This article has been withdrawn by the authors. This article did not comply with the editorial guidelines of MCP. Specifically, single peptide based protein identifications of 9-19% were included in the analysis and discussed in the results and conclusions. We wish to withdraw this article and resubmit a clarified, corrected manuscript for review.

Ion mobility can add a dimension to LC-MS based shotgun proteomics which has the potential to boost proteome coverage, quantification accuracy and dynamic range.  Required for this is suitable software that extracts the information contained in the four-dimensional (4D) data space spanned by m/z, retention time, ion mobility and signal intensity. Here we describe the ion mobility enhanced MaxQuant software, which utilizes the added data dimension. It offers an end to end computational workflow for the identification and quantification of peptides and proteins in LC-IMS-MS/MS shotgun proteomics data. We apply it to trapped ion mobility spectrometry (TIMS) coupled to a quadrupole time-of-flight (QTOF) analyzer. A highly parallelizable 4D feature detection algorithm extracts peaks which are assembled to isotope patterns. Masses are recalibrated with a non-linear m/z, retention time, ion mobility and signal intensity dependent model, based on peptides from the sample. A new matching between runs (MBR) algorithm that utilizes collisional cross section (CCS) values of MS1 features in the matching process significantly gains specificity from the extra dimension. Prerequisite for using CCS values in MBR is a relative alignment of the ion mobility values between the runs. The missing value problem in protein quantification over many samples is greatly reduced by CCS aware MBR.MS1 level label-free quantification is also implemented which proves to be highly precise and accurate on a benchmark dataset with known ground truth. MaxQuant for LC-IMS-MS/MS is part of the basic MaxQuant release and can be downloaded from http://maxquant.org.

Quantitative proteomics by mass spectrometry is widely used in biomarker research and basic biology research for investigation of phenotype level cellular events. Despite the wide application, the methodology for statistical analysis of differentially expressed proteins has not been unified. Various methods such as t-test, linear model and mixed effect models are used to define changes in proteomics experiments. However, none of these methods consider the specific structure of MS-data. Choices between methods, often originally developed for other types of data, are based on compromises between features such as statistical power, general applicability and user friendliness. Furthermore, whether to include proteins identified with one peptide in statistical analysis of differential protein expression varies between studies. Here we present DEqMS, a robust statistical method developed specifically for differential protein expression analysis in mass spectrometry data. In all datasets investigated there is a clear dependence of variance on the number of PSMs or peptides used for protein quantification. DEqMS takes this feature into account when assessing differential protein expression. This allows for a more accurate data-dependent estimation of protein variance and inclusion of single peptide identifications without increasing false discoveries. The method was tested in several datasets including E.coli proteome spike-in data, using both label-free and TMT-labelled quantification. In comparison to previous statistical methods used in quantitative proteomics, DEqMS showed consistently better accuracy in detecting altered protein levels compared to other statistical methods in both label-free and labelled quantitative proteomics data. DEqMS is available as an R package in Bioconductor.

In bottom-up mass spectrometry-based proteomics, relative protein quantification is often achieved with data-dependent acquisition (DDA), data-independent acquisition (DIA), or selected reaction monitoring (SRM). These workflows quantify proteins by summarizing the abundances of all the spectral features of the protein (e.g., precursor ions, transitions or fragments) in a single value per protein per run. When abundances of some features are inconsistent with the overall protein profile (for technological reasons such as interferences, or for biological reasons such as post-translational modifications), the protein-level summaries and the downstream conclusions are undermined. We propose a statistical approach that automatically detects spectral features with such inconsistent patterns. The detected features can be separately investigated, and if necessary removed from the dataset. We evaluated the proposed approach on a series of benchmark controlled mixtures and biological investigations with DDA, DIA and SRM data acquisitions. The results demonstrated that it can facilitate and complement manual curation of the data. Moreover, it can improve the estimation accuracy, sensitivity and specificity of detecting differentially abundant proteins, and reproducibility of conclusions across different data processing tools. The approach is implemented as an option in the open-source R-based software MSstats.

Drug resistance is a major obstacle to curative cancer therapies, and increased understanding of the molecular events contributing to resistance would enable better prediction of therapy response, as well as contribute to new targets for combination therapy. Here we have analyzed the early molecular response to epidermal growth factor receptor (EGFR) inhibition using RNA sequencing data covering 13 486 genes and mass spectrometry data covering 10 138 proteins. This analysis revealed a massive response to EGFR inhibition already within the first 24 hours, including significant regulation of hundreds of genes known to control downstream signaling, such as transcription factors, kinases, phosphatases and ubiquitin E3-ligases. Importantly, this response included upregulation of key genes in multiple oncogenic signaling pathways that promote proliferation and survival, such as ERBB3, FGFR2, JAK3 and BCL6, indicating an early adaptive response to EGFR inhibition. Using a library of more than 500 approved and experimental compounds in a combination therapy screen, we could show that several kinase inhibitors with targets including JAK3 and FGFR2 increased the response to EGFR inhibitors. Further, we investigated the functional impact of BCL6 upregulation in response to EGFR inhibition using siRNA-based silencing of BCL6. Proteomics profiling revealed that BCL6 inhibited transcription of multiple target genes including p53, resulting in reduced apoptosis which implicates BCL6 upregulation as a new EGFR inhibitor treatment escape mechanism. Finally, we demonstrate that combined treatment targeting both EGFR and BCL6 act synergistically in killing lung cancer cells. In conclusion, or data indicates that multiple different adaptive mechanisms may act in concert to blunt the cellular impact of EGFR inhibition, and we suggest BCL6 as a potential target for EGFR inhibitor-based combination therapy.

