Reviews & Perspectives
Deciphering Spatial Protein–Protein Interactions in Brain Using Proximity Labeling
In Brief PL has emerged as a powerful tool to identify proteomes in distinct cell types in brain as well as proteomes and protein–protein interaction networks in structures difficult to isolate, such as the synaptic cleft, axonal projections, or astrocyte–neuron junctions. Here, we review recent advances in PL methods and their application to neurobiology.New Views of Old Proteins: Clarifying the Enigmatic Proteome
In Brief Capturing the biology of proteins will require improved technologies to readout their composition in space and time. Developing these improved technologies presents a major opportunity for biomedical research. How might we proceed in the decades ahead?MHC Class I Immunopeptidome: Past, Present, and Future
In Brief A pioneer in studying CD8+ T-cell immunosurveillance of viruses and tumors reviews the critical contributions of MS-based studies to early, current, and future understanding of how cells generate the MHC class I immunopeptidome: the repertoire of foreign and self-peptides presented by MHC class I molecules for T-cell recognition.An Expanding Repertoire of Protein Acylations
In Brief In this work, we give a general overview of the 12 main protein acylations, also including novel acylations, such as benzoylation and 2-hydroxyisobutyrylation. We summarize the recent advances in protein acylation, mainly focus on their substrates, enzymes, biological functions, and novel detecting methods and related diseases, especially in cancer. We believe that the review will provide an unprecedented and comprehensive view of protein acylations and bring important reference significance for future research.Subcellular Transcriptomics and Proteomics: A Comparative Methods Review
In Brief The interior of the cell is molecularly crowded. Its compartmentalization within organelles enables the regulation of biochemical processes and allows multifunctionality of proteins and RNAs. Subcellular information can thus give insights into the function of these biomolecules. Multiple techniques to measure such information have been established, with ever-increasing throughput and sensitivity. These techniques are covered in this review, and demonstrating their application is providing valuable insights into cellular biology, such as aiding our understanding of single-cell heterogeneity and posttranslational modifications.Scaling Up Single-Cell Proteomics
In Brief Single-cell proteomics will drive the next wave of single-cell biology. This requires broad adoption of existing methods, the application of rigorous quality control standards, and the continuous advancement of the technology. The advancement will be driven by numerous innovations, including highly parallelized analysis, and will increase the throughput, quantitative accuracy, and the accessibility of the single-cell proteomics.Ethical Principles, Constraints, and Opportunities in Clinical Proteomics
In Brief We introduce bioethical principles and use these as operational definitions to carry out a systematic review of the literature on ethical issues in clinical proteomics. We identify 10 ethical themes across 16 studies, many of which are familiar from other fields. We therefore survey how genomics has dealt with ethical issues and regulation. We also add our own perspectives on the ethical aspects of study design and sample treatment as well as the ethical potential of preventive proteomics profiling.Reflections on the HUPO Human Proteome Project, the Flagship Project of the Human Proteome Organization, at 10 Years
In Brief Starting from several organ-oriented projects, HUPO in 2010 launched the Human Proteome Project to identify and characterize the protein parts list and integrate proteomics into multiomics research. Key steps were partnerships with neXtProt, PRIDE, PeptideAtlas, Human Protein Atlas, and instrument makers; global engagement of researchers; creation of ProteomeXchange; adoption of HPP Guidelines for Interpretation of MS Data and SRMAtlas for proteotypic peptides; annual metrics of finding “missing proteins” and functionally annotating proteins; and initiatives for early career scientists.Data Management of Sensitive Human Proteomics Data: Current Practices, Recommendations, and Perspectives for the Future
In Brief Availability of proteomics data in the public domain has become the norm, as it has been the case in genomics and transcriptomics for many years. Analogously to sequencing data, there are increasing ethical issues and legal requirements related to sensitive human clinical proteomics data. We review the current state of the art and make concrete recommendations to address these issues in the proteomics field, which are summarized in four different areas.Neuroproteomics of the Synapse: Subcellular Quantification of Protein Networks and Signaling Dynamics
In Brief Advancements in MS-based proteomics have increased the study of synaptic proteins using neuroproteomics. The development of proximity, genetic labeling and bio-orthogonal amino acid labeling approaches now allow for the study of synaptic protein–protein interactions and protein signaling dynamics. In this review, we highlight studies from the last 5 years, with a focus on synapse structure, composition, functioning, or signaling and finally discuss some recent developments that could further advance the field of neuroproteomics.Are There Indeed Spliced Peptides in the Immunopeptidome?
