Targeted mass spectrometry (MS) is becoming widely used in academia and in pharmaceutical and biotechnology industries for sensitive and quantitative detection of proteins, peptides and ...post-translational modifications. Here we describe the increasing importance of targeted MS technologies in clinical proteomics and the potential key roles these techniques will have in bridging biomedical discovery and clinical implementation.
Obtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are ...therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it was insufficiently specific when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine newly discovered mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.
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•Ratiometric tagging with an engineered peroxidase gives nanometer spatial resolution•Mitochondrial intermembrane space proteome mapped with >94% specificity•Nine newly discovered mitochondrial proteins confirmed by imaging and western blotting•Data support MICU1 and MICU2 localization in the mitochondrial intermembrane space
APEX is an engineered ascorbate peroxidase that can tag endogenous proteins for identification by mass spectrometry. Here, Hung et al. improve spatial specificity of APEX and analyze protein composition of the mitochondrial intermembrane space (IMS). The resulting IMS proteome of 127 proteins has >94% mitochondrial specificity and includes 9 newly discovered, validated mitochondrial proteins.
RNA–protein interactions underlie a wide range of cellular processes. Improved methods are needed to systematically map RNA–protein interactions in living cells in an unbiased manner. We used two ...approaches to target the engineered peroxidase APEX2 to specific cellular RNAs for RNA-centered proximity biotinylation of protein interaction partners. Both an MS2-MCP system and an engineered CRISPR-Cas13 system were used to deliver APEX2 to the human telomerase RNA hTR with high specificity. One-minute proximity biotinylation captured candidate binding partners for hTR, including more than a dozen proteins not previously linked to hTR. We validated the interaction between hTR and the N⁶-methyladenosine (m⁶A) demethylase ALKBH5 and showed that ALKBH5 is able to erase the m⁶A modification on endogenous hTR. ALKBH5 also modulates telomerase complex assembly and activity. MS2- and Cas13-targeted APEX2 may facilitate the discovery of novel RNA–protein interactions in living cells.
Isobaric labeling using tandem mass tags (TMTs) is increasingly applied for deep-scale proteomic studies in a multitude of organisms and addressing diverse research questions. The cost of labeling ...reagents represents a substantial proportion of the total expenses for conducting such experiments. Here, Zecha et al. present an economically optimized TMT labeling approach that reduces the quantity of required labeling reagent by a factor of eight and reproducibly achieves complete labeling.
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Highlights
•TMT labeling protocol with excellent intra- and interlaboratory reproducibility.•Complete in-solution labeling of peptides using 1/8 of recommended TMT quantities.•Demonstration of utility for deep-scale (phospho)proteome analysis.
Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mm and 2 g/l, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlaboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities.
The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins providing both biophysical and biochemical cues that are important regulators of cell proliferation, survival, ...differentiation, and migration. We present here a proteomic strategy developed to characterize the in vivo ECM composition of normal tissues and tumors using enrichment of protein extracts for ECM components and subsequent analysis by mass spectrometry. In parallel, we have developed a bioinformatic approach to predict the in silico “matrisome” defined as the ensemble of ECM proteins and associated factors. We report the characterization of the extracellular matrices of murine lung and colon, each comprising more than 100 ECM proteins and each presenting a characteristic signature. Moreover, using human tumor xenografts in mice, we show that both tumor cells and stromal cells contribute to the production of the tumor matrix and that tumors of differing metastatic potential differ in both the tumor- and the stroma-derived ECM components. The strategy we describe and illustrate here can be broadly applied and, to facilitate application of these methods by others, we provide resources including laboratory protocols, inventories of ECM domains and proteins, and instructions for bioinformatically deriving the human and mouse matrisome.
N6-methyladenosine (m6A) is a common modification of mRNA with potential roles in fine-tuning the RNA life cycle. Here, we identify a dense network of proteins interacting with METTL3, a component of ...the methyltransferase complex, and show that three of them (WTAP, METTL14, and KIAA1429) are required for methylation. Monitoring m6A levels upon WTAP depletion allowed the definition of accurate and near single-nucleotide resolution methylation maps and their classification into WTAP-dependent and -independent sites. WTAP-dependent sites are located at internal positions in transcripts, topologically static across a variety of systems we surveyed, and inversely correlated with mRNA stability, consistent with a role in establishing “basal” degradation rates. WTAP-independent sites form at the first transcribed base as part of the cap structure and are present at thousands of sites, forming a previously unappreciated layer of transcriptome complexity. Our data shed light on the proteomic and transcriptional underpinnings of this RNA modification.
