The high affinity of biotin to streptavidin has made it one of the most widely used affinity tags in proteomics. Early methods used biotin for enrichment alone and mostly ignored the biotin-labeled ...peptide. Recent advances in labeling have led to an increase in biotinylation efficiency and shifted the interest to the detection of the site of biotinylation. This has increased the confidence in identification and provided additional structural information, yet it requires the efficient release of the biotinylated protein/peptide and the sensitive separation and detection of biotinylated peptides by LC–MS/MS. Despite its long use in affinity proteomics, the effect of biotinylation on the chromatographic, ionization, and fragmentation behavior and the ultimate detection of peptides is not well understood. To address this, we compare two commercially available biotin labels, EZ-Link Sulfo-NHS-Biotin and Sulfo-NHS-SS-Biotin, the latter containing a labile linker to efficiently release biotin to determine the effects of peptide modification on peptide detection. We describe an increase in the hydrophobicity and charge reduction with an increasing number of biotin labels attached. On the basis of our data, we recommend gradient optimization to account for more hydrophobic biotinylated peptides and include singly charged precursors to account for charge reduction by biotin.
Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological ...pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8-/- versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and validated evidence for the existence of the protease web, a network that affects the activity of most proteases and thereby influences the functional state of the proteome and cell activity.
The nonlinear signal response of electrospray ionization (ESI) presents a critical limitation for mass spectrometry (MS)-based quantitative analysis. In the field of metabolomics research, this issue ...has largely remained unaddressed; MS signal intensities are usually directly used to calculate fold changes for quantitative comparison. In this work, we demonstrate that, due to the nonlinear ESI response, signal intensity ratios of a metabolic feature calculated between two samples may not reflect their real metabolic concentration ratios (i.e., fold-change compression), implying that conventional fold-change calculations directly using MS signal intensities can be misleading. In this regard, we developed a quality control (QC) sample-based signal calibration workflow to overcome the quantitative bias caused by the nonlinear ESI response. In this workflow, calibration curves for every metabolic feature are first established using a QC sample injected in serial injection volumes. The MS signals of each metabolic feature are then calibrated to their equivalent QC injection volumes for comparative analysis. We demonstrated this novel workflow in a targeted metabolite analysis, showing that the accuracy of fold-change calculations can be significantly improved. Furthermore, in a metabolomic comparison of the bone marrow interstitial fluid samples from leukemia patients before and after chemotherapy, an additional 59 significant metabolic features were found with fold changes larger than 1.5, and an additional 97 significant metabolic features had fold changes corrected by more than 0.1. This work enables high-quality quantitative analysis in untargeted metabolomics, thus providing more confident biological hypotheses generation.
The knowledgebase TopFIND is an analysis platform focussed on protein termini, their origin, modification and hence their role on protein structure and function. Here, we present a major update to ...TopFIND, version 3, which includes a 70% increase in the underlying data to now cover a 90,696 proteins, 165,044 N-termini, 130,182 C-termini, 14,382 cleavage sites and 33,209 substrate cleavages in H. sapiens, M. musculus, A. thaliana, S. cerevisiae and E. coli. New features include the mapping of protein termini and cleavage entries across protein isoforms and significantly, the mapping of protein termini originating from alternative transcription and alternative translation start sites. Furthermore, two analysis tools for complex data analysis based on the TopFIND resource are now available online: TopFINDer, the TopFIND ExploRer, characterizes and annotates proteomics-derived N- or C-termini sets for their origin, sequence context and implications for protein structure and function. Neo-termini are also linked to associated proteases. PathFINDer identifies indirect connections between a protease and list of substrates or termini thus supporting the evaluation of complex proteolytic processes in vivo. To demonstrate the utility of the tools, a recent N-terminomics data set of inflamed murine skin has been re-analyzed. In re-capitulating the major findings originally performed manually, this validates the utility of these new resources. The point of entry for the resource is http://clipserve.clip.ubc.ca/topfind from where the graphical interface, all application programming interfaces (API) and the analysis tools are freely accessible.
A goal of the Chromosome-centric Human Proteome Project is to identify all human protein species. With 3844 proteins annotated as “missing”, this is challenging. Moreover, proteolytic processing ...generates new protein species with characteristic neo-N termini that are frequently accompanied by altered half-lives, function, interactions, and location. Enucleated and largely void of internal membranes and organelles, erythrocytes are simple yet proteomically challenging cells due to the high hemoglobin content and wide dynamic range of protein concentrations that impedes protein identification. Using the N-terminomics procedure TAILS, we identified 1369 human erythrocyte natural and neo-N-termini and 1234 proteins. Multiple semitryptic N-terminal peptides exhibited improved mass spectrometric identification properties versus the intact tryptic peptide enabling identification of 281 novel erythrocyte proteins and six missing proteins identified for the first time in the human proteome. With an improved bioinformatics workflow, we developed a new classification system and the Terminus Cluster Score. Thereby we described a new stabilizing N-end rule for processed protein termini, which discriminates novel protein species from degradation remnants, and identified protein domain hot spots susceptible to cleavage. Strikingly, 68% of the N-termini were within genome-encoded protein sequences, revealing alternative translation initiation sites, pervasive endoproteolytic processing, and stabilization of protein fragments in vivo. The mass spectrometry proteomics data have been deposited to ProteomeXchange with the data set identifier .
Protein N termini reveal fundamental regulatory mechanisms and their perturbation in disease. Regulatory proteolysis is often spatially and temporally confined, thus accessible only in minimal ...specimen incompatible with established protocols. We developed a robust, sensitive, scalable and automatable method for system-wide identification of thousands of N termini from minute samples. Applications revealed distinct N-terminal profiles in sorted immune cells and mitochondria from pediatric cancer patient cells, protease substrates in Arabidopsis seedlings and effects of chemotherapy on proteolytic proteoforms in clinical liquid biopsies.