In Brief Peptide splicing was suggested to significantly contribute ligands to the immunopeptidome. This article argues that peptide splicing is at most very rare, even if it happens at all. Considerations against peptide splicing are based on bioinformatics calculations related to the analysis of the LC-MS/MS data, and on the abundance of water in the cells, which should compete effectively with the transpeptidation reaction, needed for peptide splicing.Proteomics-Based Insights Into the SARS-CoV-2–Mediated COVID-19 Pandemic: A Review of the First Year of Research
In Brief SARS-CoV-2, the betacoronavirus that caused the COVID-19 pandemic, became a major source of human disease and death in 2020. The fundamental constituents of a virus being its genome and proteome, characterizing the proteome is essential to understanding its biology. In this review article, we survey the proteomics literature from the first year of the COVID-19 pandemic, including protein–protein interaction studies, post-translational modification studies, and work using proteomics technologies to probe host response, which collectively inform efforts to ameliorate the pandemic.MS-Based HLA-II Peptidomics Combined With Multiomics Will Aid the Development of Future Immunotherapies
In Brief Although challenges remain in leveraging MS-based HLA-II peptidomics, investigations into the interplay between disease pathologies and the presentation of HLA-II peptides to CD4+ T cells will enable the development of future immunotherapies. In this Review article, we discuss our current understanding of HLA-II peptidomics and outstanding questions in the field and how MS-based innovations will enable us to fill knowledge gaps and help improve our ability to select HLA-II-presented antigens as targets for personalized immunotherapies.Decoding Post-Translational Modification Crosstalk With Proteomics
In Brief We provide an overview of current experimental and computational proteomic methods, as well as a perspective on emerging technologies to study PTM crosstalk.Uncovering the Depths of the Human Proteome: Antibody-based Technologies for Ultrasensitive Multiplexed Protein Detection and Quantification
In Brief Probing the human plasma proteome is attractive for biomarker and drug target discovery. Recent breakthroughs in multiplex proteomics technologies enable the simultaneous and sensitive quantification of thousands of proteins in biofluids. We provide a comprehensive guide to the methodologies, performance, advantages, and disadvantages of established and emerging technologies for the multiplexed ultrasensitive measurement of proteins. Gaining knowledge on these innovations is crucial for choosing the right multiplexed proteomics tool to critically complement traditional proteomics methods.An Introduction to Advanced Targeted Acquisition Methods
In Brief The analytical power of targeted proteomics depends on how efficiently the mass spectrometer detects target peptides. A number of “smart” acquisition approaches have been developed that enable more targets per run and improve analytical performance such as sensitivity, specificity, and quantitative accuracy. This review provides an introduction to these methods and highlights their inherent strengths and weaknesses.Recent Advances in Software Tools for More Generic and Precise Intact Glycopeptide Analysis
In Brief This article provides a systematic review of the most recent MS-based strategies and corresponding software tools for the analysis of intact glycopeptides, particularly intact N-glycopeptides, reported in the last decade, including the process of identifying N-glycopeptides from MS data, the existing methods of MS data acquisition and interpretation, the quality control methods, the display of results, and the software applications.Recent Advances in Analytical Approaches for Glycan and Glycopeptide Quantitation
In Brief Recent years have seen an explosion in novel strategies for quantitative glycomics and glycoproteomics. Whether through metabolic incorporation of stable isotopes, deposition of custom isotopic labels, or high-throughput isobaric chemical tags, these numerous novel strategies provide ease of access to glycomic and glycoproteomic investigation. This review highlights the recent innovations in labeling methods, label-free strategies, acquisition modes, and bioinformatic tools for glycan and glycopeptide quantitation, while providing critical evaluations and technical considerations to enable effective analysis.The Role of Data-Independent Acquisition for Glycoproteomics
In Brief As a highly abundant and diverse post-translational modification, protein glycosylation is challenging to characterize in various approaches including MS. In MS-based proteomics, data-independent acquisition (DIA) has been advanced rapidly and showed outstanding analytical performances. DIA now started to be applied in different facets of glycoproteomics, including deglycosylated and intact N-linked and O-linked glycopeptides, and screening of oxonium ions. We summarized current applications of DIA in glycoproteomics and discussed its limitations and perspectives.Chromatin Proteomics to Study Epigenetics — Challenges and Opportunities
