Proteogenomic Features of the Highly Polymorphic Histidine-rich Glycoprotein Arose Late in Evolution.

Histidine-rich glycoprotein (HRG) is a liver-produced protein circulating in human serum at high concentrations of around 125 μg/ml. HRG belongs to the family of type-3 cystatins and has been implicated in a plethora of biological processes, albeit that its precise function is still not well understood. Human HRG is a highly polymorphic protein, with at least five variants with minor allele frequencies of more than 10%, variable in populations from different parts of the world.

Identification of Protein Complexes by Integrating Protein Abundance and Interaction Features Using a Deep Learning Strategy.

Many essential cellular functions are carried out by multi-protein complexes that can be characterized by their protein-protein interactions. The interactions between protein subunits are critically dependent on the strengths of their interactions and their cellular abundances, both of which span orders of magnitude. Despite many efforts devoted to the global discovery of protein complexes by integrating large-scale protein abundance and interaction features, there is still room for improvement.

Schistosoma mansoni infection affects the proteome and lipidome of circulating extracellular vesicles in the host.

Eggs, schistosomula and adult Schistosoma worms are known to release extracellular vesicles (EV) during in vitro incubations and these EVs are postulated to affect the host responses. So far only those EVs released during in vitro incubations of schistosomes have been studied and it is unknown whether in blood of infected hosts the schistosomal EVs can be detected amidst all the circulating EVs of the host itself. In this study we analyzed the protein as well as the phospholipid composition of EVs circulating in blood plasma of S.

An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas.

Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti(4+)-IMAC-based enrichment.

Spatial Organization in Protein Kinase A Signaling Emerged at the Base of Animal Evolution.

In phosphorylation-directed signaling, spatial and temporal control is organized by complex interaction networks that diligently direct kinases toward distinct substrates to fine-tune specificity. How these protein networks originate and evolve into complex regulatory machineries are among the most fascinating research questions in biology. Here, spatiotemporal signaling is investigated by tracing the evolutionary dynamics of each functional domain of cAMP-dependent protein kinase (PKA) and its diverse set of A-kinase anchoring proteins (AKAPs).

ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma.

Treatment of BRAF mutant melanomas with specific BRAF inhibitors leads to tumor remission. However, most patients eventually relapse due to drug resistance. Therefore, we designed an integrated strategy using (phospho)proteomic and functional genomic platforms to identify drug targets whose inhibition sensitizes melanoma cells to BRAF inhibition.

Proteome adaptation of Saccharomyces cerevisiae to severe calorie restriction in Retentostat cultures.

Stationary-phase, carbon-starved shake-flask cultures of Saccharomyces cerevisiae are popular models for studying eukaryotic chronological aging. However, their nutrient-starved physiological status differs substantially from that of postmitotic metazoan cells. Retentostat cultures offer an attractive alternative model system in which yeast cells, maintained under continuous calorie restriction, hardly divide but retain high metabolic activity and viability for prolonged periods of time.

Facilitating protein disulfide mapping by a combination of pepsin digestion, electron transfer higher energy dissociation (EThcD), and a dedicated search algorithm SlinkS.

Disulfide bond identification is important for a detailed understanding of protein structures, which directly affect their biological functions. Here we describe an integrated workflow for the fast and accurate identification of authentic protein disulfide bridges. This novel workflow incorporates acidic proteolytic digestion using pepsin to eliminate undesirable disulfide reshuffling during sample preparation and a novel search engine, SlinkS, to directly identify disulfide-bridged peptides isolated via electron transfer higher energy dissociation (EThcD).

qcML: an exchange format for quality control metrics from mass spectrometry experiments.

Quality control is increasingly recognized as a crucial aspect of mass spectrometry based proteomics. Several recent papers discuss relevant parameters for quality control and present applications to extract these from the instrumental raw data. What has been missing, however, is a standard data exchange format for reporting these performance metrics.

Identification of enriched PTM crosstalk motifs from large-scale experimental data sets.

Post-translational modifications (PTMs) play an important role in the regulation of protein function. Mass spectrometry based proteomics experiments nowadays identify tens of thousands of PTMs in a single experiment. A wealth of data has therefore become publically available.

Deep proteome profiling of circulating granulocytes reveals bactericidal/permeability-increasing protein as a biomarker for severe atherosclerotic coronary stenosis.

Coronary atherosclerosis represents the major cause of death in Western societies. As atherosclerosis typically progresses over years without giving rise to clinical symptoms, biomarkers are urgently needed to identify patients at risk. Over the past decade, evidence has accumulated suggesting cross-talk between the diseased vasculature and cells of the innate immune system.

The PRoteomics IDEntification (PRIDE) Converter 2 framework: an improved suite of tools to facilitate data submission to the PRIDE database and the ProteomeXchange consortium.

