Mass spectrometry is the method of choice for deep and reliable exploration of the (human) proteome. Targeted mass spectrometry reliably detects and quantifies pre-determined sets of proteins in a complex biological matrix and is used in studies that rely on the quantitatively accurate and reproducible measurement of proteins across multiple samples. It requires the one-time, a priori generation of a specific measurement assay for each targeted protein. SWATH-MS is a mass spectrometric method that combines data-independent acquisition (DIA) and targeted data analysis and vastly extends the throughput of proteins that can be targeted in a sample compared to selected reaction monitoring (SRM). Here we present a compendium of highly specific assays covering more than 10,000 human proteins and enabling their targeted analysis in SWATH-MS datasets acquired from research or clinical specimens. This resource supports the confident detection and quantification of 50.9% of all human proteins annotated by UniProtKB/Swiss-Prot and is therefore expected to find wide application in basic and clinical research. Data are available via ProteomeXchange (PXD000953-954) and SWATHAtlas (SAL00016-35).

Clinical specimens are each inherently unique, limited and non-renewable. As such, small samples such as tissue biopsies are often completely consumed after a limited number of analyses. Here we present a method that enables fast and reproducible conversion of a small amount of tissue (approximating the quantity obtained by a biopsy) into a single, permanent digital file representing the mass spectrometry-measurable proteome of the sample. The method combines pressure cycling technology (PCT) and SWATH mass spectrometry (MS), and the resulting proteome maps can be analyzed, re-analyzed, compared and mined in silico to detect and quantify specific proteins across multiple samples. We used this method to process and convert 18 biopsy samples from 9 renal cell carcinoma patients into SWATH-MS fragment ion maps. From these proteome maps we detected and quantified more than 2,000 proteins with a high degree of reproducibility across all samples. The identified proteins clearly separated tumorous kidney tissues from healthy tissue, and differentiated distinct histomorphological kidney cancer subtypes.

Small ubiquitin-like modifier (SUMO) is covalently conjugated to its target proteins thereby altering their activity. The mammalian SUMO protein family includes four members (SUMO-1-4) of which SUMO-2 and SUMO-3 are conjugated in a stress-inducible manner. The vast majority of known SUMO substrates are recognized by the single SUMO E2-conjugating enzyme Ubc9 binding to a consensus tetrapeptide (⌿KXE where ⌿ stands for a large hydrophobic amino acid) or extended motifs that contain phosphorylated or negatively charged amino acids called PDSM (phosphorylation-dependent sumoylation motif) and NDSM (negatively charged amino acid-dependent sumoylation motif), respectively. We identified 382 SUMO-2 targets using a novel method based on SUMO protease treatment that improves separation of SUMO substrates on SDS-PAGE before LC-ESI-MS/MS. We also implemented a software SUMOFI (SUMO motif finder) to facilitate identification of motifs for SUMO substrates from a user-provided set of proteins and to classify the substrates according to the type of SUMOtargeting consensus site. Surprisingly more than half of the substrates lacked any known consensus site, suggesting that numerous SUMO substrates are recognized by a yet unknown consensus site-independent mechanism. Gene ontology analysis revealed that substrates in distinct functional categories display strikingly different prevalences of NDSM sites. Given that different types of motifs are bound by Ubc9 using alternative mechanisms, our data suggest that the preference of SUMO-2 targeting mechanism depends on the biological function of the substrate.

T helper (Th) cells differentiate into functionally distinct effector cell subsets of which Th1 and Th2 cells are best characterized. Besides T cell receptor signaling, IL-12-induced STAT4 and T-bet-and IL-4-induced STAT6 and GATA3 signaling pathways are the major players regulating the Th1 and Th2 differentiation process, respectively. However, there are likely to be other yet unknown factors or pathways involved. In this study we used quantitative proteomics exploiting cleavable ICAT labeling and LC-MS/MS to identify IL-4-regulated proteins from the microsomal fractions of CD4 ؉ cells extracted from umbilical cord blood. We were able to identify 557 proteins of which 304 were also quantified. This study resulted in the identification of the down-regulation of small GTPases GIMAP1 and GIMAP4 by IL-4 during Th2 differentiation. We also showed that both GIMAP1 and GI-