In Brief MS-based analysis of chromatin has emerged as a powerful tool to identify proteins associated with gene regulation. Total chromatin isolated from cells can be directly analyzed using MS, further fractionated into transcriptionally active and inactive chromatin, enriched for specific compartment or regions, and potentially used for single-locus isolation. This review highlights recent advances and discusses current challenges that should be addressed to further advance the field of chromatin proteomics.Calculating Glycoprotein Similarities From Mass Spectrometric Data
In Brief To understand the roles of glycoproteins in biological processes, it is necessary to quantify the changes that occur to glycosylation at individual sites and to the whole molecule. That glycoprotein glycosylation is inherently heterogeneous means that the distribution of glycoforms at each glycosite must be quantified in order to inform calculation of molecular similarities. We review analytical and statistical methods for determining glycoprotein molecular similarities from glycoproteomics data.Glycomics, Glycoproteomics, and Glycogenomics: An Inter-Taxa Evolutionary Perspective
In Brief This review article i) assesses the utility of current glycomic, glycoproteomic, and glycogenomic methods to characterize protein glycosylation in less-well-studied eukaryotes; ii) assembles a plausible evolutionary lineage of eukaryotic glycan–protein linkages from the last eukaryotic common ancestor through protists to multicellular plants, invertebrates, and vertebrates; and iii) highlights the diversity of peripheral glycan specializations and modifications with an emphasis on available information from diverse protist kingdoms and invertebrate animals.Developments in Mass Spectrometry for Glycosaminoglycan Analysis: A Review
In Brief Glycosaminoglycans (GAGs) participate in a variety of biological functions and have a multitude of medicinal properties. Due to their non template driven biosynthesis, GAGs are produced as nonuniform complex mixtures. Mass spectrometry paired with on-line separation techniques has been utilized to determine the composition of these complex mixtures. Advances in tandem mass spectrometry have also made determining sequence information such as sulfation location and C-5 epimerization possible. This review covers recent developments in the analysis of GAGs using mass spectrometry.The Peptide Vaccine of the Future
In Brief Therapeutic peptide-based vaccination approaches for the treatment of cancer patients have shown first glimmers of success. However, to achieve broad clinical efficacy and implement peptide vaccinations in the standard treatment of cancer patients future peptide vaccines need further optimization in terms of target antigen selection, adjuvant choice, vaccination schedules, delivery routes, biomarkers, and combinatorial drugs.Methods for Enrichment and Assignment of N-Acetylglucosamine Modification Sites
In Brief This review article summarizes methods for O-GlcNAc enrichment and different mass spectrometric approaches for acquiring data on modified peptides and describes software strategies for analyzing data, including the challenges of reliably identifying modification sites and differentiating between other potential HexNAc modifications. It then presents a new dataset to exemplify what is currently achievable.A Pragmatic Guide to Enrichment Strategies for Mass Spectrometry–Based Glycoproteomics
In Brief Interest in mass spectrometry–based glycoproteomics analysis is increasing because of recent advances in instrumentation and data analysis tools. Such studies can provide a wealth of information across a wide spectrum of glycan classes and biological systems. However, many studies require the choice of an enrichment strategy for glycosylated species prior to analysis to obtain the maximum amount of analytical information. Here, common enrichment strategies are reviewed with strengths and weaknesses, and the practical considerations for various methods are discussed.Meta-heterogeneity: Evaluating and Describing the Diversity in Glycosylation Between Sites on the Same Glycoprotein
In Brief Diversity in protein glycosylation can be described in terms of micro-heterogeneity and macro-heterogeneity, respectively, referring to the variation and occupancy of glycans at a given glycosylation site. However, these terms are not sufficient to describe a higher level of regulation when proteins are multiply glycosylated. For this, we propose the term meta-heterogeneity: variation in glycosylation across multiple sites of a given glycoprotein. In this review, we describe several remarkable examples of glycoprotein meta-heterogeneity and underline the need for its investigation.Accelerating the Field of Epigenetic Histone Modification Through Mass Spectrometry–Based Approaches
