DIA-NN: neural networks and interference correction enable deep proteome coverage in high throughput

Nature Methods, Journal Year: 2019, Volume and Issue: 17(1), P. 41 - 44

Published: Nov. 25, 2019

Language: Английский

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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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Revisiting biomarker discovery by plasma proteomics

Molecular Systems Biology, Journal Year: 2017, Volume and Issue: 13(9)

Published: Sept. 1, 2017

Clinical analysis of blood is the most widespread diagnostic procedure in medicine, and biomarkers are used to categorize patients support treatment decisions. However, existing far from comprehensive often lack specificity new ones being developed at a very slow rate. As described this review, mass spectrometry (MS)-based proteomics has become powerful technology biological research it now poised allow characterization plasma proteome great depth. Previous "triangular strategies" aimed discovering single biomarker candidates small cohorts, followed by classical immunoassays much larger validation cohorts. We propose "rectangular" profiling strategy, which patterns large cohorts correlated with their phenotypes health disease. Translating such concepts into clinical practice will require restructuring several aspects decision-making, we discuss some first steps direction.

Language: Английский

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Ultra-High-Throughput Clinical Proteomics Reveals Classifiers of COVID-19 Infection

Cell Systems, Journal Year: 2020, Volume and Issue: 11(1), P. 11 - 24.e4

Published: June 2, 2020

The COVID-19 pandemic is an unprecedented global challenge, and point-of-care diagnostic classifiers are urgently required. Here, we present a platform for ultra-high-throughput serum plasma proteomics that builds on ISO13485 standardization to facilitate simple implementation in regulated clinical laboratories. Our low-cost workflow handles up 180 samples per day, enables high precision quantification, reduces batch effects large-scale longitudinal studies. We use our collected from cohort of early hospitalized cases the SARS-CoV-2 identify 27 potential biomarkers differentially expressed depending WHO severity grade COVID-19. They include complement factors, coagulation system, inflammation modulators, pro-inflammatory factors upstream downstream interleukin 6. All protocols software implementing approach freely available. In total, this work supports development routine proteomic assays aid decision making generate hypotheses about therapeutic targets.

Language: Английский

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Extracellular Vesicles Provide a Means for Tissue Crosstalk during Exercise

Cell Metabolism, Journal Year: 2018, Volume and Issue: 27(1), P. 237 - 251.e4

Published: Jan. 1, 2018

Language: Английский

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Plasma proteomic signature of age in healthy humans

Aging Cell, Journal Year: 2018, Volume and Issue: 17(5)

Published: July 11, 2018

Abstract To characterize the proteomic signature of chronological age, 1,301 proteins were measured in plasma using SOMAscan assay (SomaLogic, Boulder, CO, USA) a population 240 healthy men and women, 22–93 years old, who disease‐ treatment‐free had no physical cognitive impairment. Using p ≤ 3.83 × 10 −5 significance threshold, 197 positively associated, 20 negatively associated with age. Growth differentiation factor 15 (GDF15) strongest, positive association age (GDF15; 0.018 ± 0.001, = 7.49 −56 ). In our sample, GDF15 was not other cardiovascular risk factors such as cholesterol or inflammatory markers. The functional pathways enriched 217 age‐associated included blood coagulation, chemokine pathways, axon guidance, peptidase activity, apoptosis. elastic net regression models, we created based on relative concentrations 76 that highly correlated ( r 0.94). generalizability findings needs replication an independent cohort.