Stimulating brown adipose tissue (BAT) activity represents a promising therapy for overcoming metabolic diseases. mTORC2 is important for regulating BAT metabolism, but its downstream targets have not been fully characterized. In this study, we apply proteomics and phosphoproteomics to investigate the downstream effectors of mTORC2 in brown adipocytes. We compare wild-type controls to isogenic cells with an induced knockout of the mTORC2 subunit RICTOR (Rictor-iKO) by stimulating each with insulin for a 30-minute time course. In Rictor-iKO cells, we identify decreases to the abundance of glycolytic and de novo lipogenesis enzymes, and increases to mitochondrial proteins as well as a set of proteins known to increase upon interferon stimulation. We also observe significant differences to basal phosphorylation due to chronic RICTOR loss including decreased phosphorylation of the lipid droplet protein perilipin-1 in Rictor-iKO cells, suggesting that RICTOR could be involved with regulating basal lipolysis or droplet dynamics. Finally, we observe mild dampening of acute insulin signaling response in Rictor-iKO cells, and a subset of AKT substrates exhibiting statistically significant dependence on RICTOR.

Glioblastoma (GBM) is one of the most aggressive human cancers with a median survival of less than two years. A distinguishing pathological feature of GBM is a high degree of inter- and intratumoral heterogeneity. Intertumoral heterogeneity of GBM has been extensively investigated on genomic, methylomic, transcriptomic, proteomic and metabolomics levels, however only a few studies describe intratumoral heterogeneity due to the lack of methods allowing to analyze GBM samples with high spatial resolution. Here, we applied TOF-SIMS (Time-of-flight secondary ion mass spectrometry) for the analysis of single cells and clinical samples such as paraffin and frozen tumor sections obtained from 57 patients. We developed a technique that allows us to simultaneously detect the distribution of proteins and metabolites in glioma tissue with 800 nm spatial resolution. Our results demonstrate that according to TOF-SIMS data glioma samples can be subdivided into clinically relevant groups and distinguished from the normal brain tissue. In addition, TOF-SIMS was able to elucidate differences between morphologically distinct regions of GBM within the same tumor. By staining GBM sections with gold-conjugated antibodies against Caveolin-1 we could visualize border between zones of necrotic and cellular tumor and subdivide glioma samples into groups characterized by different survival of the patients. Finally, we demonstrated that GBM contains cells that are characterized by high levels of Caveolin-1 protein and cholesterol. This population may partly represent a glioma stem cells. Collectively, our results show that the technique described here allows to analyze glioma tissues with a spatial resolution beyond reach of most of other omics approaches and the obtained data may be used to predict clinical behavior of the tumor.

The onset of obesity-linked type 2 diabetes (T2D) is marked by an eventual failure in pancreatic β-cell function and mass that is no longer able to compensate for the inherent insulin resistance and increased metabolic load intrinsic to obesity. However, in a commonly used model of T2D, the db/db mouse, β-cells have an inbuilt adaptive flexibility enabling them to effectively adjust insulin production rates relative to the metabolic demand. Pancreatic β-cells from these animals have markedly reduced intracellular insulin stores, yet high rates of (pro)insulin secretion, together with a substantial increase in proinsulin biosynthesis highlighted by expanded rough endoplasmic reticulum and Golgi apparatus. However, when the metabolic overload and/or hyperglycemia is normalized, β-cells from db/db mice quickly restore their insulin stores and normalize secretory function. This demonstrates the β-cell’s adaptive flexibility and indicates that therapeutic approaches applied to encourage β-cell rest are capable of restoring endogenous β-cell function. However, mechanisms that regulate β-cell adaptive flexibility are essentially unknown. To gain deeper mechanistic insight into the molecular events underlying β-cell adaptive flexibility in db/db β-cells, we conducted a combined proteomic and post-translational modification specific proteomic (PTMomics) approach on islets from db/db mice and wild-type controls (WT) with or without prior exposure to normal glucose levels. We identified differential modifications of proteins involved in redox homeostasis, protein refolding, K48-linked deubiquitination, mRNA/protein export, focal adhesion, ERK1/2 signaling, and renin-angiotensin-aldosterone signaling, as well as sialyltransferase activity, associated with β-cell adaptive flexibility. These proteins are all related to proinsulin biosynthesis and processing, maturation of insulin secretory granules, and vesicular trafficking—core pathways involved in the adaptation of insulin production to meet metabolic demand. Collectively, this study outlines a novel and comprehensive global PTMome signaling map that highlights important molecular mechanisms related to the adaptive flexibility of β-cell function, providing improved insight into disease pathogenesis of T2D.

