Building high-quality assay libraries for targeted analysis of SWATH MS data.

Targeted proteomics by selected/multiple reaction monitoring (S/MRM) or, on a larger scale, by SWATH (sequential window acquisition of all theoretical spectra) MS (mass spectrometry) typically relies on spectral reference libraries for peptide identification. Quality and coverage of these libraries are therefore of crucial importance for the performance of the methods. Here we present a detailed protocol that has been successfully used to build high-quality, extensive reference libraries supporting targeted proteomics by SWATH MS.

RNF168 promotes noncanonical K27 ubiquitination to signal DNA damage.

Ubiquitination regulates numerous cellular processes by generating a versatile communication system based on eight structurally and functionally different chains linked through distinct residues. Except for K48 and K63, the biological relevance of different linkages is largely unclear. Here, we show that RNF168 ubiquitin ligase promotes noncanonical K27-linked ubiquitination both in vivo and in vitro.

Reproducible and consistent quantification of the Saccharomyces cerevisiae proteome by SWATH-mass spectrometry.

Targeted mass spectrometry by selected reaction monitoring (S/MRM) has proven to be a suitable technique for the consistent and reproducible quantification of proteins across multiple biological samples and a wide dynamic range. This performance profile is an important prerequisite for systems biology and biomedical research. However, the method is limited to the measurements of a few hundred peptides per LC-MS analysis.

Mass spectrometry-based proteomic quest for diabetes biomarkers.

Diabetes mellitus (DM) is a metabolic disorder characterized by chronic hyperglycemia, which affects hundreds of millions of individuals worldwide. Early diagnosis and complication prevention of DM are helpful for disease treatment. However, currently available DM diagnostic markers fail to achieve the goals.

Chemical cross-linking/mass spectrometry maps the amyloid β peptide binding region on both apolipoprotein E domains.

Apolipoprotein E (apoE) binds the amyloid β peptide (Aβ), one of the major culprits in Alzheimer's disease development. The formation of apoE:Aβ complexes is implicated in both Aβ clearance and fibrillization. However, the binding interface between apoE and Aβ is poorly defined despite substantial previous research efforts, and the exact role of apoE in the pathology of Alzheimer's disease remains largely elusive.

mzDB: a file format using multiple indexing strategies for the efficient analysis of large LC-MS/MS and SWATH-MS data sets.

The analysis and management of MS data, especially those generated by data independent MS acquisition, exemplified by SWATH-MS, pose significant challenges for proteomics bioinformatics. The large size and vast amount of information inherent to these data sets need to be properly structured to enable an efficient and straightforward extraction of the signals used to identify specific target peptides. Standard XML based formats are not well suited to large MS data files, for example, those generated by SWATH-MS, and compromise high-throughput data processing and storing.

Using data-independent, high-resolution mass spectrometry in protein biomarker research: perspectives and clinical applications.

In medicine, there is an urgent need for protein biomarkers in a range of applications that includes diagnostics, disease stratification, and therapeutic decisions. One of the main technologies to address this need is MS, used for protein biomarker discovery and, increasingly, also for protein biomarker validation. Currently, data-dependent analysis (also referred to as shotgun proteomics) and targeted MS, exemplified by SRM, are the most frequently used mass spectrometric methods.

DIGESTIF: a universal quality standard for the control of bottom-up proteomics experiments.

In bottom-up mass spectrometry-based proteomics analyses, variability at any step of the process, particularly during sample proteolysis, directly affects the sensitivity, accuracy, and precision of peptide detection and quantification. Currently, no generic internal standards are available to control the quality of sample processing steps. This makes it difficult to assess the comparability of MS proteomic data obtained under different experimental conditions.

Phosphoproteomic analyses reveal novel cross-modulation mechanisms between two signaling pathways in yeast.

Cells respond to environmental stimuli via specialized signaling pathways. Concurrent stimuli trigger multiple pathways that integrate information, predominantly via protein phosphorylation. Budding yeast responds to NaCl and pheromone via two mitogen-activated protein kinase cascades, the high osmolarity, and the mating pathways, respectively.

Architecture and conformational switch mechanism of the ryanodine receptor.

Muscle contraction is initiated by the release of calcium (Ca(2+)) from the sarcoplasmic reticulum into the cytoplasm of myocytes through ryanodine receptors (RyRs). RyRs are homotetrameric channels with a molecular mass of more than 2.2 megadaltons that are regulated by several factors, including ions, small molecules and proteins.

Checkpoint blockade cancer immunotherapy targets tumour-specific mutant antigens.

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells.

