Nature Immunology, Journal Year: 2017, Volume and Issue: 18(5), P. 583 - 593
Published: March 6, 2017
Language: Английский
Nature Methods, Journal Year: 2019, Volume and Issue: 17(1), P. 41 - 44
Published: Nov. 25, 2019
Language: Английский
Citations
1817
Cell, Journal Year: 2020, Volume and Issue: 182(1), P. 59 - 72.e15
Published: May 28, 2020
Language: Английский
Citations
1418
Nature Protocols, Journal Year: 2018, Volume and Issue: 14(1), P. 68 - 85
Published: Nov. 16, 2018
Language: Английский
Citations
1345
Nature Reviews Genetics, Journal Year: 2018, Volume and Issue: 20(2), P. 71 - 88
Published: Nov. 8, 2018
Language: Английский
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1182
Nature Reviews Genetics, Journal Year: 2020, Volume and Issue: 21(10), P. 630 - 644
Published: July 24, 2020
Language: Английский
Citations
939
Molecular Systems Biology, Journal Year: 2018, Volume and Issue: 14(8)
Published: Aug. 1, 2018
Review13 August 2018Open Access Data-independent acquisition-based SWATH-MS for quantitative proteomics: a tutorial Christina Ludwig Corresponding Author [email protected] orcid.org/0000-0002-6131-7322 Bavarian Center Biomolecular Mass Spectrometry (BayBioMS), Technical University of Munich (TUM), Freising, Germany Search more papers by this author Ludovic Gillet Department Biology, Institute Molecular Systems ETH Zurich, Switzerland George Rosenberger orcid.org/0000-0002-1655-6789 Columbia University, New York, NY, USA Sabine Amon Ben C Collins orcid.org/0000-0003-0827-3495 Ruedi Aebersold Faculty Science, Information *,1,‡, Gillet2,‡, Rosenberger2,3, Amon2, Collins2 and Aebersold2,4 1Bavarian 2Department 3Department 4Faculty ‡These authors contributed equally to work *Corresponding author. Tel: +49 8161 71 6130; E-mail: Mol Syst Biol (2018)14:e8126https://doi.org/10.15252/msb.20178126 PDFDownload PDF article text main figures. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Many research questions in fields such as personalized medicine, drug screens or systems biology depend on obtaining consistent quantitatively accurate proteomics data from many samples. is specific variant data-independent acquisition (DIA) methods emerging technology that combines deep proteome coverage capabilities with consistency accuracy. In measurement, all ionized peptides given sample fall within specified mass range are fragmented systematic unbiased fashion using rather large precursor isolation windows. To analyse data, strategy based peptide-centric scoring has been established, which typically requires prior knowledge about the chromatographic spectrometric behaviour interest form spectral libraries peptide query parameters. This provides guidelines how set up plan experiment, perform measurement scoring. Furthermore, concepts improve acquisition, potential trade-offs parameter settings alternative analysis strategies discussed. Introduction Over last decades, liquid chromatography coupled tandem spectrometry (LC-MS/MS) become choice high-throughput characterization proteins proteomes (Aebersold Mann, 2016). Recent developments field have moved beyond enumerating proteins, post-translational modifications detected one few samples towards delivering high quality quantification large-scale projects comprise 100s Especially areas biomarker research, screens, genetic association studies biology, numbers individuals, conditions perturbations need be investigated draw meaningful biological conclusions. For purpose, matrices generated must reproducible, complete possible. order address these needs, several different proteomic developed over years. An strategy, focus tutorial, sequential window theoretical spectra (SWATH-MS), was described et al (2012). detailed introduction into history, basic principles well general advantages limitations (also summarized Table 1), we refer Appendix. Briefly, non-labelled protein digested trypsin resulting analysed spectrometer operating so-called mode. mode, compounds fashion. Figure 1A–C shows DIA scheme initial implementation (Gillet al, 2012), 32 consecutive, slightly overlapping windows, width 25 m/z each. Depending complexity, will lead co-fragmentation co-eluting concurrently selected ion ultimately highly multiplexed complex fragment (Fig 1D). deal proposed novel scoring, relies querying coordinates parameters (PQPs). PQPs derived previously libraries. 