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Highlights
•Single-pot workflow for manual or automated enrichment of N-terminal peptides.•Sensitive enrichment of protein N termini from 10,000 cells or 2 μg crude proteome.•Data independent acquisition improves precision of peptide level quantification.•First degradomic analyses of sorted immune cells, single seedlings, and mitochondria from patient cells.
Protein N termini unambiguously identify truncated, alternatively translated or modified proteoforms with distinct functions and reveal perturbations in disease. Selective enrichment of N-terminal peptides is necessary to achieve proteome-wide coverage for unbiased identification of site-specific regulatory proteolytic processing and protease substrates. However, many proteolytic processes are strictly confined in time and space and therefore can only be analyzed in minute samples that provide insufficient starting material for current enrichment protocols. Here we present High-efficiency Undecanal-based N Termini EnRichment (HUNTER), a robust, sensitive and scalable method for the analysis of previously inaccessible microscale samples. HUNTER achieved identification of >1000 N termini from as little as 2 μg raw HeLa cell lysate. Broad applicability is demonstrated by the first N-terminome analysis of sorted human primary immune cells and enriched mitochondrial fractions from pediatric cancer patients, as well as protease substrate identification from individual Arabidopsis thaliana wild type and Vacuolar Processing Enzyme-deficient mutant seedlings. We further implemented the workflow on a liquid handling system and demonstrate the feasibility of clinical degradomics by automated processing of liquid biopsies from pediatric cancer patients.
Resolution of inflammation reduces pathological tissue destruction and restores tissue homeostasis. Here, we used a proteomic protease substrate discovery approach, terminal amine isotopic labeling ...of substrates (TAILS), to analyze the role of the macrophage-specific matrix metalloproteinase-12 (MMP12) in inflammation. In murine peritonitis, MMP12 inactivates antithrombin and activates prothrombin, prolonging the activated partial thromboplastin time. Furthermore, MMP12 inactivates complement C3 to reduce complement activation and inactivates the chemoattractant anaphylatoxins C3a and C5a, whereas iC3b and C3b opsonin cleavage increases phagocytosis. Loss of these anti-inflammatory activities in collagen-induced arthritis in Mmp12−/− mice leads to unresolved synovitis and extensive articular inflammation. Deep articular cartilage loss is associated with massive neutrophil infiltration and abnormal DNA neutrophil extracellular traps (NETs). The NETs are rich in fibrin and extracellular actin, which TAILS identified as MMP12 substrates. Thus, macrophage MMP12 in arthritis has multiple protective roles in countering neutrophil infiltration, clearing NETs, and dampening inflammatory pathways to prepare for the resolution of inflammation.
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•Macrophages resolve inflammation via matrix metalloproteinase 12 (MMP12)•MMP12 dampens neutrophil infiltration and clears actin and fibrin from NETs•MMP12 terminates complement activation and increases phagocytosis•By activation of prothrombin in vivo, MMP12 exhibits procoagulant activity
Using TAILS proteomics, Bellac et al. now demonstrate that macrophage MMP12 executes multiple protective roles in vivo in inflammation resolution. In arthritis, MMP12 counters neutrophil influx, terminates complement activation, accelerates coagulation, and clears NETs of actin and fibrin to dampen and prepare for the resolution of inflammation.
Positional proteomics provides proteome-wide information on protein termini and their modifications, uniquely enabling unambiguous identification of site-specific, limited proteolysis. Such ...proteolytic cleavage irreversibly modifies protein sequences resulting in new proteoforms with distinct protease-generated neo-N and C-termini and altered localization and activity. Misregulated proteolysis is implicated in a wide variety of human diseases. Protein termini, therefore, constitute a huge, largely unexplored source of specific analytes that provides a deep view into the functional proteome and a treasure trove for biomarkers.
We briefly review principal approaches to define protein termini and discuss recent advances in method development. We further highlight the potential of positional proteomics to identify and trace specific proteoforms, with a focus on proteolytic processes altered in disease. Lastly, we discuss current challenges and potential for applying positional proteomics in biomarker and pre-clinical research.
Recent developments in positional proteomics have provided significant advances in sensitivity and throughput. In-depth analysis of proteolytic processes in clinical cohorts thus appears feasible in the near future. We argue that this will provide insights into the functional state of the proteome and offer new opportunities to utilize proteolytic processes altered or targeted in disease as specific diagnostic, prognostic and companion biomarkers.
Breast cancer risk for carriers of BRCA1 pathological variants is modified by genetic factors. Genetic variation in HMMR may contribute to this effect. However, the impact of risk modifiers on cancer ...biology remains undetermined and the biological basis of increased risk is poorly understood. Here, we depict an interplay of molecular, cellular, and tissue microenvironment alterations that increase BRCA1-associated breast cancer risk. Analysis of genome-wide association results suggests that diverse biological processes, including links to BRCA1-HMMR profiles, influence risk. HMMR overexpression in mouse mammary epithelium increases Brca1-mutant tumorigenesis by modulating the cancer cell phenotype and tumor microenvironment. Elevated HMMR activates AURKA and reduces ARPC2 localization in the mitotic cell cortex, which is correlated with micronucleation and activation of cGAS-STING and non-canonical NF-κB signaling. The initial tumorigenic events are genomic instability, epithelial-to-mesenchymal transition, and tissue infiltration of tumor-associated macrophages. The findings reveal a biological foundation for increased risk of BRCA1-associated breast cancer.
Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging ...for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows, and analysis pipelines, yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for automated sonication-free acid-assisted proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens, achieving similar proteome coverage as established methods using expensive sonicators, resulting in reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time- and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens.
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