The human leucocyte antigen (HLA)-B*27:05 allele and the endoplasmic reticulum-resident aminopeptidases are strongly associated with ankylosing spondylitis (AS), a chronic inflammatory spondyloarthropathy. This study examined the effect of endoplasmic reticulum aminopeptidase 2 (ERAP2) in the generation of the natural HLA-B*27:05 ligandome in live cells. Complexes of HLA-B*27:05-bound peptide pools were isolated from human ERAP2-edited cell clones and the peptides were identified using high throughput mass spectrometry analyses. The relative abundance of thousand ligands was established by quantitative tandem mass spectrometry and bioinformatics analysis. The residue frequencies at different peptide position, identified in presence or absence of ERAP2, determined structural features of ligands and their interactions with specific pockets of antigen binding site of HLA-B*27:05 molecule. Sequence alignment of ligands identified with species of bacteria associated with HLA-B*27-dependent reactive arthritis was performed. In the absence of ERAP2, peptides with N-terminal basic residues, and minority canonical P2 residues are enriched in the natural ligandome. Further, alterations of residue frequencies and hydrophobicity profile at P3, P7, and P positions were detected. In addition, several ERAP2-dependent cellular peptides were highly similar to protein sequences of arthritogenic bacteria, including one human HLA-B*27:05 ligand fully conserved in a protein from Campylobacter jejuni. These findings highlight the pathogenic role of this aminopeptidase in the triggering of AS autoimmune disease.

Post-translational modifications play a critical and diverse role in regulating cellular activities. Despite their fundamentally important role in cellular function, there has been no report to date of an effective generalized approach to the targeting, extraction and characterization of the critical c-terminal regions of natively prenylated proteins. Various chemical modification and metabolic labelling strategies in cell culture have been reported. However, their applicability is limited to cell culture systems and does not allow for analysis of tissue samples. The chemical characteristics (hydrophobicity, low abundance, highly basic charge) of many of the c-terminal regions of prenylated proteins have impaired the use of standard proteomic workflows. In this context, we sought a direct approach to the problem in order to examine these proteins in tissue without the use of labelling.  Here we demonstrate that prenylated proteins can be captured on chromatographic resins functionalized with mixed disulfide functions. Protease treatment of resin-bound proteins using chymotryptic digestion revealed peptides from many known prenylated proteins. Exposure of the protease-treated resin to reducing agents and hydro organic mixtures released c-terminal peptides with intact prenyl groups along with other enzymatic modifications expected in this protein family. Database and search parameters were selected to allow for c-terminal modifications unique to these molecules such as CAAX box processing and c-terminal methylation. In summary, we present a direct approach to enrich and obtain information at a molecular level of detail about prenylation of proteins from tissue and cell extracts using high performance LCMS without the need for metabolic labeling and derivatization.

Genetic and genomic research has greatly advanced our understanding of heart disease. Yet, comprehensive, in-depth, quantitative maps of protein expression in hearts of living humans are still lacking. Using samples obtained during valve replacement surgery in patients with mitral valve prolapse (MVP), we set out to define inter-chamber differences, the intersect of proteomic data with genetic or genomic datasets, and the impact of left atrial dilation on the proteome of patients with no history of atrial fibrillation (AF).  We collected biopsies from right atria (RA), left atria (LA) and left ventricle (LV) of seven male patients with mitral valve regurgitation with dilated LA but no history of AF. Biopsy samples were analyzed by high-resolution mass spectrometry (MS), where peptides were pre-fractionated by reverse phase high-pressure liquid chromatography prior to MS measurement on a Q-Exactive-HF Orbitrap instrument. We identified 7,314 proteins based on 130,728 peptides. Results were confirmed in an independent set of biopsies collected from three additional individuals. Comparative analysis against data from post-mortem samples showed enhanced quantitative power and confidence level in samples collected from living hearts. Our analysis, combined with data from genome wide association studies suggested candidate gene associations to MVP, identified higher abundance in ventricle for proteins associated with cardiomyopathies and revealed the dilated LA proteome, demonstrating differential representation of molecules previously associated with AF, in non-AF hearts. This is the largest dataset of cardiac protein expression from human samples collected in vivo. It provides a comprehensive resource that allows insight into molecular fingerprints of MVP and facilitates novel inferences between genomic data and disease mechanisms. We propose that over-representation of proteins in ventricle is consequent not to redundancy but to functional need, and conclude that changes in abundance of proteins known to associate with AF are not sufficient for arrhythmogenesis.

Accumulation and propagation of hyperphosphorylated tau (p-tau) is a common neuropathological hallmark associated with neurodegeneration of Alzheimer's disease (AD), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), and related tauopathies. Extracellular vesicles, specifically exosomes, have recently been demonstrated to participate in mediating tau propagation in brain. Exosomes produced by human induced pluripotent stem cell (iPSC)-derived neurons expressing mutant Tau (mTau), containing the P301L and V337M Tau mutations of FTDP-17, possess the ability to propagate p-tau pathology after injection into mouse brain.  To gain an understanding of the mTau exosome cargo involved in tau pathogenesis, these pathogenic exosomes were analyzed by proteomics and bioinformatics. The data showed that mTau expression dysregulates the exosome proteome to result in (1) proteins uniquely present only in mTau, and not control exosomes, (2) the absence of proteins in mTau exosomes, uniquely present in control exosomes, and (3) shared proteins which were significantly up-regulated or down-regulated in mTau compared to control exosomes. Notably, mTau exosomes (not control exosomes) contain ANP32A (also known as I1PP2A), an endogenous inhibitor of the PP2A phosphatase which regulates the phosphorylation state of p-tau.  Several of the mTau exosome-specific proteins have been shown to participate in AD mechanisms involving lysosomes, inflammation, secretases, and related processes.  Furthermore, the mTau exosomes lacked a substantial portion of proteins present in control exosomes involved in pathways of localization, vesicle transport, and protein binding functions. The shared proteins present in both mTau and control exosomes represented exosome functions of vesicle-mediated transport, exocytosis, and secretion processes. These data illustrate mTau as a dynamic regulator of the biogenesis of exosomes to result in acquisition, deletion, and up- or down-regulation of protein cargo to result in pathogenic mTau exosomes capable of in vivo propagation of p-tau neuropathology in mouse brain. 