1. Advantages comparison data-dependent (DDA) targeted (SRM, PRM) Principle sequentially windowed SWATH-MS(A) measurements performed fast scanning hybrid spectrometers, employing quadrupole first analyser TOF Orbitrap second analyser. single (MS1) spectrum recorded, followed series (MS2) wide windows (for example m/z). Through repeated cycling consecutive defined range, comprehensive includes continuous information detectable ions. Hence, extracted chromatograms can MS2 MS1 level. employed, queried (B) The (2012) Q-TOF uses scans increments m/z, starting at 400 ending 1,200 m/z. One full scan recorded beginning. By applying an time 100 ms per scan, total cycle ~3.3 s achieved. (C) detects precursors eluting point. example, 925 950 three species (green, red blue). (D) corresponding 925–950 represents mixed fragments species. Download figure PowerPoint addition method, wealth other schemes established. overview, see Appendix Fig S1, recent reviews (Chapman 2014; Bilbao 2015). While term “SWATH” became registered trademark SCIEX context instrumentation, company Biognosys trademarked name “Hyper Reaction Monitoring” (HRM) analogous mode instrumentation (Box 1). Throughout only use “SWATH-MS”, independently underlying instrument type, generic “DIA” when referring breadth strategies. Box 1: Definitions frequently used terms Term Definition Data-Independent Acquisition (DIA)—here, umbrella continuously acquire fashion, without requiring detection ions survey (as DDA) nor values SRM PRM). Specific family include SWATH-MS, Shotgun-CID, MSE, XDIA, MSX, AIF FT-ARM others Sequential Windowed All Theoretical Fragment Ion Spectra (SWATH-MS)—represents DIA, full-scan instruments (preferably Q-Orbitrap). SWATH SCIEX. successive pre-defined ranges isolated subjected 2012). Peptide-centric library HRM Hyper Monitoring (HRM)—Synonym SWATH-MS. implemented analysers A starts list target tests whether those certain confidence. It applied individual (XICs). analysis, readily available. Typically, acquired PRM but also methods, way. equivalent literature “targeted extraction” Spectrum-centric type aims finding sequence(s) user-specified explain(s) best. discovery-driven (DDA), Peptide (PQPs) Compendium required identification stored table format (i) optimal (proteotypic) protein, (ii) elution times setup, (iii) most intense (typically four six) under fragmentation conditions, (iv) charge state(s) (v) relative intensity fragments. previous experiments, identifications (Targeted) assay Synonym mainly SRM, where tier levels (1–3) analytical validation (Carr 2014) Spectral confidently assigned sequence, acquisition. case, same sequence either best average consensus gets reported. retention file normalized through re-alignment reference peptides. Alternatively, deconvoluted pseudo-MS/MS directly (DIA-Umpire; Tsou 2015) Transition Pair value. Mainly Targeted Umbrella aim quantifying PTM-peptides interest. two classical approaches, PRM, itself However, “untargeted”, measurements, classifies approaches When method my study? major advantage it supports analyses covering 1,000s ideally suited entail number require reproducible fraction expressed peptidome each sample. Typical exactly properties (Liu Muntel 2015; Kulkarni 2016; Ortea 2016), Okada Williams clinical drug/perturbation (preprint: Litichevskiy 2018; Tan 2017; Keam 2018) exploratory (Collins 2013; Lambert Parker 2015a; Schubert 2015b). particularly LC gradient lengths below 60 min (Vowinckel 2018). Proteome coverages ~50% MS-detectable achieved mammalian single-shot (Bruderer Kelstrup current drawback compared (SRM still three- 10-fold less sensitive 2012; Liu