Mouse Quantitative Proteomics Knowledgebase: reference protein concentration ranges in 20 mouse tissues using 5000 quantitative proteomics assays.

MotivationLaboratory mouse is the most used animal model in biological research, largely due to its high conserved synteny with human. Researchers use mice to answer various questions ranging from determining a pathological effect of knocked out/in gene to understanding drug metabolism. Our group developed >5000 quantitative targeted proteomics assays for 20 mouse tissues and determined the concentration ranges of a total of >1600 proteins using heavy labeled internal standards. We describe here MouseQuaPro; a knowledgebase that hosts this collection of carefully curated experimental data. ResultsThe web-based application includes protein concentrations from >700 mouse tissue samples from three common research strains, corresponding to >200k experimentally determined concentrations. The knowledgebase integrates the assay and protein concentration information with their human orthologs, functional and molecular annotations, biological pathways, related human diseases and known gene expressions. At its core are the protein concentration ranges, which provide insights into (dis)similarities between tissues, strains and sexes. MouseQuaPro implements advanced search as well as filtering functionalities with a simple interface and interactive visualization. This information-rich resource provides an initial map of protein absolute concentration in mouse tissues and allows guided design of proteomics phenotyping experiments. The knowledgebase is available on mousequapro.proteincentre.com. AVAILABILITY AND IMPLEMENTATION: The knowledgebase is available free of charge on http://mousequapro.proteincentre.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

An Update on MRMAssayDB: A Comprehensive Resource for Targeted Proteomics Assays in the Community.

Precise multiplexed quantification of proteins in biological samples can be achieved by targeted proteomics using multiple or parallel reaction monitoring (MRM/PRM). Combined with internal standards, the method achieves very good repeatability and reproducibility enabling excellent protein quantification and allowing longitudinal and cohort studies. A laborious part of performing such experiments lies in the preparation steps dedicated to the development and validation of individual protein assays. Several public repositories host information on targeted proteomics assays, including NCI's Clinical Proteomic Tumor Analysis Consortium assay portals, PeptideAtlas SRM Experiment Library, SRMAtlas, PanoramaWeb, and PeptideTracker, with all offering varying levels of details. We introduced MRMAssayDB in 2018 as an integrated resource for targeted proteomics assays. The Web-based application maps and links the assays from the repositories, includes comprehensive up-to-date protein and sequence annotations, and provides multiple visualization options on the peptide and protein level. We have extended MRMAssayDB with more assays and extensive annotations. Currently it contains >828 000 assays covering >51 000 proteins from 94 organisms, of which >17 000 proteins are present in >2400 biological pathways, and >48 000 mapping to >21 000 Gene Ontology terms. This is an increase of about four times the number of assays since introduction. We have expanded annotations of interaction, biological pathways, and disease associations. A newly added visualization module for coupled molecular structural annotation browsing allows the user to interactively examine peptide sequence and any known PTMs and disease mutations, and map all to available protein 3D structures. Because of its integrative approach, MRMAssayDB enables a holistic view of suitable proteotypic peptides and commonly used transitions in empirical data. Availability: http://mrmassaydb.proteincentre.com.

MRMAssayDB: an integrated resource for validated targeted proteomics assays.

Motivation: Multiple Reaction Monitoring (MRM)-based targeted proteomics is increasingly being used to study the molecular basis of disease. When combined with an internal standard, MRM allows absolute quantification of proteins in virtually any type of sample but the development and validation of an MRM assay for a specific protein is laborious. Therefore, several public repositories now host targeted proteomics MRM assays, including NCI's Clinical Proteomic Tumor Analysis Consortium assay portals, PeptideAtlas SRM Experiment Library, SRMAtlas, PanoramaWeb and PeptideTracker, with all of which contain different levels of information. Results: Here we present MRMAssayDB, a web-based application that integrates these repositories into a single resource. MRMAssayDB maps and links the targeted assays, annotates the proteins with information from UniProtKB, KEGG pathways and Gene Ontologies, and provides several visualization options on the peptide and protein level. Currently MRMAssayDB contains >168K assays covering more than 34K proteins from 63 organisms; >13.5K of these proteins are present in >2.3K KEGG biological pathways corresponding to >300 master pathways, and mapping to >13K GO biological processes. MRMAssayDB allows comprehensive searches for a targeted-proteomics assay depending on the user's interests, by using target-protein name or accession number, or using annotations such as subcellular localization, biological pathway, or disease or drug associations. The user can see how many data repositories include a specific peptide assay, and the commonly used transitions for each peptide in all empirical data from the repositories. Availability and implementation: http://mrmassaydb.proteincentre.com. Supplementary information: Supplementary data are available at Bioinformatics online.