The degradation of intra- and extracellular proteins is essential in all cell types and mediated by two systems, the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway. This study investigates the changes in autophagosomal and lysosomal proteomes upon inhibition of proteasomes by bortezomib (BTZ) or MG132. We find an increased abundance of more than 50 proteins in lysosomes of cells in which the proteasome is inhibited. Among those are dihydrofolate reductase (DHFR), ß-Catenin and 3-hydroxy-3-methylglutaryl-coenzym-A (HMGCoA)-reductase. Since these proteins are known to be degraded by the proteasome they seem to be compensatorily delivered to the autophagosomal pathway when the proteasome is inactivated. Surprisingly, most of the proteins which show increased amounts in the lysosomes of BTZ or MG132 treated cells are proteasomal subunits. Thus an inactivated, non-functional proteasome is delivered to the autophagic pathway. Native gel electrophoresis shows that the proteasome reaches the lysosome intact and not disassembled. Adaptor proteins, which target proteasomes to autophagy, have been described in Arabidopsis, Saccharomyces and upon starvation in mammalians. However, in cell lines deficient of these proteins or their mammalian orthologues, respectively, the transfer of proteasomes to the lysosome is not impaired. Obviously, these proteins do not play a role as autophagy adaptor proteins in mammalian cells. We can also show that chaperone-mediated autophagy (CMA) does not participate in the proteasome delivery to the lysosomes. In autophagy-related (ATG)-5 and ATG7 deficient cells the delivery of inactivated proteasomes to the autophagic pathway was only partially blocked, indicating the existence of at least two different pathways by which inactivated proteasomes can be delivered to the lysosome in mammalian cells.

Protein microarrays are crucial tools in the study of proteins in an unbiased, high-throughput manner, as they allow for characterization of up to thousands of individually purified proteins in parallel. The adaptability of this technology has enabled its use in a wide variety of applications, including the study of proteome-wide molecular interactions, analysis of post-translational modifications, identification of novel drug targets, and examination of pathogen-host interactions. In addition, the technology has also been shown to be useful in profiling antibody specificity, as well as in the discovery of novel biomarkers, especially for autoimmune diseases and cancers. In this review, we will summarize the developments that have been made in protein microarray technology in both in basic and translational research over the past decade. We will also introduce a novel membrane protein array, the GPCR-VirD array, and discuss the future directions of functional protein microarrays.

Data independent acquisition (DIA) is an attractive alternative to standard shotgun proteomics methods for quantitative experiments. However, most DIA methods require collecting exhaustive, sample-specific spectrum libraries with data dependent acquisition (DDA) to detect and quantify peptides. In addition to working with non-human samples, studies of splice junctions, sequence variants, or simply working with small sample yields can make developing DDA-based spectrum libraries impractical. Here we illustrate how to acquire, queue, and validate DIA data without spectrum libraries, and provide a workflow to efficiently generate DIA-only chromatogram libraries using gas-phase fractionation (GPF). We present best-practice methods for collecting DIA data using Orbitrap-based instruments, and develop an understanding for why DIA using an Orbitrap mass spectrometer should be approached differently than when using time-of-flight instruments. Finally, we discuss several methods for analyzing DIA data without libraries.

Molecular mechanisms underlying sperm motility have not been fully explained, particularly in chickens. The objective was to identify seminal plasma proteins associated with chicken sperm motility by comparing the seminal plasma proteomic profile of roosters with low sperm motility (LSM, n = 4) and high sperm motility (HSM, n = 4). Using a label-free MS-based method, a total of 522 seminal plasma proteins were identified, including 386 (~74%) previously reported and 136 novel ones. A total of 70 differentially abundant proteins were defined, including 48 more-abundant, 15 less-abundant, and seven proteins unique to the LSM group (specific proteins). Key secretory proteins like less-abundant ADGRG2 and more-abundant SPINK2 in the LSM suggested that the corresponding secretory tissues played a crucial role in maintaining sperm motility. Majority (80%) of the more-abundant and five specific proteins were annotated to the cytoplasmic domain which might be a result of higher plasma membrane damage and acrosome dysfunction in LSM. Additionally, more-abundant mitochondrial proteins were detected in LSM seminal plasma associated with lower spermatozoa mitochondrial membrane potential (m) and ATP concentrations. Further studies showed that the spermatozoa might be suffering from oxidative stress, as the amount of spermatozoa reactive oxygen species (ROS) were largely enhanced, seminal malondialdehyde (MDA) concentrations were increased, and the seminal plasma total antioxidant capacity (T-AOC) were decreased. Our study provides an additional catalog of chicken seminal plasma proteome and supports the idea that seminal plasma could be as an indicator of spermatozoa physiology. More-abundant of acrosome, mitochondria and sperm cytoskeleton proteins in the seminal plasma could be a marker of sperm dysfunction and loss of motility. The degeneration of spermatozoa caused the reduced seminal T-AOC and enhanced oxidative stress might be potential determinants of low sperm motility. These results could extend our understanding of sperm motility and sperm physiology regulation.