Schmidlin remains better option involve low-abundant maximal further DDA-based upfront effort experimental silico PQP generation optimization (Table very analyses, currently successfully label-free DDA workflow, intensities counts, possibly combined fractionation techniques (Lawrence Geyer Frejno 2017). important improvement application sets development tools allow transfer between thereby completeness matrix (Prakash 2006; Mueller 2007; Cox 2014). even tools, missing higher than especially low concentration 2015, direct comparisons MS1/DDA workflow versus challenging, demonstrated injected operated once outperforms associated reproducibility popular isobaric labelling, using, tags (TMT) (Thompson 2003) absolute quantitation (iTRAQ) (Unwin 2005). Frequently, labelling extensive procedure. fractions then individually (Chick Roumeliotis With state-of-the-art TMT reagents 10 (McAlister 2012) 11, simultaneously, leading minimal preparation biases early implementations suffered ratio compression due interferences reporter co-fragmenting peptides, addressed some extent optimized (Ting 2011; Savitski McAlister Ahrne O'Brien Sonnett Labelling comparative medium-sized projects; however, if analysed, issues incompleteness batch effects apparent again across date, compare tagging missing. overall intent guide readers performing their own measurements. We give Setting planning experiment If study progresses eventually samples, particular attention should paid feasibility producing comparable good quality, both longitudinally instrument, multiple conceivably platforms. Encouraging progress made demonstrating comparability laboratories standard Cumulative contamination during series, caused contaminants lipids, polymers detergents, concern extra care taken produce mostly devoid contaminants. Contaminants issue because observed faster severe performance loss (such charging sensitivity issues) instrument. possible explanation observation substantially flux (which compose > 90% time), turn means impurities might enter contaminate loss. Therefore, monitoring maintaining acceptable level prerequisite. efforts undertaken develop software enable tracking (Rudnick 2010; Wang Bereman Chiva statistical considerations design, group size, technical variability achievable selectivity, account (Krzywinski Altman, 2014b). suggest proper randomization blocking 2014a) sufficient replicates (Blainey ensure power 2013) downstream process. Prior concept behind empirically selectively extract peptide-specific convoluted referred “peptide parameters” worth noting “prior” indicates available prerequisite before undertaken, while actual does not availability PQPs. What kind do contain? detail, monitor dominant value(s) peptide(s) thus state distribution, six expected pattern i.e. intensities, v) signals, reference. commonly obtained [or potentially chromatogram (Sharma 2014)] shown 2. Computational pipelines integrating steps extraction simplify standardize process Skyline (Egertson 2015), PeakView (SCIEX), Spectronaut Pulsar (further Spectronaut) (Biognosys) Trans-Proteomic Pipeline (TPP) (Deutsch 2010). Further prepare convert [specL (Panse Fraggle/Tramler/Franklin (Teleman 2017)] extend [SwathXtend (Wu 2016)] Particularly constrained MSPLIT-DIA (Wang come from?PQPs contain peptide, exemplified here AAHTEDINACTLTTSPR. Various input types purpose generation. results files. From file(s), relevant filtering identified indicated criteria. about: charge, (retention iRT). details create protocol paper (Schubert 2015a). following paragraphs, discuss sources generate extracted. acquiring runs spectrum-centric data. algorithms schemes, recently DIA-Umpire (Tsou Group-DIA (Li make specifically improved latest instruments. Generally, cumulative related achieve similar unfractionated Representative Here, choose endogenous interest, synthetic analogues recombinant full-length 2). majority date side-by-side These step beneficial perspective post-acquisition queries (Rosenberger Zi Selevsek