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

Marking exodus: death and funerals in the religious paintings of Clementine Hunter

Previous scholarship on artist Clementine Hunter of Natchitoches, Louisiana, has been predominantly biographical and stylistic. Through analysis of her religious scenes, I propose that Hunter’s paintings concerning death reflect concepts of exodus that have influenced Christianity and its practice since its beginning. Considering specifically Hunter's mid-1970s Frenchie Goin' to Heaven, I explore ideas of exodus first through the concept of death as a doorway or transitional space, an association that extends back historically to the Early Christian period. I then link the theme of exodus to the variety of religious influences apparent in Hunter's work, considering especially her depictions of angels, baptisms, and the possible influence of African-American connections between exodus, death, and baptism. These conceptual and historical considerations provide new ways of discussing the complexity of Hunter's work, whether intended by Hunter or not, that place her work in a continuum of religious thought both mainstream and idiosyncratic

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

Separating Golgi proteins from cis to trans reveals underlying properties of cisternal localization

The order of enzymatic activity across Golgi cisternae is essential for complex molecule biosynthesis. However, an inability to separate Golgi cisternae has meant the cisternal distribution of most resident proteins, and their underlying localization mechanisms, are unknown. Here, we exploit differences in surface charge of intact cisternae to perform the first separation of early to late Golgi sub-compartments. We determine protein and glycan abundance profiles across the Golgi; over 390 resident proteins are identified, including 136 new additions, with over 180 cisternal assignments. These assignments provide a means to better understand the functional roles of Golgi proteins and how they operate sequentially. Protein and glycan distributions are validated in-vivo, using high resolution microscopy. Results reveal distinct functional compartmentalization among resident Golgi proteins. Analysis of transmembrane proteins shows several sequence-based characteristics relating to pI, hydrophobicity, Ser abundance and Phe bilayer asymmetry that change across the Golgi. Overall this suggests a continuum of TM features, rather than discrete rules, which guide proteins to earlier or later locations within the Golgi stack.Marie Curie fellowship PIEF-GA-2011-301401 1058 (FP7-PEOPLE-2011) MR

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)

Protein neighbors and proximity proteomics

Within cells, proteins can co-assemble into functionally integrated and spatially restricted multicomponent complexes. Often, the affinities between individual proteins are relatively weak, and proteins within such clusters may interact only indirectly with many of their other protein neighbors. This makes proteomic characterization difficult using methods such as immunoprecipitation or cross-linking. Recently, several groups have described the use of enzyme-catalyzed proximity labeling reagents that covalently tag the neighbors of a targeted protein with a small molecule such as fluorescein or biotin. The modified proteins can then be isolated by standard pulldown methods and identified by mass spectrometry. Here we will describe the techniques as well as their similarities and differences. We discuss their applications both to study protein assemblies and to provide a new way for characterizing organelle proteomes. We stress the importance of proteomic quantitation and independent target validation in such experiments. Furthermore, we suggest that there are biophysical and cell-biological principles that dictate the appropriateness of enzyme-catalyzed proximity labeling methods to address particular biological questions of interest.This work was supported by Biotechnology and Biological Sciences Research Council (UK) Grant BB/J021091/1. XWL & SP supported by Chinese Ministry of Science and Technology 973 Program Grants 2012CB911000 and 2013CB910700 and National Natural Science Foundation of China Grant 31110103914.This is the final version of the article. It was first available from ASBMB via http://dx.doi.org/10.1074/mcp.R115.05290

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