Assembly factors play a critical role in the biogenesis of mitochondrial respiratory chain complexes I-IV where they assist in the membrane insertion of subunits, attachment of co-factors, and stabilization of assembly intermediates. The major fraction of complexes I, III and IV are present together in large molecular structures known as respiratory chain supercomplexes. A number of assembly factors have been proposed as required for supercomplex assembly, including the hypoxia inducible gene 1 domain family member HIGD2A. Using gene-edited human cell lines and extensive steady state, translation and affinity enrichment proteomics techniques we show that loss of HIGD2A leads to defects in the de novo biogenesis of mtDNA-encoded COX3, subsequent accumulation of complex IV intermediates and turnover of COX3 partner proteins. Deletion of HIGD2A also leads to defective complex IV activity. The impact of HIGD2A loss on complex IV was not altered by growth under hypoxic conditions, consistent with its role being in basal complex IV assembly. While in the absence of HIGD2A we show that mitochondria do contain an altered supercomplex assembly, we demonstrate it to harbor a crippled complex IV lacking COX3. Our results redefine HIGD2A as a classical assembly factor required for building the COX3 module of complex IV.

Label-Free Quantitative mass spectrometry based workflows for differential expression (DE) analysis of proteins impose important challenges on the data analysis due to peptide-specific effects and context dependent missingness of peptide intensities. Peptide-based workflows, like MSqRob, test for DE directly from peptide intensities and outperform summarization methods which first aggregate MS1 peptide intensities to protein intensities before DE analysis. However, these methods are computationally expensive, often hard to understand for the non-specialised end-user, and do not provide protein summaries, which are important for visualisation or downstream processing. In this work, we therefore evaluate state-of-the-art summarization strategies using a benchmark spike-in dataset and discuss why and when these fail compared to the state-of-the-art peptide based model, MSqRob. Based on this evaluation, we propose a novel summarization strategy, MSqRobSum, which estimates MSqRob’s model parameters in a two-stage procedure circumventing the drawbacks of peptide-based workflows. MSqRobSum maintains MSqRob’s superior performance, while providing useful protein expression summaries for plotting and downstream analysis. Summarising peptide to protein intensities considerably reduces the computational complexity, the memory footprint and the model complexity, and makes it easier to disseminate DE inferred on protein summaries. Moreover, MSqRobSum provides a highly modular analysis framework, which provides researchers with full flexibility to develop data analysis workflows tailored towards their specific applications.

The vessel wall is continuously exposed to hemodynamic forces generated by blood flow. Endothelial mechanosensors perceive and translate mechanical signals via cellular signaling pathways into biological processes that control endothelial development, phenotype and function. To assess the hemodynamic effects on the endothelium on a system-wide level, we applied a quantitative mass spectrometry approach combined with cell surface chemical footprinting. SILAC-labeled endothelial cells were subjected to flow-induced shear stress for 0, 24 or 48 hours, followed by chemical labeling of surface proteins using a non-membrane permeable biotin label, and analysis of the whole proteome and the cell surface proteome by LC-MS/MS analysis. These studies revealed that of the >5000 quantified proteins 104 were altered, which were highly enriched for extracellular matrix proteins and proteins involved in cell-matrix adhesion. Cell surface proteomics indicated that LAMA4 was proteolytically processed upon flow-exposure, which corresponded to the decreased LAMA4 mass observed on immunoblot. Immunofluorescence microscopy studies highlighted that the endothelial basement membrane was drastically remodeled upon flow exposure. We observed a network-like pattern of LAMA4 and LAMA5, which corresponded to the localization of laminin-adhesion molecules ITGA6 and ITGB4. Furthermore, the adaptation to flow-exposure did not affect the inflammatory response to tumor necrosis factor α, indicating that inflammation and flow trigger fundamentally distinct endothelial signaling pathways with limited reciprocity and synergy. Taken together, this study uncovers the blood flow-induced remodeling of the basement membrane and stresses the importance of the subendothelial basement membrane in vascular homeostasis.

Protein cross-linking and the analysis of cross-linked peptides by mass spectrometry is currently receiving much attention. Not only is this approach applied to isolated complexes to provide information about spatial arrangements of proteins but it is also increasingly applied to entire cells and their organelles. As in quantitative proteomics, the application of isotopic labelling further makes it possible to monitor quantitative changes in the protein-protein interactions between different states of a system. Here, we cross-linked mitochondria from Saccharomyces cerevisiae grown on either glycerol- or glucose-containing medium to monitor protein-protein interactions under non-fermentative and fermentative conditions. We investigated qualitatively the protein-protein interactions of the 400 most abundant proteins applying stringent data-filtering criteria, i.e. a minimum of two cross-linked peptide spectrum matches and a cut-off in the spectrum scoring of the used search engine. The cross-linker BS3 proved to be equally suited for connecting proteins in all compartments of mitochondria when compared with its water-insoluble but membrane-permeable derivative DSS. We also applied quantitative cross-linking to mitochondria of both the growth conditions using stable-isotope labelled BS3. Significant differences of cross-linked proteins under glycerol and glucose conditions were detected, however, mainly due to the different copy numbers of these proteins in mitochondria under both the conditions. Results obtained from the glycerol condition indicate that the internal NADH:ubiquinone oxidoreductase Ndi1 is part of an electron transport chain supercomplex. We have also detected several hitherto uncharacterized proteins and identified their interaction partners. Among those, Min8 was found to be associated with cytochrome c oxidase. BN-PAGE analyses of min8 mitochondria suggest that Min8 promotes the incorporation of Cox12 into cytochrome c oxidase.