lower would fully covered “single shot” strategy. focusing non-fractionated sometimes favoured straightforward implement; primarily reasons, considered. Chemically synthesized long source knowledge, particular, respect assays (Kuster 2005; Picotti 2010), synthesize measure organisms proteome-wide scale reported (Picotti Kusebauch Zolg approach advantages: represented library, irrespective they observed, high-quality generated, concentrations represent ground truth, error rate close zero. cases, selection driven empirical observation. since there so far no proteotypic computationally predicted (Mallick 2007). Since computational prediction turned out reliable (Searle preferred prediction. More purposes extended (> 330,000 human peptides) synthesizing much larger including PTMs common variants (www.proteometools.org; useful extension vitro transcription/translation systems. way, suitable determined (Stergachis Matsumoto MS resources yet exploited seems likely going forward. Hybrid consisting increase may attractive proved case organism-scale Mycobacterium tuberculosis Comprehensive sample-specific natural ideas discussed above attempt characterize
Language: Английский
Citations
891
Molecular & Cellular Proteomics, Journal Year: 2018, Volume and Issue: 17(12), P. 2534 - 2545
Published: Nov. 5, 2018
In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra acquired about 100 microseconds time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension separation. Among several formats, trapped spectrometry (TIMS) is attractive because its small size, low voltage requirements and high efficiency utilization. We have recently demonstrated scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies sequencing speed without any loss sensitivity (Meier et al., PMID: 26538118). Here we introduce timsTOF Pro instrument, optimally implements online PASEF. It features an orthogonal path into device, limiting amount debris entering instrument making it very robust daily operation. investigate different precursor selection schemes for shotgun proteomics to allocate excess events per second. More than 600,000 standard 120 min LC runs achievable, can be used near exhaustive complex mixtures or accumulating signal weak precursors. single HeLa digest, MaxQuant identified more 6,000 proteins matching library quantitative reproducibility (R > 0.97). Online PASEF achieves remarkable 2,500 30 only 10 ng digest. also show that highly reproducible collisional cross sections on large scale 0.99). valuable addition technological toolbox number unique operating modes beginning explored. Jointly, form cellular machinery—the proteome—that orchestrates essentially all biological processes health disease. Studying system-wide holds great promise advance our understanding biology disease mechanisms (1Altelaar A.F.M. Munoz J. Heck A.J.R. Next-generation proteomics: towards integrative view proteome dynamics.Nat. Rev. Genet. 2012; 14: 35-48Crossref PubMed Scopus (521) Google Scholar, 2Larance M. Lamond A.I. Multidimensional cell biology.Nat. Mol. Cell Biol. 2015; 16: 269-280Crossref (291) 3Aebersold R. Mann Mass-spectrometric exploration structure function.Nature. 2016; 537: 347-355Crossref (1113) Scholar). However, compared genomics transcriptomics technologies, still lags terms coverage, throughput, sensitivity. Virtually complete measurements mammalian proteomes become possible (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. An Optimized Shotgun Strategy Rapid Generation Comprehensive Human Proteomes.Cell Syst. 2017; 4: 587-599.e4Abstract Full Text PDF (259) Scholar), but mostly involved laborious sample preparation workflows, days measurement time substantial amounts starting material. Furthermore, current high-performance instrumentation often requires expert knowledge extensive maintenance, impedes widespread adaptation nonspecialized laboratories. extracted from interest enzymatically cleaved make them amenable spectrometric (MS) analysis. The resulting peptide typically