mRNAs, proteins and the emerging principles of gene expression control

Nature Reviews Genetics, Journal Year: 2020, Volume and Issue: 21(10), P. 630 - 644

Published: July 24, 2020

Language: Английский

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Medulloblastoma

Nature Reviews Disease Primers, Journal Year: 2019, Volume and Issue: 5(1)

Published: Feb. 14, 2019

Language: Английский

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TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach

Molecular & Cellular Proteomics, Journal Year: 2019, Volume and Issue: 18(7), P. 1468 - 1478

Published: April 10, 2019

Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such often require hundreds micrograms that cost reagents represents a major contribution to overall an experiment. Here, we describe and evaluate robust cost-effective protocol TMT reduces quantity required reagent by factor eight achieves complete labeling. Under- overlabeling derived complex digests tissues cell lines were systematically evaluated using peptide quantities between 12.5 800 μg TMT-to-peptide ratios (wt/wt) ranging 8:1 1:2 at different concentrations. When reaction volumes reduced maintain concentrations least 10 mm 2 g/l, respectively, as low 1:1 resulted efficiencies > 99% excellent intra- interlaboratory reproducibility. The utility optimized was further demonstrated deep-scale proteome phosphoproteome patient-derived xenograft tumor tissue benchmarked against procedure recommended vendor. Finally, discuss impact parameters N-hydroxysuccinimide ester-based chemistry provide guidance adopting efficient protocols quantities. In bottom-up proteomics, variety strategies can be followed determine quantitative differences abundance proteins posttranslational modifications (PTMs) (1Bantscheff M. Lemeer S. Savitski M.M. Kuster B. Quantitative spectrometry proteomics: Critical review update 2007 present.Anal. Bioanalytical Chem. 2012; 404: 939-965Crossref PubMed Scopus (581) Google Scholar). Among those, isobaric enables multiplexing up 11 samples (2Thompson A. Schäfer J. Kuhn K. Kienle Schwarz Schmidt G. Neumann T. Hamon C. Tandem tags: A novel strategy comparative protein mixtures MS/MS.Anal. 2003; 75: 1895-1904Crossref (1709) Each these used label primary amines via with NHS reactive group. Subsequently, all are pooled processed together, thus reducing technical variation experimental workflow. Inside spectrometer, nature leads summation each signal labeled combined MS1 spectrum. Following fragmentation, sample-specific reporter ions mass-to-charge (m/z) values generated owing combinations heavy carbon nitrogen isotopes present ions. This differentiation relative conditions same MS2 scan. Multiplexing this manner number missing Further, reproducibility less sensitive performance variations liquid chromatography (LC) 1The abbreviations are:LCliquid chromatographyMSmass spectrometryNCEnormalized collision energyNHSN-hydroxysuccinimidePDXpatient-derived xenograftPSMpeptide spectrum matchRPreversed phaseTMTtandem tags. (MS) systems than label-free measurements. addition, capability achieving deep coverage multiple reasonable amount measurement time. Together advantages render attractive MS-based studies, including analyses depend (3Zecha Meng Zolg D.P. Samaras P. Wilhelm Peptide level turnover measurements enable study dynamics.Mol. Cell Proteomics. 2018; 17: 974Abstract Full Text PDF (66) high workflow has recently been (PDX) models breast cancer (4Mertins Tang L.C. Krug Clark D.J. Gritsenko M.A. Chen L. Clauser K.R. Clauss T.R. Shah Gillette Petyuk V.A. Thomas S.N. Mani D.R. Mundt F. Moore R.J. Hu Y. Zhao R. Schnaubelt Keshishian H. Monroe M.E. Zhang Z. Udeshi N.D. D. Davies S.R. Townsend R.R. Chan D.W. Smith R.D. Liu Carr S.A. Reproducible multiplexed chromatography–mass spectrometry.Nat. Protoc. 13: 1632-1661Crossref (198) Scholar), motivates use larger patients (5Archer T.C. Ehrenberger Gold M.P. Mah C.K. Mahoney E.L. Daniel C.J. LeNail Ramamoorthy Mertins Noble Pierre-François Silterra Jensen Tamayo Korshunov Pfister S.M. Kool Northcott P.A. Sears R.C. Lipton J.O. Mesirov J.P. Pomeroy S.L. Fraenkel E. Proteomics, post-translational modifications, integrative reveal molecular heterogeneity within medulloblastoma subgroups.Cancer Cell. 34: 396-410.e398Abstract (90) normalized energy match reversed phase One substantial shortcoming reagents. We others have previously reported which vendor (6Böhm Prefot Jung Selzer Mitra V. Britton Pike I. Thompson A.H. Low-pH solid-phase