Protein subcellular localization is an essential and highly regulated determinant of protein function. Major advances in mass spectrometry and imaging have allowed the development of powerful spatial proteomics approaches for determining protein localization at the whole cell scale. Here, a brief overview of current methods is presented, followed by a detailed discussion of organellar mapping through proteomic profiling. This relatively simple yet flexible approach is rapidly gaining popularity, due to its ability to capture the localizations of thousands of proteins in a single experiment. It can be used to generate high-resolution cell maps, and as a tool for monitoring protein localization dynamics. This review highlights the strengths and limitations of the approach, and provides guidance to designing and interpreting profiling experiments. 

Protein-protein interactions are often mediated by short linear motifs (SLiMs) that are located in intrinsically disordered regions (IDRs) of proteins. Interactions mediated by SLiMs are notoriously difficult to study, and many functionally relevant interactions likely remain to be uncovered. Recently, pull-downs with synthetic peptides in combination with quantitative mass spectrometry emerged as a powerful screening approach to study protein-protein interactions mediated by SLiMs. Specifically, arrays of synthetic peptides immobilized on cellulose membranes provide a scalable means to identify the interaction partners of many peptides in parallel. In this minireview we briefly highlight the relevance of SLiMs for protein-protein interactions, outline existing screening technologies, discuss unique advantages of peptide-based interaction screens and provide practical suggestions for setting up such peptide-based screens.

The cyclic GMP-AMP synthase (cGAS) protein is a pattern-recognition receptor of the mammalian innate immune system that is recognized as a main cytosolic sensor of pathogenic or damaged DNA. cGAS DNA binding initiates catalytic production of the second messenger, cyclic GMP-AMP, which activates the STING-TBK1-IRF3 signaling axis to induce cytokine expression. Post-translational modification (PTM) has started to be recognized as a critical component of cGAS regulation, yet the extent of these modifications remains unclear. Here, we report the identification and functional analysis of cGAS phosphorylations and acetylations in several cell types under basal and immune-stimulated conditions. cGAS was enriched by immunoaffinity purification from human primary fibroblasts prior to and after infection with herpes simplex virus type 1 (HSV-1), as well as from immune-stimulated STING-HEK293T cells. Six phosphorylations and eight acetylations were detected, of which eight PTMs were not previously documented. PTMs were validated by parallel reaction monitoring (PRM) mass spectrometry in fibroblasts, HEK293T cells, and THP-1 macrophage-like cells. Primary sequence and structural analysis of cGAS highlighted a subset of PTM sites with elevated surface accessibility and high evolutionary sequence conservation. To assess the functional relevance of each PTM, we generated a series of single-point cGAS mutations. Stable cell lines were constructed to express cGAS with amino acid substitutions that prevented phosphorylation (Ser-to-Ala) and acetylation (Lys-to-Arg) or that mimicked the modification state (Ser-to-Asp and Lys-to-Gln). cGAS-dependent apoptotic and immune signaling activities were then assessed for each mutation. Our results show that acetyl-mimic mutations at Lys384 and Lys414 inhibit the ability of cGAS to induce apoptosis. In contrast, the Lys198 acetyl-mimic mutation increased cGAS-dependent interferon signaling when compared to the unmodified charge-mimic. Moreover, targeted PRM quantification showed that Lys198 acetylation is decreased upon infections with two herpesviruses—HSV-1 and human cytomegalovirus (HCMV), highlighting this residue as a regulatory point during virus infection.

The presentation of post-translationally modified (PTM) peptides by cell surface HLA molecules has the potential to increase the diversity of targets for surveilling T cells. Whilst immunopeptidomics studies routinely identify thousands of HLA-bound peptides from cell lines and tissue samples, in-depth analyses of the proportion and nature of peptides bearing one or more PTMs remains challenging. Here we have analyzed HLA-bound peptides from a variety of allotypes and assessed the distribution of mass spectrometry-detected PTMs, finding deamidation of asparagine or glutamine to be highly prevalent. Given that asparagine deamidation may arise either spontaneously or through enzymatic reaction, we assessed allele-specific and global motifs flanking the modified residues. Notably, we found that the N-linked glycosylation motif NX(S/T) was highly abundant across asparagine-deamidated HLA-bound peptides. This finding, demonstrated previously for a handful of deamidated T cell epitopes, implicates a more global role for the retrograde transport of nascently N-glycosylated polypeptides from the ER and their subsequent degradation within the cytosol to form HLA-ligand precursors. Chemical inhibition of Peptide:N-Glycanase (PNGase), the endoglycosidase responsible for the removal of glycans from misfolded and retrotranslocated glycoproteins, greatly reduced presentation of this subset of deamidated HLA-bound peptides. Importantly, there was no impact of PNGase inhibition on peptides not containing a consensus NX(S/T) motif. This indicates that a large proportion of HLA-I bound asparagine deamidated peptides are generated from formerly glycosylated proteins that have undergone deglycosylation via the ER-associated protein degradation (ERAD) pathway. The information herein will help train deamidation prediction models for HLA-peptide repertoires and aid in the design of novel T cell therapeutic targets derived from glycoprotein antigens.