Pectobacterium atrosepticum is a Gram-negative plant-pathogenic bacterium that rots potato stems and tubers. Microarray analysis was used to identify genes that were differentially expressed when host extracts were added to the growth medium. Potato extracts downregulated the expression of ribosomal genes and genes related to uptake and metabolism of nutrients, and upregulated genes needed for nitrate or phosphonate use. Some of the observed changes in gene expression in host-extract-induced cultures are similar to those during attachment of the bacterium to host tissues. Other responses indicated defence against toxic metabolites in the extract. Tuber extract induced a large gene cluster having homology to type VI secretion genes shown to be virulence determinants in many, but not all, animal and human pathogens. Two of the genes in the type VI cluster were found to be expressed during infection in potato tubers and stems, and mutants with knockouts of the corresponding genes had increased virulence on potato. One of the type VI secretion mutants was further characterized and found to grow to higher cell density in culture in the presence of host extract and to produce slightly more extracellular tissue-macerating enzymes than the wild-type strain. Analysis of secreted proteins showed that this type VI mutant was affected in the production of haemolysin-coregulated proteins (Hcps), which have been suggested to be secreted by the type VI pathway in other bacteria. The results suggest that the type VI secretion system of P. atrosepticum is needed for secretion of Hcps but not for virulence on its host plant, potato.

SummaryOrganellar reactive oxygen species (ROS) signalling is a key mechanism that promotes the onset of defensive measures in stress-exposed plants. The underlying molecular mechanisms and feedback regulation loops, however, still remain poorly understood. Our previous work has shown that a specific regulatory B 0 c subunit of protein phosphatase 2A (PP2A) is required to control organellar ROS signalling and associated metabolic adjustments in Arabidopsis thaliana. Here, we addressed the mechanisms through which PP2A-B 0 c impacts on organellar metabolic crosstalk and ROS homeostasis in leaves. Genetic, biochemical and pharmacological approaches, together with a combination of data-dependent acquisition (DDA) and selected reaction monitoring (SRM) MS techniques, were utilized to assess PP2A-B 0 c-dependent adjustments in Arabidopsis thaliana. We show that PP2A-B'c physically interacts with the cytoplasmic form of aconitase, a central metabolic enzyme functionally connected with mitochondrial respiration, oxidative stress responses and regulation of cell death in plants. Furthermore, PP2A-B'c impacts ROS homeostasis by controlling the abundance of specific alternative oxidase isoforms, AOX1A and AOX1D, in leaf mitochondria.We conclude that PP2A-B'c-dependent regulatory actions modulate the functional status of metabolic enzymes that essentially contribute to intracellular ROS signalling and metabolic homeostasis in plants.

Metagenomic approaches focus on taxonomy or gene annotation but lack power in defining functionality of gut microbiota. Therefore, metaproteomics approaches have been introduced to overcome this limitation. However, the common metaproteomics approach uses data-dependent acquisition mass spectrometry, which is known to have limited reproducibility when analyzing samples with complex microbial composition. In this work, we provide a proof of concept for data-independent acquisition (DIA) metaproteomics. To this end, we analyze metaproteomes using DIA mass spectrometry and introduce an open-source data analysis software package, diatools, which enables accurate and consistent quantification of DIA metaproteomics data. We demonstrate the feasibility of our approach in gut microbiota metaproteomics using laboratoryassembled microbial mixtures as well as human fecal samples.

BackgroundA key feature of clear cell renal cell carcinoma (ccRCC) is the inactivation of the von Hippel-Lindau tumour suppressor protein (pVHL) that leads to the activation of hypoxia-inducible factor (HIF) pathway also in well-oxygenated conditions. Important regulator of HIF-α, prolyl hydroxylase PHD3, is expressed in high amounts in ccRCC. Although several functions and downstream targets for PHD3 in cancer have been suggested, the role of elevated PHD3 expression in ccRCC is not clear.MethodsTo gain insight into the functions of high PHD3 expression in ccRCC, we used PHD3 knockdown by siRNA in 786-O cells under normoxic and hypoxic conditions and performed discovery mass spectrometry (LC-MS/MS) of the purified peptide samples. The LC-MS/MS results were analysed by label-free quantification of proteome data using a peptide-level expression-change averaging procedure and subsequent gene ontology enrichment analysis.ResultsOur data reveals an intriguingly widespread effect of PHD3 knockdown with 91 significantly regulated proteins. Under hypoxia, the response to PHD3 silencing was wider than under normoxia illustrated by both the number of regulated proteins and by the range of protein expression levels. The main cellular functions regulated by PHD3 expression were glucose metabolism, protein translation and messenger RNA (mRNA) processing. PHD3 silencing led to downregulation of most glycolytic enzymes from glucose transport to lactate production supported by the reduction in extracellular acidification and lactate production and increase in cellular oxygen consumption rate. Moreover, upregulation of mRNA processing-related proteins and alteration in a number of ribosomal proteins was seen as a response to PHD3 silencing. Further studies on upstream effectors of the translational machinery revealed a possible role for PHD3 in regulation of mTOR pathway signalling.ConclusionsOur findings suggest crucial involvement of PHD3 in the maintenance of key cellular functions including glycolysis and protein synthesis in ccRCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s40170-017-0167-y) contains supplementary material, which is available to authorized users.