via nano-flow (LC) 1The abbreviations are:LCliquid chromatographyCCScollisional sectionIMSion spectrometryPASEFparallel accumulation–serial fragmentationTIMStrapped spectrometry. spectrometry., ionized electrospray analyzed. "data-dependent" "topN" acquisition schemes, spectrometer detects suitable ions full scans selects N consecutive MS/MS scans. High resolution accuracy analyzers detect hundreds thousands distinct molecular LC-MS experiments, minority quantified (5Michalski A. Cox 100,000 detectable species elute majority inaccessible data-dependent LC-MS/MS.J. Proteome Res. 2011; 10: 1785-1793Crossref (480) These co-eluting abundances ranging over many orders magnitude present formidable analytical challenge, has constantly pushed development faster sensitive last decades 6Aebersold Mass spectrometry-based proteomics.Nature. 2003; 422: 198-207Crossref (5596) 7Eliuk Makarov Evolution Orbitrap Spectrometry Instrumentation.Annu. Anal. Chem. 8: 61-80Crossref (252) section Time-of-flight instruments desirable properties analysis consequently been employed long (8Domon B. Aebersold protein analysis.Science. 2006; 312: 212-217Crossref (1619) 9Han X. Aslanian Yates J.R. proteomics.Curr. Opin. 2008; 12: 483-490Crossref (504) Instrumental performance steadily improved years, groups described at 35,000 within μs impact II (10Beck Michalski Raether O. Lubeck Kaspar Goedecke N. Baessmann Hornburg D. Meier F. Paron I. Kulak N.A. Impact II, high-resolution quadrupole (QTOF) deep proteomics.Mol. Cell. Proteomics. 2014-2029Abstract (121) predecessor subject this paper. rate TOF coupling fast separation techniques, such (IMS) (11Kanu A.B. Dwivedi P. Tam Matz L. Hill H.H. Ion mobility-mass spectrometry.J. Spectrom. 43: 1-22Crossref (885) 12Cumeras Figueras E. Davis C.E. Baumbach J.I. Gràcia Review Mobility Spectrometry. Part 2: hyphenated methods effects experimental parameters.Analyst. 140: 1391-1410Crossref 13May J.C. McLean J.A. spectrometry: time-dispersive instrumentation.Anal. 87: 1422-1436Crossref (279) IMS separates gas phase based their size shape, precisely (CCS, Ω), 10s 100s milliseconds (14Eiceman G.A. Karpas Z. H.H.J. 3rd Ed. CRC Press, 2013Crossref As emerge they efficiently sampled ms sub-ms frame analyzers. Nested between MS, technology provides additional (15Valentine S.J. Counterman A.E. Hoaglund C.S. Reilly J.P. Clemmer D.E. Gas-phase separations protease digests.J. Am. Soc. 1998; 9: 1213-1216Crossref (97) 16Srebalus Barnes C.A. Hilderbrand Valentine Resolving isomeric mixtures: A combined HPLC/ion mobility-TOFMS 4000-component combinatorial library.Anal. 2002; 74: 26-36Crossref (90) 17Ewing M.A. Glover M.S. Hybrid tool.J. Chromatogr. : 27-29Google Scholar) increase selectivity (18Lanucara Holman S.W. Gray C.J. Eyers power structural characterization study conformational 2014; 6: 281-294Crossref (659) samples (19Valentine Plasencia M.D. Liu Krishnan Naylor Udseth H.R. Smith R.D. Toward plasma profiling 5: 2977-2984Crossref (128) 20Baker E.S. Livesay E.A. Orton D.J. Moore R.J. Danielson W.F. Prior D.C. Ibrahim Y.M. LaMarche B.L. Mayampurath A.M. Schepmoes A.A. Hopkins D.F. Tang K. Belov M.E. LC-IMS-MS platform providing increased dynamic high-throughput proteomic studies.J. 2010; 997-1006Crossref (106) 21Geromanos Hughes Ciavarini Vissers J.P.C. Langridge Using purity scores enhancing precision samples.Anal. Bioanal. 404: 1127-1139Crossref (42) 22Helm Hahne H. Ruprecht Pachl Grzyb Richardson Wildgoose Maier S.K. Marx Wilhelm Becher Lemeer Bantscheff Kuster tandem enhances 13: 3709-3715Abstract (73) 23Distler U. Kuharev Navarro Levin Y. Schild Tenzer Drift time-specific collision energies enable deep-coverage data-independent proteomics.Nat. Methods. 11: 167-170Crossref (306) implementations IMS, drift tubes, challenging device sizes voltages may limit proportion continuous incoming beam utilized (12Cumeras 24Cumeras 1: instrumentation.Analyst. 1376-1390Crossref Trapped (25Fernandez-Lima F.A. Kaplan D.A. Park Note: Integration spectrometry.Rev. Sci. Instrum. 