptional regulatory and physical interaction networks of yeast genes possessing such functional coherence to gain novel biological insight. The present analysis identified only a few transcriptional regulators amongst a large gene cohort associated with the protein metabolism and processing in yeast. These transcription factors are not functionally required for the maintenance of these tasks in growing cells. Rather, they are involved in rewiring gene transcription in response to such major challenges as starvation, hypoxia, DNA damage, heat shock or the accumulation of unfolded proteins. Indeed, only a subset of these proteins were captured empirically in the nuclear-enriched fraction of non-stressed yeast cells, suggesting that the transcriptional regulation of protein metabolism and processing in yeast is primarily concerned with maintaining cellular robustness in the face of threat by either internal or external stressors