The original PRIDE Converter tool greatly simplified the process of submitting mass spectrometry (MS)-based proteomics data to the PRIDE database. However, after much user feedback, it was noted that the tool had some limitations and could not handle several user requirements that were now becoming commonplace. This prompted us to design and implement a whole new suite of tools that would build on the successes of the original PRIDE Converter and allow users to generate submission-ready, well-annotated PRIDE XML files.

Current challenges in software solutions for mass spectrometry-based quantitative proteomics.

Mass spectrometry-based proteomics has evolved as a high-throughput research field over the past decade. Significant advances in instrumentation, and the ability to produce huge volumes of data, have emphasized the need for adequate data analysis tools, which are nowadays often considered the main bottleneck for proteomics development. This review highlights important issues that directly impact the effectiveness of proteomic quantitation and educates software developers and end-users on available computational solutions to correct for the occurrence of these factors.

High confidence and sensitivity four-dimensional fractionation for human plasma proteome analysis.

Reducing the complexity of plasma proteome through complex multidimensional fractionation protocols is critical for the detection of low abundance proteins that have the potential to be the most specific disease biomarkers. Therefore, we examined a four dimension profiling method, which includes low abundance protein enrichment, tryptic digestion and peptide fractionation by IEF, SCX and RP-LC. The application of peptide pI filtering as an additional criterion for the validation of the identifications allows to minimize the false discovery rate and to optimize the best settings of the protein identification database search engine.

Database independent proteomics analysis of the ostrich and human proteome.

Mass spectrometry (MS)-based proteome analysis relies heavily on the presence of complete protein databases. Such a strategy is extremely powerful, albeit not adequate in the analysis of unpredicted postgenome events, such as posttranslational modifications, which exponentially increase the search space. Therefore, it is of interest to explore "database-free" approaches.

Evaluating experimental bias and completeness in comparative phosphoproteomics analysis.

Unraveling the functional dynamics of phosphorylation networks is a crucial step in understanding the way in which biological networks form a living cell. Recently there has been an enormous increase in the number of measured phosphorylation events. Nevertheless, comparative and integrative analysis of phosphoproteomes is confounded by incomplete coverage and biases introduced by different experimental workflows.

Improving SRM assay development: a global comparison between triple quadrupole, ion trap, and higher energy CID peptide fragmentation spectra.

In proteomics, selected reaction monitoring (SRM) is rapidly gaining importance for targeted protein quantification. The triple quadrupole mass analyzers used in SRM assays allow for levels of specificity and sensitivity hard to accomplish by more standard shotgun proteomics experiments. Often, an SRM assay is built by in silico prediction of transitions and/or extraction of peptide precursor and fragment ions from a spectral library.

Deconvolution of overlapping isotopic clusters improves quantification of stable isotope-labeled peptides.

High-resolution mass spectrometry and the use of stable isotopes have greatly improved our ability to quantify proteomes. Typically, the relative abundance of peptides is estimated by identifying the isotopic clusters and by comparing the peak intensities of peptide pairs. However, when the mass shift between the labeled peptides is small, there can be the possibility for overlap of the isotopic clusters which will hamper quantification accuracy with a typical upwards bias for the heavier peptide.

A high-throughput processing service for retention time alignment of complex proteomics and metabolomics LC-MS data.

Unlabelled: Warp2D is a novel time alignment approach, which uses the overlapping peak volume of the reference and sample peak lists to correct misleading peak shifts. Here, we present an easy-to-use web interface for high-throughput Warp2D batch processing time alignment service using the Dutch Life Science Grid, reducing processing time from days to hours. This service provides the warping function, the sample chromatogram peak list with adjusted retention times and normalized quality scores based on the sum of overlapping peak volume of all peaks.

RockerBox: analysis and filtering of massive proteomics search results.

A major problem in the analysis of mass spectrometry-based proteomics data is the vast growth of data volume, caused by improvements in sequencing speed of mass spectrometers. This growth affects analysis times and storage requirements so severely that many analysis tools are no longer able to cope with the increased file sizes. We present a tool, RockerBox, to address size problems for search results obtained from the widely used Mascot search engine.

Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels.

Background: The addition of an acetyl group to protein N-termini is a widespread co-translational modification. NatB is one of the main N-acetyltransferases that targets a subset of proteins possessing an N-terminal methionine, but so far only a handful of substrates have been reported. Using a yeast nat3Δ strain, deficient for the catalytic subunit of NatB, we employed a quantitative proteomics strategy to identify NatB substrates and to characterize downstream effects in nat3Δ.

LysNDeNovo: an algorithm enabling de novo sequencing of Lys-N generated peptides fragmented by electron transfer dissociation.

Recently, we introduced a novel proteomics method employing a metalloendopeptidase with Lys-N specificity to produce proteolytic peptides. Fragmentation spectra generated by electron transfer dissociation, for a large proportion of the Lys-N proteolytic peptides, were found to be dominated by extensive series of c-type ions. Taking advantage of this unique spectral property, we developed an algorithm, LysNDeNovo, to facilitate de novo sequencing of these peptides.