82: 126106Crossref (163) 26Fernandez-Lima Suetering using spectrometer.Int. Mobil. 93-98Crossref (214) reverses concept traditional tube bringing rest positions tunnel balanced electrical field against constant stream (27Ridgeway Jordens short review.Int. 2018; 425: 22-35Crossref (159) Once enough separated, lowering potential releases time-resolved TIMS downstream analyzer. design reduces analyzer dimensions centimeters length—allowing two implemented series 100% duty cycle operation (28Silveira Ridgeway Laukien F.H. Parallel spectrometry.Int. 413: 168-175Crossref (44) furthermore offers flexibility users tune resolving (Ω/ΔFWHMΩ) up 200 higher simply (29Silveira peptides.Anal. 86: 5624-5627Crossref (132) 30Ridgeway Silveira J.E. Microheterogeneity states ubiquitin revealed spectrometry.Analyst. 6964-6972Crossref introduced "Parallel Accumulation SErial Fragmentation" (PASEF) (31Meier Beck Grassl (PASEF): multiplying synchronized device.J. 5378-5387Crossref (179) synchronizes scheme one increases severalfold accumulated parallel, overcomes diminishing returns increasingly acquisition, otherwise necessarily implied less spectrum. Our first iteration was laboratory prototype, required manual programming limited electronics involved. Here, describe construction fully integrates concept, Bruker Pro. cervical cancer cells (HeLa S3, ATCC, Manassas, VA) were grown Dulbecco's modified Eagle's medium 10% fetal bovine serum, 20 mm glutamine 1% penicillin-streptomycin (all Life Technologies Ltd., Paisley, UK). Escherichia coli (strain: XL1 blue) cultured 37 °C LB until logarithmic (optical density = 0.5, λ 600 nm). Cells collected centrifugation. Following washing step cold phosphate buffered saline, pelleted flash frozen nitrogen stored −80 °C. One-device lysis, reduction, alkylation performed sodium deoxycholate (SDC) buffer chloroacetamide (PreOmics GmbH, Martinsried, Germany) according previously published protocol (32Kulak Pichler G. Nagaraj Minimal, encapsulated proteomic-sample processing applied copy-number estimation eukaryotic cells.Nat. 319-324Crossref (1002) Briefly, suspension twice boiled 95 subsequently sonicated 15 maximum energy (Bioruptor, Diagenode, Seraing, Belgium). Proteins hydrolyzed overnight LysC trypsin (1:100 enzyme:protein (wt/wt) both). To stop digestion, reaction mixture acidified five volumes isopropanol trifluoroacetic acid (TFA). Peptides de-salted purified steps, styrenedivinylbenzene-reversed sulfonate (SDB-RPS), second C18 sorbent. dried eluates re-constituted water 2% acetonitrile (ACN) 0.1% TFA direct pH reversed-phase fractionation. For experiments Evosep One (see below), pellets re-suspended lysed water/trifluoroethanol. Disulfide bonds reduced dithiothreitol alkylated iodoacetamide ammonium bicarbonate buffer. tryptic fractionation EASY-nLC 1000 (Thermo Fisher Scientific, Bremen, coupled "spider fractionator" (PreOmics) detailed ref (33Kulak Geyer P.E. Loss-less nano-fractionator sensitivity, coverage (mcp.O116.065136)Abstract (105) Purified cm × 250 μm column flow 2 μl/min 10. binary gradient started 3% B (PreOmics), followed linear 30% 45 min, 60% 17 finally 95% 5 min. Each automatically concatenated 48 fractions 90 s intervals. vacuum-centrifuge reconstituted ACN 1200 Scientific) ultra-high pressure system hybrid flight (timsTOF Pro, Daltonics, nano-electrospray source (CaptiveSpray, Daltonics). Liquid 60 400 nL/min (50 75 i.d.) pulled emitter tip, packed 1.9 C18-coated porous silica beads (Dr. Maisch, Ammerbuch-Entringen, Germany). Mobile phases formic (v/v) 80/20/0.1% ACN/water/formic (v/v/vol), respectively. 