Design Principles Involving Protein Disorder Facilitate Specific Substrate Selection and Degradation by the Ubiquitin-Proteasome System.

The ubiquitin-proteasome system (UPS) regulates diverse cellular pathways by the timely removal (or processing) of proteins. Here we review the role of structural disorder and conformational flexibility in the different aspects of degradation. First, we discuss post-translational modifications within disordered regions that regulate E3 ligase localization, conformation, and enzymatic activity, and also the role of flexible linkers in mediating ubiquitin transfer and reaction processivity. Next we review well studied substrates and discuss that substrate elements (degrons) recognized by E3 ligases are highly disordered: short linear motifs recognized by many E3s constitute an important class of degrons, and these are almost always present in disordered regions. Substrate lysines targeted for ubiquitination are also often located in neighboring regions of the E3 docking motifs and are therefore part of the disordered segment. Finally, biochemical experiments and predictions show that initiation of degradation at the 26S proteasome requires a partially unfolded region to facilitate substrate entry into the proteasomal core.

Bioinformatics Approaches for Predicting Disordered Protein Motifs.

Short, linear motifs (SLiMs) in proteins are functional microdomains consisting of contiguous residue segments along the protein sequence, typically not more than 10 consecutive amino acids in length with less than 5 defined positions. Many positions are 'degenerate' thus offering flexibility in terms of the amino acid types allowed at those positions. Their short length and degenerate nature confers evolutionary plasticity meaning that SLiMs often evolve convergently. Further, SLiMs have a propensity to occur within intrinsically unstructured protein segments and this confers versatile functionality to unstructured regions of the proteome. SLiMs mediate multiple types of protein interactions based on domain-peptide recognition and guide functions including posttranslational modifications, subcellular localization of proteins, and ligand binding. SLiMs thus behave as modular interaction units that confer versatility to protein function and SLiM-mediated interactions are increasingly being recognized as therapeutic targets. In this chapter we start with a brief description about the properties of SLiMs and their interactions and then move on to discuss algorithms and tools including several web-based methods that enable the discovery of novel SLiMs (de novo motif discovery) as well as the prediction of novel occurrences of known SLiMs. Both individual amino acid sequences as well as sets of protein sequences can be scanned using these methods to obtain statistically overrepresented sequence patterns. Lists of putatively functional SLiMs are then assembled based on parameters such as evolutionary sequence conservation, disorder scores, structural data, gene ontology terms and other contextual information that helps to assess the functional credibility or significance of these motifs. These bioinformatics methods should certainly guide experiments aimed at motif discovery.

Multiple reaction monitoring assays for large-scale quantitation of proteins from 20 mouse organs and tissues.

Mouse is the mammalian model of choice to study human health and disease due to its size, ease of breeding and the natural occurrence of conditions mimicking human pathology. Here we design and validate multiple reaction monitoring mass spectrometry (MRM-MS) assays for quantitation of 2118 unique proteins in 20 murine tissues and organs. We provide open access to technical aspects of these assays to enable their implementation in other laboratories, and demonstrate their suitability for proteomic profiling in mice by measuring normal protein abundances in tissues from three mouse strains: C57BL/6NCrl, NOD/SCID, and BALB/cAnNCrl. Sex- and strain-specific differences in protein abundances are identified and described, and the measured values are freely accessible via our MouseQuaPro database: http://mousequapro.proteincentre.com . Together, this large library of quantitative MRM-MS assays established in mice and the measured baseline protein abundances represent an important resource for research involving mouse models.