Language: Английский

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A Novel LC System Embeds Analytes in Pre-formed Gradients for Rapid, Ultra-robust Proteomics

Molecular & Cellular Proteomics, Journal Year: 2018, Volume and Issue: 17(11), P. 2284 - 2296

Published: Aug. 13, 2018

To further integrate mass spectrometry (MS)-based proteomics into biomedical research and especially clinical settings, high throughput robustness are essential requirements. They largely met in high-flow rate chromatographic systems for small molecules but these not sufficiently sensitive applications. Here we describe a new concept that delivers on requirements while maintaining the sensitivity of current nano-flow LC systems. Low-pressure pumps elute sample from disposable trap column, simultaneously forming gradient is stored long storage loop. An auxiliary creates an offset, ensuring re-focusing peptides before separation analytical column by single high-pressure pump. This simplified design enables robust operation over thousands injections. Furthermore, steps between injections performed parallel, reducing overhead time to few minutes allowing analysis more than 200 samples per day. From fractionated HeLa cell lysates, deep proteomes covering 130,000 sequence unique close 10,000 proteins were rapidly acquired. Using this data as library, demonstrate quantitation 5200 only 21 min. Thus, system - termed Evosep One analyzes extremely manner, without sacrificing depth coverage. Bottom-up highly successful generic technology, which now allows complex ranging bacteria through line even human tissue (1Aebersold R. Mann M. Mass-spectrometric exploration proteome structure function.Nature. 2016; 537: 347-355Crossref PubMed Scopus (1105) Google Scholar). State-of-the-art workflows begin with preparation digest harvest purified (2Kulak N.A. Pichler G. Paron I. Nagaraj N. Minimal, encapsulated proteomic-sample processing applied copy-number estimation eukaryotic cells.Nat. Methods. 2014; 11: 319-324Crossref (991) Scholar), separated liquid chromatography (LC) 1The abbreviation used is:LCliquid chromatography. they analyzed spectrometer (MS). Established software solutions automatically interpret acquired spectra, generating lists quantified (3Cox J. MaxQuant peptide identification rates, individualized p.p.b.-range accuracies proteome-wide protein quantification.Nat. Biotechnol. 2008; 26: 1367-1372Crossref (9154) Scholar, 4Bekker-Jensen D.B. Kelstrup C.D. Batth T.S. Larsen S.C. Haldrup C. Bramsen J.B. Sørensen K.D. Høyer S. Ørntoft T.F. Andersen C.L. Nielsen M.L. Olsen J.V. optimized shotgun strategy rapid generation comprehensive proteomes.Cell Systems. 2017; 4: 587-599Abstract Full Text PDF (255) 5Kelstrup Bekker-Jensen Arrey T.N. Hogrebe A. Harder Performance evaluation Q Exactive HF-X proteomics.J Proteome Res,. 2018; 17: 727-738Crossref (159) 6Kulak Geyer P.E. Loss-less nano-fractionator sensitivity, coverage proteomics.Mol. Cell. Proteomics. (Manuscript Press, 2017)Abstract (104) 7Bruderer Bernhardt OM Gandhi T Xuan Y Sondermann J Schmidt M Gomez-Varela D Reiter L Optimization experimental parameters data-independent significantly increases reproducibility results.Mol. 16: 2296-2309Abstract (189) 8Meier F. Virreira Winter Cox BoxCar acquisition method single-shot at 100 minutes.Nat. 10.1038/s41592-018-0003-5Crossref (218) The performance level result improvements spectrometric components also part LC-MS workflow. In quest ever increasing power, columns have become longer particle sizes smaller reaching sub 2 μm range. may require pump pressures 1000 bar, presenting great engineering challenges both entire system, often limiting routine operation. remains weak link MS-based workflows, leading calls approaches (9Riley N.M. Hebert A.S. Coon J.J. Proteomics Moves Fast Lane.Cell Syst,. 2: 142-143Abstract (33) irreproducibility retention times within laboratories severely limits strategies rely transfer accurate times, targeted (10Picotti P. Aebersold Selected reaction monitoring-based proteomics: potential, pitfalls future directions.Nat. 2012; 9: 555-566Crossref independent (11Gillet L.C. Leitner Mass bottom-up entering high-throughput era hypothesis testing.Annu. Rev. Anal. Chem. 449-472Crossref (207) Scholar) "match runs" MS (12Cox Hein M.Y. Luber C.A. Accurate label-free quantification delayed normalization maximal ratio extraction, MaxLFQ.Mol. 13: 2513-2526Abstract (2688) 13Geiger T. Wehner A Schaab C Comparative proteomic eleven common lines reveals ubiquitous varying expression most proteins.Mol. 11M111.014050Abstract (577) There interest applying power diagnostic questions (14Geyer Holdt L.M. Teupser D. Revisiting biomarker discovery plasma proteomics.Mol Syst Biol,. 