120-min 7.5 27.5% 37.5% further 55% re-equilibration. separations, 40% 57.5% before initial 10–30% (7.5 min) (2.5 some (Evosep, Odense, Denmark), new HPLC employing embedded capable turnaround analyses (34Bache Bekker-Jensen Hoerning Falkenby Treit P.V. Doll Müller Vorm novel embeds analytes pre-formed gradients rapid, ultra-robust (mcp.TIR118.000853)Abstract (140) Samples eluted Evotips storage loop offset lower percentage organic Separation customized 5.6 (200 samples/day method) 2.0 4 x 150 i.d. 3 (PepSep, Denmark). successor redesign hardware firmware. Apart incorporating TIMS, goals included achievement similar better (>35,000) robustness through path. here, operated mode. Desolvated entered vacuum region glass capillary deflected 90°, focused electrodynamic funnel, front consisting stacked printed circuit boards (PCBs) inner diameter 8 total length mm. PCB electrodes multipole direction transfer, RF 350 Vpp confined radially. electrically parts (dual TIMS), where trap primarily stores spectrometer, while part performs soon finished, transferred filled again. If equal times both regions, enables cycles close 100%. transfer regions takes therefore does not affect overall utilization typical ramp around 25 ms. superimposed (from entrance exit) increasing longitudinal gradient, simultaneously experience drag repulsion field. ambient air Depending charge states, come closer (high mobility) exit (low mobility). achieved ramping −207 V −77 V. TIMS-MS composed individual 110 each. reported systematically varied 25, 50, 100, 150, keeping fixed All topN cycle, except four cycle. spectrometry, signal-to-noise ratios conveniently summation low-abundance precursors intensity below 'target value' repeatedly scheduled PASEF-MS/MS summed count reached target value (e.g. 5000 arbitrary units (a.u.) 20,000 a.u.). set a.u. methods, 24,000 MS recorded m/z 1700. Suitable selected real survey sophisticated scheduling algorithm Results). polygon filter plane select most likely representing rather singly charged background ions. isolation width Th < 700 700, ramped stepwise function mobility: 52 eV 0–19% time; 47 19–38%; 42 38–57%; 57–76%; 32 remainder. calibrated linearly obtain coefficients (1/K0) three Agilent ESI-L Tuning Mix (m/z 622, 922, 1222) (35Stow S.M. Causon T.J. Zheng Kurulugama R.T. Mairinger T. May Rennie E.E. Baker Hann Fjeldsted interlaboratory evaluation measurements.Anal. 89: 9048-9055Crossref (282) Collisional calculated Mason Schamp equation (36Mason McDaniel E.W. Transport Properties Ions Gases. John Wiley & Sons, Inc., New York, NY1988Crossref CCS=3ze161K02πμkbT z ion, e elemental charge, kb Boltzman's constant, μ mass, T temperature (305 K). calculations, assumed pure N2 gas. raw files processed (37Cox identification rates, individualized p.p.b.-range accuracies proteome-wide quantification.Nat. Biotechnol. 26: 1367-1372Crossref (9214) version 1.6.1.13, extended incorporate adapted handle data format. publicly available will detail separately (Cox co-workers, preparation). assembles four-dimensional isotope clusters defined m/z, retention time, extracts spectrum assigned respective values, case fragmented multiple collapsed signal-to-noise. feature detection MaxQuant, every point (TOF scan) considered ("TIMS width" 3), "TIMS half parameter (equivalent 440 μs). resolution" 32,000 peaks 1.5 discarded. matched silico derived fragment values reference (Uniprot, 2016/05, HeLa: 91,618 entries including isoforms, coli: 4313 isoforms) 245 contaminants built-in Andromeda search engine (38Cox Neuhauser Scheltema R.A. Andromeda: integrated environment.J. 1794-1805Crossref (3469) missing cleavages allowed, minimum sequence 7 amino acids, 4600 Da. Carbamidomethylation cysteine residues modification, methionine oxidation acetylation termini variable modifications. tolerances 70 ppm 35 After re-calibration, individually adjusted each pattern local measurement. reversed control false discovery (FDR) matches group identifications. Decoy database hits, contaminants,
Language: Английский
Citations
831
Nature Methods, Journal Year: 2019, Volume and Issue: 16(6), P. 509 - 518
Published: May 27, 2019
Language: Английский
Citations
786
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
Citations
767
Nature Methods, Journal Year: 2020, Volume and Issue: 17(12), P. 1229 - 1236
Published: Nov. 30, 2020
Language: Английский
Citations
665