amino TMTs improves identification rates global phosphopeptide analysis.J. Proteome Res. 2015; 14: 2500-2510Crossref (24) Scholar, 7Edwards Haas W. Multiplexed proteomics high-throughput comprehensive comparisons human lines.in: Reinders Proteomics Systems Biology: Methods Protocols. Springer New York, York2016: 1-13Google 8Ruprecht Zecha High pH reversed-phase micro-columns simple, sensitive, fractionation (TMT labeled) digests.in: Comai Katz J.E. Mallick Proteomics: York2017: 83-98Google 9Navarrete-Perea Yu Q. Gygi S.P. Paulo J.A. Streamlined tag (SL-TMT) protocol: An (phospho)proteome profiling tag-synchronous precursor selection-MS3.J. 2226-2236Crossref (133) 10Stepanova Filter-based digestion (FPD): detergent-free scaffold-based workflows.J. 1227-1234Crossref (13) Scholar) successfully economically workflows address biological questions 11Koch Ruprecht Beck Frejno Klaeger Phosphoproteome reveals mechanisms growth-factor-mediated kinase inhibitor resistance EGFR-overexpressing cells.J. 2016; 15: 4490-4504Crossref 12Paulo O'Connell J.D. Everley R.A. O'Brien spectrometry-based compares 5000 cerevisiae sources.J. 148: 85-93Crossref (102) 13Mirzaei Gupta V.B. Chick J.M. Greco T.M. Wu Chitranshi N. Wall R.V. Hone Deng Dheer Abbasi Rezaeian Braidy You Salekdeh G.H. Haynes Molloy Martins Cristea I.M. Graham V.K. Age-related neurodegenerative disease associated pathways identified retinal vitreous glaucoma eyes.Scientific Reports. 2017; 7: 12685Crossref (75) details reactants vary widely published literature and, best our knowledge, no systematic evaluation influence date. study, established times demonstrate transferability laboratories, adoption approach quantities, show applicability improved in-depth phosphoproteomic analyses. rationale design described more detail respective results sections, detailed overview volumes, reactants, buffers, solvents listed supplement. three independent experiments, increasing (12.5 μg) concentration (100 including, total, technical, intralaboratory duplicates triplicates. Moreover, 17 experiments singlicates, applying (40 400 200 explore examine adaptability lower To assess robustness, four titrated (50 constant μg), carried out seven replicates two performed laboratories. All method optimization analyzed single-shot LC-MS/MS runs. highly fractionated samples, but (i.e. 8x reagent) comparing it original protocol. Briefly, basal (B) luminal (L) PDX (WHIM2 WHIM16) five TMT10-plex experiment (B-l-B-B-l-B-l-l-B-L) RP chromatography. After pooling enrichment, 24 whole 12 fractions measured LC-MS/MS. HeLa Jurkat cells cultured DMEM RPMI 1640 medium, supplemented 10% FBS (GibcoTM), 1% antibiotic, antimycotic solution (Sigma). WHIM2 WHIM16 (14Li Shen Shao Crowder Prat He X. Hoog Lu Ding Griffith O.L. Miller Larson Fulton R.S. Harrison Mooney McMichael J.F. Luo Tao Goncalves Schlosberg Hiken Saied Sanchez Giuntoli Bumb Cooper Kitchens R.T. Lin Phommaly Kavuri M.S. McEachern Dong Yi Ma Pluard Naughton Bose Suresh McDowell Michel Aft Gillanders DeSchryver Wilson R.K. Wang Mills G.B. Gonzalez-Angulo Edwards J.R. Maher Perou C.M. Mardis E.R. Ellis M.J. Endocrine-therapy-resistant ESR1 variants revealed genomic characterization breast-cancer-derived xenografts.Cell Rep. 2013; 4: 1116-1130Abstract (451) Cells, murine liver lysed 8 m urea 40 Tris-HCl, 7.6 (Hela), 75 NaCl 50 Tris, 8.0 (Jurkat, liver, tissue), containing protease phosphatase cocktails. Lysates incubated ice 15 30 min centrifugation 20,000 × g 4 °C remove insoluble debris. Protein supernatant determined PierceTM Coomassie BCA Assay Kit (ThermoScientific). Disulfide bridges DTT (HeLa) 5 37 1 h mouse tissue). Alkylation room temperature dark chloroacetamide iodoacetamide 45 diluted < (HeLa), Digestion either adding trypsin (Promega) 1:50 enzyme-to-substrate ratio incubating overnight 600 rpm performing double 25 LysC (Wako) Digests acidified addition neat formic acid (FA) 1%, centrifuged pellet matter, desalted tC18 extraction cartridges (Waters Corp.; wash solvent: 0.1% FA TFA; elution 50% acetonitrile (ACN)). Eluates frozen −80 dried vacuum centrifugation. Desalted reconstituted Kit. For aliquoted accordingly peptides), °C, Peptides HEPES (pH 8.5) TMTzero mix (ThermoFisher) added stocks dissolved 100% anhydrous ACN. Respective specified section peptide–TMT mixture rpm, stopped 5% hydroxylamine final 0.4% μl 8, incubation rpm. solutions 45% (v/v) ACN prior drying directly analyses, Lys-C/trypsin tumors et al. following 300 60 8.5), started (15 56.7 (20 μg/μl) ACN). Samples 