942Crossref (390) "Clinical proteomics", however, requires far stability available advanced laboratories. Note issues features se, measurement firmly established around world, routinely measure hundreds two key differences one their much larger diameters (20-fold) flow rates (1000-fold), making them easier control less error-prone. Increasing achieve greater has already been advocated context cancer (15Liu Y. Hüttenhain Collins B. maps research.Expert Mol. Diagn. 2013; 811-825Crossref (99) However, signal intensity electrospray ionization concentration dependent higher μl/min. Apart robustness, other central requirement proteomics, if it enter use. Instead, generally employ fractionation—multiplying time—or use relatively times. recent large-scale study measured our laboratory, involving thousand samples, 80% overall down was attributable HPLC rather MS. At same time, equilibration, loading washing runs limited attractiveness very short gradients (16Geyer Kulak Plasma profiling assess health disease.Cell Syst. 185-195Abstract (380) 17Geyer Wewer Albrechtsen N.J. Tyanova Grassl Iepsen E.W. Lundgren Madsbad Holst Torekov S.S. effects sustained weight loss proteome.Mol. Biol. 12: 901Crossref (135) Several years ago, some authors devised different injection approach. Termed speLC, solid-phase-extraction (nano) chromatography, intended needed application (18Falkenby L.G. Such-Sanmartín M.R. Vorm O. Bache Jensen O.N. Integrated solid-phase extraction-capillary (speLC) interfaced ESI-MS/MS fast characterization proteomes.J. Res. 6169-6175Crossref (18) speLC made StageTips commonly employed micro-scale purification crude manual fractionation (19Ishihama Rappsilber Modular stop go extraction tips stacked disks parallel multidimensional Peptide proteomics.J. 2006; 5: 988-994Crossref (224) 20Rappsilber Ishihama Stop matrix-assisted laser desorption/ionization, nanoelectrospray, LC/MS pretreatment proteomics.Anal. 2003; 75: 663-670Crossref (1796) 21Wisniewski J.R. Dus K. Proteomic workflow archival formalin-fixed paraffin-embedded 10 000 proteins.Proteomics Clin. Appl. 7: 225-233Crossref (119) Instead eluting autosampler vial low-pressure passed 5–10 min StageTip itself directly toward can analyze 192 E. coli 30 h, well identifying 500 lysate subsequent work, combined pre-fractionation such 1D gel electrophoresis or strong cation exchange (SCX), capitalizing its ability each fractions (22Binai Marino Soendergaard Mohammed Heck A.J. Rapid analyses interactomes using integrated extraction-liquid chromatography-MS/MS system.J. 2015; 14: 977-985Crossref (5) Although useful simple mixtures, elution inherently system. work reported here, aimed preserve benefits original device achieving desirable modern instruments. We realized goal coupling novel downstream design, eluted low pressure tens μl/min special EvotipTM. Notably, along analytes captured capillary then applies nano-scale column. results undiminished eliminating need form pressure. layout marries convenience large columns, narrow low-flow nano-LC detail principle development instrument investigate throughput, typical applications encountered proteomics. incorporates four stroke piston (A, B, C, D) (HP) (Fig. 1A; supplemental Fig. S1A, S1B). Together create separate low- sub-system. Each equipped sensor monitor precisely individual solvent. custom 12-port valve (operating low-pressure) diverts either solvent bottles (sol A, sol B) refilling analysis. 6-port high-pressure) refilling. path loop, connected sub-system controlled rotary 1A). way, always in-line bypasses contrast, waste bypassing loop becomes bridge sub-systems. illustrated timetable diagrams, highlighting stages run 1B, S2–S9). beginning run, XYZ-axis manipulator picks up (Evotip) ceramic needle positions A/B/C/D mixing cross 1A, second step, B primary A/B tee flows S2). organic content initial 35% ensure off unwanted compounds polymers, lipids, hydrophobic remain bound single-use, any particulate matter loaded samples. final volume μl precise minimize bleeding molecules. "partial elution" will be described RESULTS AND DISCUSSION. additional pumps, modify eluent "offset" (supplemental purpose lowering contents, initially retained offset embedded moved being switched first steps, filled S3) equilibrated S4). Subsequently, switches preformed pushed S5). prepared next ejecting needle, aligning solvents S6–S9). contains procedures state during detect parts warns potential leaks lack Evotips designated position. It built trouble shooting Chronos user interface. Moreover, carries out preparatory actions run. cells cultured glucose DMEM 10% fetal bovine serum 1% penicillin-streptomycin (Life Technologies, Inc.). Cells counted Invitrogen countess counter after snap freezing −80 °C. Protein concentrations determined 8 m urea tryptophan fluorescence emission 350 nm, excitation wavelength 295 nm. Tryptophan 0.1 μg/μl establish standard calibration curve (0–4 μl). estimated equivalent 7 extract, average accounts 1.3% amino acid composition. For iST kit starting 