The structural basis of substrate recognition by the eukaryotic chaperonin TRiC/CCT.

The eukaryotic chaperonin TRiC (also called CCT) is the obligate chaperone for many essential proteins. TRiC is hetero-oligomeric, comprising two stacked rings of eight different subunits each. Subunit diversification from simpler archaeal chaperonins appears linked to proteome expansion.

Characterization of a DNA exit gate in the human cohesin ring.

Chromosome segregation depends on sister chromatid cohesion mediated by cohesin. The cohesin subunits Smc1, Smc3, and Scc1 form tripartite rings that are thought to open at distinct sites to allow entry and exit of DNA. However, direct evidence for the existence of open forms of cohesin is lacking.

Enzymatic generation of peptides flanked by basic amino acids to obtain MS/MS spectra with 2× sequence coverage.

Rationale: Tandem mass (MS/MS) spectra generated by collision-induced dissociation (CID) typically lack redundant peptide sequence information in the form of e.g. b- and y-ion series due to frequent use of sequence-specific endopeptidases cleaving C- or N-terminal to Arg or Lys residues.

Identification of Cdk targets that control cytokinesis.

The final event of the eukaryotic cell cycle is cytokinesis, when two new daughter cells are born. How the timing and execution of cytokinesis is controlled is poorly understood. Here, we show that downregulation of cyclin-dependent kinase (Cdk) activity, together with upregulation of its counteracting phosphatase Cdc14, controls each of the sequential steps of cytokinesis, including furrow ingression, membrane resolution and cell separation in budding yeast.

The complete structure of the large subunit of the mammalian mitochondrial ribosome.

Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria. Mammalian mitoribosomes, which comprise 39S and 28S subunits, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes. We have previously determined at 4.

Multilayered genetic and omics dissection of mitochondrial activity in a mouse reference population.

The manner by which genotype and environment affect complex phenotypes is one of the fundamental questions in biology. In this study, we quantified the transcriptome--a subset of the metabolome--and, using targeted proteomics, quantified a subset of the liver proteome from 40 strains of the BXD mouse genetic reference population on two diverse diets. We discovered dozens of transcript, protein, and metabolite QTLs, several of which linked to metabolic phenotypes.

Molecular architecture of the 40S⋅eIF1⋅eIF3 translation initiation complex.

Eukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits.

Structural and biochemical characterization of the Cop9 signalosome CSN5/CSN6 heterodimer.

The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit.

Direct analytical sample quality assessment for biomarker investigation: qualifying cerebrospinal fluid samples.

Measurement of biochemical markers represents an important aid to clinicians in the early diagnosis and prognosis of neurological diseases. Many factors can contribute to increase the chances that a biomarker study becomes successful. In a cerebrospinal fluid analysis (CSF), more than 84% of laboratory errors can be attributed to several preanalytical variables that include CSF collection, storage, and freeze thawing cycles.

Quantitative ADME proteomics - CYP and UGT enzymes in the Beagle dog liver and intestine.

Purpose: Beagle dogs are used to study oral pharmacokinetics and guide development of drug formulations for human use. Since mechanistic insight into species differences is needed to translate findings in this species to human, abundances of cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) drug metabolizing enzymes have been quantified in dog liver and intestine.

Methods: Abundances of enzymes were measured in Beagle dog intestine and liver using selected reaction monitoring mass spectrometry.

Phosphoproteomic analysis identifies proteins involved in transcription-coupled mRNA decay as targets of Snf1 signaling.

Stresses, such as glucose depletion, activate Snf1, the Saccharomyces cerevisiae ortholog of adenosine monophosphate-activated protein kinase (AMPK), enabling adaptive cellular responses. In addition to affecting transcription, Snf1 may also promote mRNA stability in a gene-specific manner. To understand Snf1-mediated signaling, we used quantitative mass spectrometry to identify proteins that were phosphorylated in a Snf1-dependent manner.

Spatial proteomic and phospho-proteomic organization in three prototypical cell migration modes.

Background: Tight spatio-temporal signaling of cytoskeletal and adhesion dynamics is required for localized membrane protrusion that drives directed cell migration. Different ensembles of proteins are therefore likely to get recruited and phosphorylated in membrane protrusions in response to specific cues.

Results: HERE, WE USE AN ASSAY THAT ALLOWS TO BIOCHEMICALLY PURIFY EXTENDING PROTRUSIONS OF CELLS MIGRATING IN RESPONSE TO THREE PROTOTYPICAL RECEPTORS: integrins, recepor tyrosine kinases and G-coupled protein receptors.