1,000 quenched min; °C; rpm). pooled, TMT-labeled tC18, ACN), Zorbax Extend-C18 column (3.5 μm, 4.6 250 mm; Agilent). into down measurements, residual 95% enrichment immobilized metal affinity Enrichment Ni-nitrilotriacetic superflow agarose beads (Qiagen) loaded iron (III) phosphopeptides self-packed StageTips (wash FA; Tryptic one-shot EASY-nLC 1200 Ultimate 3000 RSLCnano system coupled Q-Exactive Plus, HF-X Fusion Lumos Tribrid spectrometer (ThermoFisher Scientific). reconstitution FA, corresponding 500 ng injected. Supplemental Table I provides experiments. brief, Ultimate3000 system, separated μm x cm analytical (packed in-house 3-μm C18 resin; Reprosil Gold, Dr. Maisch) flow rate nl/min 20-min linear gradient 34% LC solvent B1 (0.1% DMSO ACN) A1 DMSO). equipped 20 22 (Picofrit, Objective, Inc.; packed 1.9-μm operated 3 55% B2 (90% ACN, FA) A2 (3% FA), 17-min three-step 60% A2, 97-min A2. Mass spectrometers data-dependent positive ionization mode. On Q Exactive spectra recorded resolution 70k automatic gain control (AGC) target value 3e6 1e6 charges maximum injection (maxIT) ms ms. fragmentation higher collisional dissociation, precursors acquired 17.5k AGC 5e4 maxIT 120 MS above modifications: 60k Fragment 30k 4e5 charges. per cycle 15k before Lumos. over 84-min 6 30% 22-cm above. 4e5. 50k 6e4 105 Cycle set s. MaxQuant: titration MaxQuant (version 1.6.3.3) its built-in search engine, Andromeda (15Cox Neuhauser Michalski Scheltema Olsen J.V. Mann Andromeda: engine integrated environment.J. 2011; 10: 1794-1805Crossref (3450) 16Tyanova Temu Cox computational platform shotgun proteomics.Nat. 11: 2301Crossref (1872) searched reference (UP000005640, 95,936 entries, downloaded October 12, 2018) and/or (UP000000589, 62,407 common contaminants. Separate searches conducted check under- overlabeling. underlabeling evaluation, TMT10 variable modification lysine N termini. assessment, fixed ion additionally, histidine serine, threonine, tyrosine. searches, carbamidomethylated cysteine oxidation methionine N-terminal acetylation modifications. Trypsin/P proteolytic enzyme missed cleavage sites allowed. Precursor tolerance ±4.5 ppm, fragment ±20 ppm. modified peptides, default cutoffs score delta score, respectively. Results adjusted false discovery employing target–decoy sequences. Raw files (phospho)proteomes RefSeq database 37,592 27,289 entries complemented contaminants (RefSeq.20160914_Human_Mouse_ucsc_hg19_mm10_customProDBnr_mito_150contams) Spectrum Mill suite vB.06.01.202 (Broad Institute Agilent Technologies). four-cycle fixed/mix ran consecutive sets round then produced output. cycles follows: unmodified, both termini lysines labeled, only labeled. Carbamidomethylation cysteines selenocysteines additional modification. acetylation, methionine, de-amidation asparagine, hydroxylation proline (when Gly), cyclization glutamine carbamidomethyl pyroglutamic (pyroGlu) pyro-carbamidomethyl cysteine, tyrosine (limited histidine-containing peptides) analysis, phosphorylation allowed while asparagine restricted glycine, not Trypsin Allow P shift range -18 262 Da allow pyroGlu one Met-ox peptide. expanded 272 phosphorylations product tolerances ppm PSM-level <1% better dissect origin, subgroup-specific grouping option enabled, (17Huang K-l. Li Cao Gunawardena H.P. Ruggles K.V. Tanioka Usary Xie Yoon Qiao J.W. Wrobel Wyczalkowski Erdmann-Gilmore Snider Singh Niu Guo Sun S.Q. Sanati Kawaler Scott Ye McLellan M.D. Wendl M.C. Malovannaya Held Fenyö Kinsinger C.R. Mesri Rodriguez Reid Proteogenomic integration therapeutic targets xenografts.Nat. Commun. 8: 14864Crossref (87) estimation molarities functional groups digests, silico digest Simulator released Pacific Northwest National Laboratory (https://omics.pnl.gov/software/protein-digestion-simulator). obtain conservative estimate termini, trypsin/P Minimum masses 6,000 Da, duplicated sequences given included. average monoisotopic calculate acids 100 digest. data hits reverse contaminant databases removed. side chains free acetylated) counted "fully labeled." did bear any annotated "not labeled," whereas contained fully classified "partially acetylated arginine excluded overlabeled. (phospho)proteomes, signals corrected impurities, unique considered analysis. Relative abundances median intensity PSMs matching site. lacking label, having purity 50%, negative forward–reverse (half false-positive identifications) excluded. normalize intensities divided channels site protein. Ratios centering absolute deviation scaling.