106 according manufacturer's instructions (P.O. 00001, PreOmics GmbH). test optimize injected 2000 tryptic cells, exploratory batches. experiment, breadboard model coupled LTQ Orbitrap instrument. All protocolled, phase, stopped optimization hardware components. last 1500 variation wear Blood taken venipuncture commercially winged infusion set collection tubes containing EDTA centrifuged 15 × g plasma. sampled healthy donor, who provided written informed consent, prior approval ethics committee Max Planck Society. distributed 96-well plate subsequently processed automated Profiling previously "UPS1 Standard" (Sigma-Aldrich) digested indicated above samples/day (5.6 gradient) 5 cm C18 (3 size). Peptides reversed-phase Acquity CSH 1.7 1 150 mm (Waters, Milford, MA) Ultimate 3000 (HPLC) (Dionex, Sunnyvale, CA) operating Buffer (5 ammonium bicarbonate) buffer (100% ACN) used. linear 5% 55 min, followed increase 70% total, 46 collected concatenation. LC-MS/MS, amount kept constant ng Easy-nLC 1200, fraction Evotip. effect diffusion function rig mimic figure 2. Zirconium nano (Prolab Instruments, GmbH, Switzerland, A: 0.1% formic (FA) H2O, Pump B: FA, acetone acetonitrile) programed following composition profile: 0–5 5–95% 10–13 95% 13–15 95–5%, 15–18 5%, 18–23 23–25 25–27 95–5% 27–30 delivered coiled (diameter cm) fused silica (length m, i.d. μm, OD375, Polymicro Technologies). After specified had passed, third UV detector (SpectraFlow 501, SunChrom) nl) record absorption 265 three all Vici (Valco Instruments Co. Inc.) script. 250 commercial (Pierce, no. 1862824) spiked fmol PicoSure Test Standard (eight synthetic mix, New Objective PS-STDN) mix Evotips. five methods, replicates Thermo acquire full scans (resolution 35k) MS2 17.5k) eight scheduled table 6B). Skyline extract 4 6 ions (parallel monitoring) (23MacLean Tomazela D.M. Shulman Chambers Finney G.L. Frewen Kern Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: open source document editor creating analyzing experiments.Bioinformatics,. 2010; 966-968Crossref (2964) Chromatographic profiles exported peak characteristics extracted Tips activated consecutive wash 100% ACN, 50% ACN 0.5% H2O H2O. BSA tip activation protocol later 1-propanol wetting material equilibration. instrumentation consisted production version (Thermo Fisher Scientific) experiments. Purified 3 Reprosil-Pur beads (Dr. Maisch, Ammerbuch, Germany) dimensions below 6B. On MS, Top6 Top12 Orbitrap, target value scan spectra charges 300–1650 m/z range maximum 50 ms resolution 60,000 200. Fragmentation precursor higher-energy C-trap dissociation (HCD) normalized collision energy 27 eV (24Olsen et al.Higher-energy modification analysis.Nat. 2007; 709-712Crossref (719) MS/MS 15,000 ion 104 25 ms. Dynamic exclusion s avoid repeated sequencing identical peptides. harvested ∼80% confluence twice PBS adding boiling lysis (6 guanidinium hydrochloride (GndCl), tris(2-carboxyethyl)phosphine, chloroacetamide, Tris pH 8.5) plate. scraping boiled micro probe sonication (Vibra-Cell VCX130, Sonics, Newton, CT) pulses amplitude. Bradford assay, LysC (Wako) enzyme/protein 1:100 (w/w) dilution Tris, 8.5, GndCl overnight trypsin (1:100 w/w). Protease activity quenched acidification trifluoroacetic (TFA) ∼1%, resulting mixture concentrated Sep-Pak (C18 Classic Cartridge, Waters, MA). Elution done ml 40% acetonitrile (ACN), 60% ACN. eluates reduced SpeedVac (Eppendorf, Germany), measuring absorbance 280 nm NanoDrop spectrophotometer (NanoDrop 2000C, Scientific, Germany). DIA iRT (Biognosys AB, Schlieren, Switzerland) added protocol. One, in-house packed 12 cm, 1.9 used, 1200 75 specifications above. temperature maintained 40 °C oven (PRSO-V1, Sonation, Biberach, online spectrometer. raw files (version 1.5.6.8) fragments searched against Uniprot Reference isoforms (April 2017 release 21,042 sequences) Andromeda search engine (25Cox Neuhauser Michalski Scheltema R.A. Andromeda: environment.J 2011; 10: 1794-1805Crossref (3450) cysteine carbamidomethylation fixed N-terminal acetylation methionine oxidations variable modifications. experiment UPS1 FASTA file, downloaded homepage Sigma-Aldrich 2018). false (FDR) 0.01 levels minimum length acids Enzyme specificity C-terminal arginine lysine expected proteases, missed cleavages. deviation ppm fragment 20 specified. Data (DIA) Spectronaut 11.0.15038.19.19667, default settings (Biognosys, Zurich, Switzerland). project specific spectral library imported pre-fractionated fractions, settings. Information about precursors, identified Supplemental Table S1 S2. bioinformatics Perseus (26Tyanova Temu Sinitcyn Carlson Geiger computational platform (prote)omics data.Nat. Method. 731-740Crossref (3530) platform. Our idea decouple formation resolution, As strategies, (a solid phase like (20Rappsilber Scholar)). instead tips, drying remove re-suspending buffer, Evotip accomplished