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•METTL3, METTL14, WTAP, and KIAA1429 are required for mRNA methylation•Methylation maps, upon depletion of WTAP, reveal two classes of methylation•WTAP-dependent sites are mostly static and correlate with mRNA stability•Thousands of WTAP-independent sites are present at the first transcribed nucleotide
N6-methyladenosine (m6A) is a highly abundant modification of mRNA. Schwartz et al. identify and validate a network of proteins required for mRNA methylation in mammalian cells. They define two distinct classes of methylation sites. The majority of sites depend on the identified proteins, are located at internal positions in transcripts, and inversely correlate with mRNA stability. Sites independent of these proteins form at the first transcribed base as part of the cap structure, forming a previously unappreciated layer of transcriptome complexity.
Ubiquitination is essential for the regulation of cellular protein homeostasis. It also has a central role in numerous signaling events. Recent advances in the production and availability of ...antibodies that recognize the Lys-ɛ-Gly-Gly (K-ɛ-GG) remnant produced by trypsin digestion of proteins having ubiquitinated lysine side chains have markedly improved the ability to enrich and detect endogenous ubiquitination sites by mass spectrometry (MS). The following protocol describes the steps required to complete a large-scale ubiquitin experiment for the detection of tens of thousands of distinct ubiquitination sites from cell lines or tissue samples. Specifically, we present detailed, step-by-step instructions for sample preparation, off-line fractionation by reversed-phase chromatography at pH 10, immobilization of an antibody specific to K-ɛ-GG to beads by chemical cross-linking, enrichment of ubiquitinated peptides using these antibodies and proteomic analysis of enriched samples by LC-tandem MS (MS/MS). Relative quantification can be achieved by performing stable isotope labeling by amino acids in cell culture (SILAC) labeling of cells. After cell or tissue samples have been prepared for lysis, the described protocol can be completed in ∼5 d.
The extracellular matrix (ECM) is a fundamental component of multicellular organisms that provides mechanical and chemical cues that orchestrate cellular and tissue organization and functions. ...Degradation, hyperproduction or alteration of the composition of the ECM cause or accompany numerous pathologies. Thus, a better characterization of ECM composition, metabolism, and biology can lead to the identification of novel prognostic and diagnostic markers and therapeutic opportunities. The development over the last few years of high-throughput (“omics”) approaches has considerably accelerated the pace of discovery in life sciences. In this review, we describe new bioinformatic tools and experimental strategies for ECM research, and illustrate how these tools and approaches can be exploited to provide novel insights in our understanding of ECM biology. We also introduce a web platform “the matrisome project” and the database MatrisomeDB that compiles in silico and in vivo data on the matrisome, defined as the ensemble of genes encoding ECM and ECM-associated proteins. Finally, we present a first draft of an ECM atlas built by compiling proteomics data on the ECM composition of 14 different tissues and tumor types.
•The matrisome is defined as the ensemble of 1000+ genes encoding ECM and ECM-associated proteins.•Bioinformatic and experimental approaches to study the ECM/matrisome are discussed.•We introduce a novel website and database MatrisomeDB to centralize resources on the matrisome.•We present a draft of an ECM atlas compiling proteomics data on the ECM of 14 different tissues and tumors.•“Omics” data provide novel insights into ECM functions in development, homeostasis and disease.
A fundamental goal of genomics is to identify the complete set of expressed proteins. Automated annotation strategies rely on assumptions about protein-coding sequences (CDSs), e.g., they are ...conserved, do not overlap, and exceed a minimum length. However, an increasing number of newly discovered proteins violate these rules. Here we present an experimental and analytical framework, based on ribosome profiling and linear regression, for systematic identification and quantification of translation. Application of this approach to lipopolysaccharide-stimulated mouse dendritic cells and HCMV-infected human fibroblasts identifies thousands of novel CDSs, including micropeptides and variants of known proteins, that bear the hallmarks of canonical translation and exhibit translation levels and dynamics comparable to that of annotated CDSs. Remarkably, many translation events are identified in both mouse and human cells even when the peptide sequence is not conserved. Our work thus reveals an unexpected complexity to mammalian translation suited to provide both conserved regulatory or protein-based functions.
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•ORF-RATER robustly identifies and quantifies translation from ribosome profiling data•ORF-RATER reveals thousands of novel micropeptides and variants of mammalian proteins•Hundreds of novel CDSs show evidence of protein-coding conservation among mammals•Many ORFs are translated in both mice and humans but lack protein-coding conservation
Fields et al. describe a ribosome profiling-based approach for empirical annotation of protein-coding regions of the genome. Of the thousands of previously unknown translated ORFs they identify in mouse and human, many are conserved or dynamically regulated. Surprisingly, a considerable subset is translated in both species despite weak sequence conservation.
Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas ...the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.
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