Language: Английский

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Second-generation molecular subgrouping of medulloblastoma: an international meta-analysis of Group 3 and Group 4 subtypes

Acta Neuropathologica, Journal Year: 2019, Volume and Issue: 138(2), P. 309 - 326

Published: May 10, 2019

In 2012, an international consensus paper reported that medulloblastoma comprises four molecular subgroups (WNT, SHH, Group 3, and 4), each associated with distinct genomic features clinical behavior. Independently, multiple recent reports have defined further intra-subgroup heterogeneity in the form of biologically clinically relevant subtypes. However, owing to differences patient cohorts analytical methods, estimates subtype number definition been inconsistent, especially within 3 4. Herein, we aimed reconcile 3/Group 4 MB subtypes through analysis a series 1501 medulloblastomas DNA-methylation profiling data, including 852 matched transcriptome data. Using complementary bioinformatic approaches, compared concordance calls between published assessments class-definition confidence reproducibility. While lowest complexity solutions continued support original 4, our most strongly supported comprising eight robust (types I-VIII). Subtype II was consistently identified across all component studies, while others were by methods. Regardless technique, increasing cohort size did not increase Summarizing clinico-pathological these indicated enrichment specific driver gene alterations cytogenetic events amongst subtypes, highly disparate survival outcomes, supporting their biological relevance. Collectively, this study provides for Groups enabling derivation of, categorical accounting for, extensive intertumoral revealed high-resolution subclassification approaches. Furthermore, findings provide basis application emerging methods (e.g., proteomics/single-cell approaches) which may additionally inform subclassification. Outputs from will help shape next generation protocols facilitate enhanced molecularly guided risk stratification improve outcomes quality life patients families.