Language: Английский

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BoxCar acquisition method enables single-shot proteomics at a depth of 10,000 proteins in 100 minutes

Nature Methods, Journal Year: 2018, Volume and Issue: 15(6), P. 440 - 448

Published: May 4, 2018

Language: Английский

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Genetics meets proteomics: perspectives for large population-based studies

Nature Reviews Genetics, Journal Year: 2020, Volume and Issue: 22(1), P. 19 - 37

Published: Aug. 28, 2020

Language: Английский

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Blood plasma phosphorylated-tau isoforms track CNS change in Alzheimer’s disease

The Journal of Experimental Medicine, Journal Year: 2020, Volume and Issue: 217(11)

Published: July 28, 2020

Highly sensitive and specific plasma biomarkers for Alzheimer’s disease (AD) have the potential to improve diagnostic accuracy in clinic facilitate research studies including enrollment prevention treatment trials. We recently reported CSF tau hyperphosphorylation, especially on T217, is an accurate predictor of β-amyloidosis at asymptomatic symptomatic stages. In current study, we determine by mass spectrometry utility p-tau isoforms detect AD pathology investigate isoforms’ profile relationships. Plasma was truncated as previously described CSF. measures p-tau-217 p-tau-181 were correlated. No correlation found between total-tau levels pS202 measures. highly amyloid plaque discovery cohort (n = 36, AUROC 0.99 0.98 respectively). validation 92), still status (AUROC 0.92), less 0.75).

Language: Английский

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