The aim of the UniProt Knowledgebase is to provide users with a comprehensive, high-quality and freely accessible set of protein sequences annotated with functional information. In this article, we describe significant updates that we have made over the last two years to the resource. The number of sequences in UniProtKB has risen to approximately 190 million, despite continued work to reduce sequence redundancy at the proteome level. We have adopted new methods of assessing proteome completeness and quality. We continue to extract detailed annotations from the literature to add to reviewed entries and supplement these in unreviewed entries with annotations provided by automated systems such as the newly implemented Association-Rule-Based Annotator (ARBA). We have developed a credit-based publication submission interface to allow the community to contribute publications and annotations to UniProt entries. We describe how UniProtKB responded to the COVID-19 pandemic through expert curation of relevant entries that were rapidly made available to the research community through a dedicated portal. UniProt resources are available under a CC-BY (4.0) license via the web at https://www.uniprot.org/.

The aim of the UniProt Knowledgebase is to provide users with a comprehensive, high-quality and freely accessible set of protein sequences annotated with functional information. In this publication we describe enhancements made to our data processing pipeline and to our website to adapt to an ever-increasing information content. The number of sequences in UniProtKB has risen to over 227 million and we are working towards including a reference proteome for each taxonomic group. We continue to extract detailed annotations from the literature to update or create reviewed entries, while unreviewed entries are supplemented with annotations provided by automated systems using a variety of machine-learning techniques. In addition, the scientific community continues their contributions of publications and annotations to UniProt entries of their interest. Finally, we describe our new website (https://www.uniprot.org/), designed to enhance our users’ experience and make our data easily accessible to the research community. This interface includes access to AlphaFold structures for more than 85% of all entries as well as improved visualisations for subcellular localisation of proteins.

Key Points• Hypoxia induces altered platelet proteome/reactivity, which correlates with a prothrombotic phenotype.• CAPNS1-dependent calpain activity in platelet activation cascade is associated with hypoxia-induced thrombogenesis.Oxygen-compromised environments, such as high altitude, air travel, and sports, and pathological conditions, such as solid tumors, have been suggested to be prothrombotic. Despite the indispensable role of platelets in thrombus formation, the studies linking hypoxia, platelet reactivity, and thrombus formation are limited. In the present study, platelet proteome/reactivity was analyzed to elucidate the acute hypoxia-induced prothrombotic phenotype. Rats exposed to acute simulated hypoxia (282 torr/8% oxygen) demonstrated a decreased bleeding propensity and increased platelet reactivity. Proteomic analysis of hypoxic platelets revealed 27 differentially expressed proteins, including those involved in coagulation. Among these proteins, calpain small subunit 1, a 28-kDa regulatory component for calpain function, was significantly upregulated under hypoxic conditions. Moreover, intraplatelet Ca 21 level and platelet calpain activity were also found to be in accordance with calpain small subunit 1 expression. The inhibition of calpain activity demonstrated reversal of hypoxia-induced platelet hyperreactivity. The prothrombotic role for calpain was further confirmed by an in vivo model of hypoxia-induced thrombosis. Interestingly, patients who developed thrombosis while at extreme altitude had elevated plasma calpain activities and increased soluble P-selectin level. In summary, this study suggests that augmented calpain activity is associated with increased incidence of thrombosis under hypoxic environments. (Blood. 2014;123(8):1250-1260

Plasma is the most easily accessible source for biomarker discovery in clinical proteomics. However, identifying potential biomarkers from plasma is a challenge given the large dynamic range of proteins. The potential biomarkers in plasma are generally present at very low abundance levels and hence identification of these low abundance proteins necessitates the depletion of highly abundant proteins. Sample pre-fractionation using immuno-depletion of high abundance proteins using multi-affinity removal system (MARS) has been a popular method to deplete multiple high abundance proteins. However, depletion of these abundant proteins can result in concomitant removal of low abundant proteins. Although there are some reports suggesting the removal of non-targeted proteins, the predominant view is that number of such proteins is small. In this study, we identified proteins that are removed along with the targeted high abundant proteins. Three plasma samples were depleted using each of the three MARS (Hu-6, Hu-14 and Proteoprep 20) cartridges. The affinity bound fractions were subjected to gelC-MS using an LTQ-Orbitrap instrument. Using four database search algorithms including MassWiz (developed in house), we selected the peptides identified at <1% FDR. Peptides identified by at least two algorithms were selected for protein identification. After this rigorous bioinformatics analysis, we identified 101 proteins with high confidence. Thus, we believe that for biomarker discovery and proper quantitation of proteins, it might be better to study both bound and depleted fractions from any MARS depleted plasma sample.

DNA methylation is crucial for gene regulation and maintenance of genomic stability. Rat has been a key model system in understanding mammalian systemic physiology, however detailed rat methylome remains uncharacterized till date. Here, we present the first high resolution methylome of rat liver generated using Methylated DNA immunoprecipitation and high throughput sequencing (MeDIP-Seq) approach. We observed that within the DNA/RNA repeat elements, simple repeats harbor the highest degree of methylation. Promoter hypomethylation and exon hypermethylation were common features in both RefSeq genes and expressed genes (as evaluated by proteomic approach). We also found that although CpG islands were generally hypomethylated, about 6% of them were methylated and a large proportion (37%) of methylated islands fell within the exons. Notably, we obeserved significant differences in methylation of terminal exons (UTRs); methylation being more pronounced in coding/partially coding exons compared to the non-coding exons. Further, events like alternate exon splicing (cassette exon) and intron retentions were marked by DNA methylation and these regions are retained in the final transcript. Thus, we suggest that DNA methylation could play a crucial role in marking coding regions thereby regulating alternative splicing. Apart from generating the first high resolution methylome map of rat liver tissue, the present study provides several critical insights into methylome organization and extends our understanding of interplay between epigenome, gene expression and genome stability.

Data-dependent acquisition (DDA) and data-independent acquisition strategies (DIA) have both resulted in improved understanding of proteomics samples. Both strategies have advantages and disadvantages that are well-published, where DDA is typically applied for deep discovery and DIA may be used to create sample records. In this paper, we present a hybrid data acquisition and processing strategy (pSMART) that combines the strengths of both techniques and provides significant benefits for qualitative and quantitative peptide analysis. The performance of pSMART is compared to published DIA strategies in an experiment that allows the objective assessment of DIA performance with respect to interrogation of previously acquired MS data. The results of this experiment demonstrate that pSMART creates fewer decoy hits than a standard DIA strategy. Moreover, we show that pSMART is more selective, sensitive, and reproducible than either standard DIA or DDA strategies alone.

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