Language: Английский

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The emerging role of mass spectrometry-based proteomics in drug discovery

Nature Reviews Drug Discovery, Journal Year: 2022, Volume and Issue: 21(9), P. 637 - 654

Published: March 29, 2022

Language: Английский

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Medulloblastomics revisited: biological and clinical insights from thousands of patients

Nature reviews. Cancer, Journal Year: 2019, Volume and Issue: 20(1), P. 42 - 56

Published: Dec. 9, 2019

Language: Английский

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A knowledge graph to interpret clinical proteomics data

Nature Biotechnology, Journal Year: 2022, Volume and Issue: 40(5), P. 692 - 702

Published: Jan. 31, 2022

Implementing precision medicine hinges on the integration of omics data, such as proteomics, into clinical decision-making process, but quantity and diversity biomedical spread clinically relevant knowledge across multiple databases publications, pose a challenge to data integration. Here we present Clinical Knowledge Graph (CKG), an open-source platform currently comprising close 20 million nodes 220 relationships that represent experimental public literature. The graph structure provides flexible model is easily extendable new become available. CKG incorporates statistical machine learning algorithms accelerate analysis interpretation typical proteomics workflows. Using set proof-of-concept biomarker studies, show how might augment enrich help inform decision-making.

Language: Английский

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TMTpro: Design, Synthesis, and Initial Evaluation of a Proline-Based Isobaric 16-Plex Tandem Mass Tag Reagent Set

Analytical Chemistry, Journal Year: 2019, Volume and Issue: 91(24), P. 15941 - 15950

Published: Nov. 18, 2019

The design and synthesis of a proline-based reporter isobaric Tandem Mass Tag structure (TMTpro) is presented. An analysis made the performance new TMTpro tags in comparison with current commercially available dimethylpiperidine-reporter-based TMT10/11 reagents. provides set 16 for use resolution 6.3 mDa mass differences high spectrometers 9 reagents 1 Da spacing between ions single dalton using heavy nuclei per tag. We show similar terms peptide identification rates quantification 16-plex TMT10/11-plex also demonstrate suitability phosphopeptide analysis. ability to pool samples reduces overall amount sample required each channel, we anticipate that will be useful enhancement any protocol benefits from pooling should reduce missing data.

Language: Английский

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Cancer proteogenomics: current impact and future prospects

Nature reviews. Cancer, Journal Year: 2022, Volume and Issue: 22(5), P. 298 - 313

Published: March 2, 2022

Language: Английский

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Pan-cancer analysis of post-translational modifications reveals shared patterns of protein regulation

Cell, Journal Year: 2023, Volume and Issue: 186(18), P. 3945 - 3967.e26

Published: Aug. 1, 2023

Post-translational modifications (PTMs) play key roles in regulating cell signaling and physiology both normal cancer cells. Advances mass spectrometry enable high-throughput, accurate, sensitive measurement of PTM levels to better understand their role, prevalence, crosstalk. Here, we analyze the largest collection proteogenomics data from 1,110 patients with profiles across 11 types (10 National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium [CPTAC]). Our study reveals pan-cancer patterns changes protein acetylation phosphorylation involved hallmark processes. These revealed subsets tumors, different types, including those dysregulated DNA repair driven by phosphorylation, altered metabolic regulation associated immune response acetylation, affected kinase specificity crosstalk between modified histone regulation. Overall, this resource highlights rich biology governed PTMs exposes potential new therapeutic avenues.

Language: Английский

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