Campylobacter jejuni is a major cause of food-borne gastroenteritis. Proteomics by label-based two-dimensional liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) identified proteins associated with growth in 0.1% sodium deoxycholate (DOC, a component of gut bile salts), and system-wide validation was performed by data-independent acquisition (DIA-SWATH-MS). LC-MS/MS quantified 1326 proteins (~82% of the predicted C. jejuni proteome), of which 1104 were validated in additional biological replicates by DIA-SWATH-MS. DOC resulted in a profound proteome shift with 512 proteins showing significantly altered abundance. Induced proteins were associated with flagellar motility and antibiotic resistance; and these correlated with increased DOC motility and resistance to polymyxin B and ciprofloxacin. DOC also increased human Caco-2 cell adherence and invasion. Abundances of proteins involved in nutrient transport were altered by DOC and aligned with intracellular changes to their respective carbon sources. DOC increased intracellular levels of sulfur-containing amino acids (cysteine and methionine) and the dipeptide cystine (Cys-Cys), which also correlated with reduced resistance to oxidative stress. A DOC induced transport protein was Cj0025c, which has sequence similarity to bacterial Cys-Cys transporters. Deletion of cj0025c (cj0025c) resulted in proteome changes consistent with sulfur starvation, as well as attenuated invasion, reduced motility, atypical morphology, increased antimicrobial susceptibility and poor biofilm formation. Targeted metabolomics showed cj0025c was capable of utilizing known C. jejuni amino and organic acid substrates commensurate with wild-type. Medium Cys-Cys levels however, were maintained in cj0025c relative to wild-type. A toxic Cys-Cys mimic (selenocystine) inhibited wild-type growth, but not cj0025c. Provision of an alternate sulfur source (2 mM thiosulfate) restored cj0025c motility. Our data confirm that Cj0025c is a Cys-Cys transporter that we have named TcyP consistent with the nomenclature of homologous proteins in other species.

Perlecan is a critical proteoglycan found in the extracellular matrix (ECM) of cartilage. In healthy cartilage, perlecan regulates cartilage biomechanics and we previously demonstrated perlecan deficiency leads to reduced cellular and ECM stiffness in vivo. This change in mechanics may lead to the early onset osteoarthritis seen in disorders resulting from perlecan knockdown such as Schwartz-Jampel syndrome (SJS). To identify how perlecan knockdown affects the material properties of developing cartilage, we used imaging and liquid chromatography–tandem mass spectrometry (LC-MS/MS) to study the ECM in a murine model of SJS, Hspg2C1532Y-Neo. Perlecan knockdown led to defective pericellular matrix formation, whereas the abundance of bulk ECM proteins, including many collagens, increased. Post-translational modifications and ultrastructure of collagens were not significantly different; however, LC-MS/MS analysis showed more protein was secreted by Hspg2C1532Y-Neo cartilage in vitro, suggesting that the incorporation of newly synthesized ECM was impaired. In addition, glycosaminoglycan deposition was atypical, which may explain the previously observed decrease in mechanics. Overall, these findings provide insight into the influence of perlecan on functional cartilage assembly and the progression of osteoarthritis in SJS.

1 April 2020

Considerations of justice and social equity are as important for the circular economy transition as they are in the contexts of low-carbon transitions and digitalization of the economy. This paper sets out the just transition approach, and its relevance in climate change and energy transition debates.

1 January 2008 , Number 2

Voters will be heading for the polls in Pakistan on January 8. But however the ballots are cast, the people will not necessarily decide who will form the next government. Power is at stake, and all the players are calculating how best to retain or acquire it.

1 January 2008 , Number 10

At the beginning of December, media reports put the violence which had plagued Algeria since the 1990s at an all time low. Only six people had been killed in November and four of those were armed Islamist militants. Then on December 11, two car bombs exploded in the capital Algiers, killing some 67 people, including at least eleven United Nations officials in the largely destroyed offices of the UN’s High Commission for Refugees. What lies beneath this return to high-level, targeted attacks?

1 February 2008 , Number 5

1 February 2008 , Number 3

1 May 2008 , Number 3

Rowdy sessions of the Iraq High Tribunal attracted sensational daily news coverage while Saddam Hussein was being tried. However, following his grim execution in December 2006, coverage all but evaporated. The foreign press and most western monitors packed their bags and left, and television reporting in Iraq dwindled. Now even the United States Department of Justice, which initially provided key financial and political support, is quietly withdrawing its advisers. So what is happening at the Tribunal and why are the Americans pulling out?