In Brief Histone post-translational modifications play essential roles in the epigenetic regulation of chromatin-related functions. Because of its high throughput, accuracy, and flexibility, mass spectrometry has emerged as a powerful tool in the epigenetic field. In this review, we describe the contributions of mass spectrometry–based proteomics in combination with distinct labeling strategies and various biological techniques to understand the roles of histone post-translational modifications and how they regulate chromatin function.Proteome Turnover in the Spotlight: Approaches, Applications, and Perspectives
In Brief In this review, we outline historical and current approaches to measure the kinetics of protein turnover on a proteome-wide scale in both steady-state and dynamic systems, with an emphasis on metabolic tracing using stable isotope–labeled amino acids. In addition, we highlight important considerations for designing proteome turnover experiments, key biological findings regarding the conserved principles of proteome turnover regulation, and future perspectives for both technological and biological investigations.Single-cell Proteomics: Progress and Prospects
Single-cell proteomics capabilities have the potential to transform biomedical research and enable understanding of biological systems with a new level of granularity. Recent advances in sample processing, separations and MS instrumentation now make it possible to quantify >1000 proteins from individual mammalian cells.Proteomics and Metaproteomics Add Functional, Taxonomic and Biomass Dimensions to Modeling the Ecosystem at the Mucosal-luminal Interface
Proteomics and metaproteomics are important tools for studying the spatiotemporal heterogeneous ecosystem in our gut. We review strategies and their applications to gut ecology studies, such as building a dynamical model of the MLI.Organellar Maps Through Proteomic Profiling – A Conceptual Guide
Protein subcellular localization is highly regulated and critical for protein function. Spatial proteomics aims at capturing the localization dynamics of all proteins expressed in a given cell type. Among different approaches, organellar mapping through proteomic profiling stands out as the only method capable of determining the subcellular localizations of thousands of proteins in a single experiment. Importantly, it can also detect movements of proteins between subcellular compartments, providing an unbiased systems analysis tool for investigating physiological and pathological cellular processes.Peptide-based Interaction Proteomics
Protein-protein interactions that are mediated by short linear motifs (SLiMs) in intrinsically disordered regions (IDRs) of proteins are notoriously difficult to study. Recently, pull-downs with synthetic peptides in combination with quantitative mass spectrometry emerged as a powerful screening approach. Here, we briefly highlight the relevance of SLiMs for protein-protein interactions, outline existing screening technologies, discuss unique advantages of peptide-based interaction screens, and provide practical suggestions for setting up such peptide-based screens.Developments and Applications of Functional Protein Microarrays
Functional protein microarray is a crucial tool in the study of proteins in native, unbiased, and high-throughput manner. There is a wide variety of applications, including the study of proteome-wide molecular interactions, analysis of post-translational modifications, identification of novel drug targets, and examination of pathogen-host interactions. Functional protein microarray is also useful in profiling antibody specificity, as well as in the discovery of novel biomarkers, especially for autoimmune diseases, infectious diseases, and cancers. Recently, the virion display method has been applied to produce functional GPCR array for various research and pharmaceutical applications.Profiling Cell Signaling Networks at Single-cell Resolution
Signaling network responses can be highly heterogeneous across cells in a tissue because of many sources of genetic and non-genetic variance. The emergence of multiplexed single-cell technologies has made it possible to evaluate this heterogeneity. In this review, we categorize currently established single-cell signaling network profiling approaches by their methodology, coverage, and application, and we discuss the advantages and limitations of these technologies. We describe the computational tools for network characterization using single-cell data and discuss potential confounding factors that may affect single-cell analyses.Proximity Dependent Biotinylation: Key Enzymes and Adaptation to Proteomics Approaches
Proximity-dependent biotinylation approaches such as BioID and APEX overcome classical limitations of biochemical purification and have gained widespread use in recent years for revealing cellular neighborhoods. Here we focus on the structural diversity and mechanisms of the two classes of enzymes, biotin protein ligases and peroxidases, and discuss current and emerging applications of these enzymes for proximity dependent biotinylation. We provide guidelines for enzyme selection and experimental design for performing and interpreting proximity-dependent biotinylation experiments.Phosphoproteomic Approaches to Discover Novel Substrates of Mycobacterial Ser/Thr Protein Kinases