Tripartite degrons confer diversity and specificity on regulated protein degradation in the ubiquitin-proteasome system.

Specific signals (degrons) regulate protein turnover mediated by the ubiquitin-proteasome system. Here we systematically analyse known degrons and propose a tripartite model comprising the following: (1) a primary degron (peptide motif) that specifies substrate recognition by cognate E3 ubiquitin ligases, (2) secondary site(s) comprising a single or multiple neighbouring ubiquitinated lysine(s) and (3) a structurally disordered segment that initiates substrate unfolding at the 26S proteasome. Primary degron sequences are conserved among orthologues and occur in structurally disordered regions that undergo E3-induced folding-on-binding. Posttranslational modifications can switch primary degrons into E3-binding-competent states, thereby integrating degradation with signalling pathways. Degradation-linked lysines tend to be located within disordered segments that also initiate substrate degradation by effective proteasomal engagement. Many characterized mutations and alternative isoforms with abrogated degron components are implicated in disease. These effects result from increased protein stability and interactome rewiring. The distributed nature of degrons ensures regulation, specificity and combinatorial control of degradation.

Functional diversity and structural disorder in the human ubiquitination pathway.

The ubiquitin-proteasome system plays a central role in cellular regulation and protein quality control (PQC). The system is built as a pyramid of increasing complexity, with two E1 (ubiquitin activating), few dozen E2 (ubiquitin conjugating) and several hundred E3 (ubiquitin ligase) enzymes. By collecting and analyzing E3 sequences from the KEGG BRITE database and literature, we assembled a coherent dataset of 563 human E3s and analyzed their various physical features. We found an increase in structural disorder of the system with multiple disorder predictors (IUPred - E1: 5.97%, E2: 17.74%, E3: 20.03%). E3s that can bind E2 and substrate simultaneously (single subunit E3, ssE3) have significantly higher disorder (22.98%) than E3s in which E2 binding (multi RING-finger, mRF, 0.62%), scaffolding (6.01%) and substrate binding (adaptor/substrate recognition subunits, 17.33%) functions are separated. In ssE3s, the disorder was localized in the substrate/adaptor binding domains, whereas the E2-binding RING/HECT-domains were structured. To demonstrate the involvement of disorder in E3 function, we applied normal modes and molecular dynamics analyses to show how a disordered and highly flexible linker in human CBL (an E3 that acts as a regulator of several tyrosine kinase-mediated signalling pathways) facilitates long-range conformational changes bringing substrate and E2-binding domains towards each other and thus assisting in ubiquitin transfer. E3s with multiple interaction partners (as evidenced by data in STRING) also possess elevated levels of disorder (hubs, 22.90% vs. non-hubs, 18.36%). Furthermore, a search in PDB uncovered 21 distinct human E3 interactions, in 7 of which the disordered region of E3s undergoes induced folding (or mutual induced folding) in the presence of the partner. In conclusion, our data highlights the primary role of structural disorder in the functions of E3 ligases that manifests itself in the substrate/adaptor binding functions as well as the mechanism of ubiquitin transfer by long-range conformational transitions.

Germline VH/VL pairing in antibodies.

Antibodies are key molecules of the adaptive immune response and are now a major class of biopharmaceuticals. Pairing of heavy and light chains is one of the ways of generating antibody diversity and, while little is known about mechanisms governing V(H)/V(L) pairing, previous studies have suggested that the germline source from which chains are paired is random. By selecting paired antibody protein sequences from human and mouse antibodies from the KabatMan database and mapping them onto their corresponding germline sequences, we find that pairing preferences do exist in the germline, but only for a small proportion of germline gene segments; others are much more promiscuous showing no preferences. The closest equivalent human and mouse gene families were identified and pairing preferences compared. This work may impact on the ability to generate more stable antibodies for use as biopharmaceuticals.