1 June 2008 , Number 3

For the third time in less than two years, Beirut has been turned into a battleground, with Hizbollah and the opposition taking over the city. The government and its allies did not engage militarily, managing to avoid civil war and becoming a regional battlefield. The Arab League is now trying to sort things out, highlighting the regional and international dimensions.

1 June 2008 , Number 6

Dmitri Medvedev’s appearance at the G8 Summit in Hokkaido will be his first step on the wide international stage that Vladimir Putin occupied with such a swagger. Expectations of change will be high, but they are likely to be unrealistic at such an early stage in Medvedev’s presidency. Putin’s foreign policy legacy is a heavy one and he has made it clear that Medvedev will be no soft touch, but will his approach ultimately be more productive?

1 July 2008 , Number 3

Ahead of the French holiday rush for relaxation in warm waters, President Nicolas Sarkozy has flung himself into the Mediterranean policy pool. Not content with existing arrangements, plans and processes for dealing with those on the other shore, the President has built his very own scheme, now adopted by the European Union. But the tides have not been altogether kind.

1 July 2008 , Number 9

Oil people call them pipe dreams: plans to transport energy across vast distances to places where it is needed most. That need is clear in Pakistan and India, but can the obstacles be overcome at this time of high prices, to turn the recurring dreams into reality?

1 August 2008 , Number 10

As ever between NATO and Ukraine, the process advances, the destination is in doubt. Or so it seemed until the Bucharest summit declaration of April 3. The statement that Ukraine and Georgia ‘will become members of NATO’ was designed to remove doubt. Yet it was also designed to alleviate pressure on the organisation. Four months later, as the European Union prepares for its summit with Ukraine, pressure remains and doubt has returned.

1 October 2008 , Number 3

Dilemmas and dangers abound for the west as it tries to come to terms with the ‘new’ Russia. There is a mood of defiance and injury, which can only be answered with firmness and prudence.

1 October 2008 , Number 4

Russia’s military action in Georgia was driven by Moscow’s domestic agenda. Foreign policy has become a tool of collective control back home. While the ‘ruling tandem’ has gained popularity, the new consensus does not provide for modernisation. Will the predatory state reform or collapse?

1 October 2008 , Number 11

Nuclear, biological or chemical weapons and acts of terror may make the headlines, but it is conventional arms that take the lives in large numbers; maybe around a thousand a day. This month, a United Nations committee will try to find a way to limit the arms trade with a new treaty. For those facing the barrel of a gun, it cannot come a moment too soon.

20 April 2020

Nuclear deterrence theory, with its roots in the Cold War era, may not account for all eventualities in the 21st century. Researchers at Chatham House have worked with eight experts to produce this collection of essays examining four contested themes in contemporary policymaking on deterrence.

1 November 2008 , Number 1

The new American president will not be inaugurated until January 20. He will certainly face the most difficult economic conditions since Franklin Roosevelt entered the White House in March 1933. The politics of presidential transition – in this year, as seventy-six years ago – seem likely only to exacerbate the global crisis.

1 November 2008 , Number 4

Russia is caught in the global crisis and cannot escape its impact. The crucial question is how the Dmitri Medvedev-Vladimir Putin leadership will respond. Putin has presided over a steadily strengthening economy; he now appears ill-equipped to handle crisis and contraction. The signs are not encouraging. Trust and confidence, two essential ingredients vital to resolving any financial crisis, are in short supply. The public could pay a heavy price for the hubris and schadenfreude of their leaders, still ‘dizzy with success’ from years of economic revival and what they perceive as a successful reaffirmation of the country’s great power status.

1 November 2008 , Number 7

An air of lassitude pervades the Iranian nuclear talks. Will things look different after the American election? What should be done to raise the chances of a good outcome?

1 November 2008 , Number 8

Ghana’s President John Kufuor has successfully promoted his country as a haven of political and economic stability, but as he comes to the end of his two terms in office, the cracks are beginning to appear. Can Ghana escape the election traumas that have been so destructive in Kenya and Zimbabwe?

1 November 2008 , Number 1

The race is on to ensure that relations between the United States and its European allies are set on the right track from the outset of Barack Obama’s presidency. Although they may not like it, the main responsibility for ensuring that the transatlantic relationship does not stumble into a series of disappointed expectations in its first critical year lies more in European capitals than in Washington.

1 November 2008 , Number 6

A new climate is likely at the United Nations climate change conference in Poland early this month and not just because of the election of Barack Obama as President of the United States. The international financial crisis has highlighted the cost of poor policies and the scale of banking bailouts has made dealing with climate change seem less formidable. Besides, such schemes could create new jobs and give an edge to the competitive economies of tomorrow.

1 January 2007 , Number 2

For all the cynicism it has evoked, the United States’ Iraq Study Group report might still lead to a regional diplomatic process, which could ease Iran and the US into negotiations. A grand bargain is very unlikely, but with Washington at the table, there might be enough advantage for both sides in a gradual process to halt the current slide towards a deeper confrontation.

1 February 2007 , Number 1

Russia is playing a risky game in Iran, continuing to build a nuclear power plant while supporting United Nations sanctions on Tehran for its nuclear policy. Moscow may be hoping for a deal with the west, but if restraint fails and Iran goes nuclear, the missiles would be too close for comfort. Equally a pre-emptive American or Israeli strike on Iranian nuclear sites might unleash serious consequences for Russia too.