Mycobacterial STPKs are responsible for orchestrating phosphorylation-dependent signaling cascades that mediate bacterial growth and environmental adaptation. Recent advances in MS-based phosphoproteomics have significantly expanded the candidate substrate lists for individual mycobacterial STPKs. Integration of the available phosphoproteomic datasets provide new insights into the functional roles of specific STPKs in cell physiology. Future research should focus on in vivo phosphorylation network reconstruction to expose the fundamental signaling pathways in mycobacteria. Linking STPKs with their physiological substrates may reveal novel antimycobacterial agents.Advances in Tools to Determine the Glycan-Binding Specificities of Lectins and Antibodies
Lectins and glycan-binding antibodies are powerful tools in biological research, provided detailed information is available about their glycan-binding specificities. Glycan-arrays, in combination with bioinformatics tools to mine the data, offer the ability to obtain such information. This review focuses on the bioinformatics tools and resources that are available for the analysis of glycan-array data. The tools are enabling new insights into protein-glycan interactions and enhancing the value of glycan-binding proteins in research.Next-generation Interactomics: Considerations for the Use of Co-elution to Measure Protein Interaction Networks
Interactome studies are necessary to understand cellular processes and co-elution methods are well suited for the simultaneous and global exploration of the interactome, as well as the assessment of biological perturbations of the network. These methods rely on the fundamental idea that proteins from the same complex migrate together during fractionation. We review the different separation techniques along with the quantification and bioinformatic approaches used for co-elution methods and provide design considerations to choose between them.Why Glycosylation Matters in Building a Better Flu Vaccine
Immunodominant influenza A virus (IAV) antigens mutate rapidly, allowing the virus to escape host antibodies. The question remains how to design vaccines that recognize conserved but subdominant IAV antigens for broader immune protection. Glycosylation is a mechanism whereby IAV evades the innate and adaptive immune systems. However, its influence on immunodominance remains poorly understood. Although mass spectrometry methods for identifying glycopeptides are maturing, quantifying glycosylation variation among sets of IAV mutants remains a technical challenge.Proteomics, Glycomics, and Glycoproteomics of Matrisome Molecules
Extracellular networks of matrisome proteins and their binding partners give rise to dynamic cell and tissue-specific microenvironments. The extreme complexity of matrisome molecule glycosylation and other post-translational modifications belies the need for specialized omics methods. It is necessary to map the modifications of matrisome molecules in detail in order to understand their roles in normal and pathological physiology. We review proteomics, glycomics and glycoproteomics methods for matrisome molecules toward the goal of achieving detailed matrisome maps.Exploiting Interdata Relationships in Next-generation Proteomics Analysis
Mass spectrometry-based proteomics and other technologies have matured to enable routine acquisition of system-wide data sets that describe concentrations, modifications, and interactions of proteins, mRNAs, and other molecules. Productive integrative studies differ from parallel data analysis by quantitative modeling of the relationships between data. We outline steps and considerations towards integromic studies to exploit the synergy between data sets.E3 Ubiquitin Ligases: Key Regulators of Hormone Signaling in Plants
Ubiquitin-mediated control of protein stability is central to most aspects of plant hormone signaling. Attachment of ubiquitin to target proteins occurs via an enzymatic cascade with the final step being catalyzed by a family of enzymes known as E3 ubiquitin ligases, which have been classified based on their protein domains and structures. Although E3 ubiquitin ligases are conserved among eukaryotes, in plants they are well-known to fulfill unique roles as central regulators of phytohormone signaling, including hormone perception and regulation of hormone biosynthesis.Analysis of Mammalian O-Glycopeptides—We Have Made a Good Start, but There is a Long Way to Go
Glycosylation is perhaps the most common post-translational modification. Recently there has been growing interest in cataloging the glycan structures, glycoproteins, and specific sites modified and deciphering the biological functions of glycosylation. Although the results are piling up for N-glycosylation, O-glycosylation is seriously trailing behind. In our review we reiterate the difficulties researchers have to overcome in order to characterize O-glycosylation. We describe how an ingenious cell engineering method delivered exciting results, and what could we gain from “wild-type” samples.
