A Compact Quadrupole-Orbitrap Mass Spectrometer with FAIMS Interface Improves Proteome Coverage in Short LC Gradients

Molecular & Cellular Proteomics, Год журнала: 2020, Номер 19(4), С. 716 - 729

Опубликована: Фев. 13, 2020

State-of-the-art proteomics-grade mass spectrometers can measure peptide precursors and their fragments with ppm accuracy at sequencing speeds of tens peptides per second attomolar sensitivity. Here we describe a compact robust quadrupole-orbitrap spectrometer equipped front-end High Field Asymmetric Waveform Ion Mobility Spectrometry (FAIMS) Interface. The performance the Orbitrap Exploris 480 is evaluated in data-dependent acquisition (DDA) data-independent (DIA) modes combination FAIMS. We demonstrate that different compensation voltages (CVs) for FAIMS are optimal DDA DIA, respectively. Combining DIA using single CVs, instrument surpasses 2500 identified minute. This enables quantification >5000 proteins short online LC gradients delivered by Evosep One system allowing 60 samples day. raw sensitivity analyzing 5 ng HeLa digest from which >1000 were reproducibly min DIA-FAIMS. To versatility instrument, recorded an organ-wide map proteome expression across 12 rat tissues quantified tandem tags label-free to depth >10,000 proteins.

Язык: Английский

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Recent advances in mass spectrometry based clinical proteomics: applications to cancer research

Clinical Proteomics, Год журнала: 2020, Номер 17(1)

Опубликована: Май 24, 2020

Abstract Cancer biomarkers have transformed current practices in the oncology clinic. Continued discovery and validation are crucial for improving early diagnosis, risk stratification, monitoring patient response to treatment. Profiling of tumour genome transcriptome now established tools novel biomarkers, but alterations proteome expression more likely reflect changes pathophysiology. In past, clinical diagnostics strongly relied on antibody-based detection strategies, these methods carry certain limitations. Mass spectrometry (MS) is a powerful method that enables increasingly comprehensive insights into advance personalized medicine. this review, recent improvements MS-based proteomics highlighted with focus oncology. We will provide detailed overview clinically relevant samples types, as well as, consideration sample preparation methods, protein quantitation MS configurations, data analysis pipelines currently available researchers. Critical each step necessary address pressing questions cancer diagnosis prognosis. While majority studies clinically-relevant there growing demand rigorous biomarker validation. These high-throughput targeted assays multi-centre standardized protocols. Additionally, sensitivity opening door new classes tumour-specific proteoforms including post-translational modifications variants originating from genomic aberrations. Overlaying proteomic complement transcriptomic datasets forges field proteogenomics, which shows great potential improve our understanding biology. Overall, advancements not only solidify proteomics’ integral position research, also accelerate shift towards becoming regular component routine practice.

Язык: Английский

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Ultra-fast proteomics with Scanning SWATH

Nature Biotechnology, Год журнала: 2021, Номер 39(7), С. 846 - 854

Опубликована: Март 25, 2021

Язык: Английский

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Automated sample preparation with SP 3 for low‐input clinical proteomics

Molecular Systems Biology, Год журнала: 2020, Номер 16(1)

Опубликована: Янв. 1, 2020

Method16 January 2020Open Access Source DataTransparent process Automated sample preparation with SP3 for low-input clinical proteomics Torsten Müller German Cancer Research Center (DKFZ), Heidelberg, Germany Medical Faculty, Heidelberg University, Search more papers by this author Mathias Kalxdorf EMBL, Rémi Longuespée Department of Clinical Pharmacology and Pharmacoepidemiology, Daniel N Kazdal orcid.org/0000-0001-8187-3281 Institute Pathology, Albrecht Stenzinger Jeroen Krijgsveld Corresponding Author [email protected] orcid.org/0000-0001-7549-9326 Information Müller1,2, Kalxdorf1,3, Longuespée4, Kazdal5, Stenzinger5 *,1,2 1German 2Medical 3EMBL, 4Department 5Institute *Corresponding author. Tel: +49 6221 421720; E-mail: Molecular Systems Biology (2020)16:e9111https://doi.org/10.15252/msb.20199111 PDFDownload PDF article text main figures. Peer ReviewDownload a summary the editorial decision including letters, reviewer comments responses to feedback. ToolsAdd favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract High-throughput streamlined workflows are essential in standardized processing samples from variety sources, fresh-frozen tissue, FFPE or blood. To reach goal, we have implemented single-pot solid-phase-enhanced (SP3) on liquid handling robot automated (autoSP3) tissue lysates 96-well format. AutoSP3 performs unbiased protein purification digestion, delivers peptides that can be directly analyzed LCMS, thereby significantly reducing hands-on time, variability quantification, improving longitudinal reproducibility. We demonstrate distinguishing ability autoSP3 samples, reproducibly quantifying 500–1,000 proteins 100 1,000 cells. Furthermore, applied approach cohort pulmonary adenocarcinoma (ADC) recapitulated their separation into known histological growth patterns. Finally, integrated AFA ultrasonication end-to-end LCMS analysis 96 intact samples. Collectively, constitutes generic, scalable, cost-effective workflow minimal manual intervention, enabling reproducible broad range non-clinical applications. Synopsis The study presents an pipeline based method. seamless integration lysis format features low variability, high sensitivity reproducibility studies. An automated, allows is interference. reduced quantification increased Minimal losses facilitate applications workflow. Introduction Mass spectrometry (MS)-based proteomic technologies matured allow robust, reliable, comprehensive proteome profiling across thousands cells tissues. This result parallel developments mass spectrometric instrumentation continues gain speed sensitivity, chromatographic technology separate interfaced MS, data pipelines reliable identification quantification. In addition, various been developed comparative analyses many e.g., using isobaric labels allowing multiplexing, label-free approaches, short chromatography (LC) gradients. has propelled studies multiple areas basic mechanistic biology, deep quantitative profiles understand spatial temporal aspects organization dynamics wide conditions (Schubert et al, 2017). speed, robustness, general accessibility present-day increasing appeal applications, reasons: (i) Underlying mechanisms diseases still unclear, where proteome-level information will increase insight (patho)physiological processes; (ii) primary targets almost all current drugs, function help how drugs impact cellular (iii) diseases, there persistent lack powerful biomarkers diagnostic, prognostic, predictive purposes. Still, successful implementation environment not materialized yet, primarily because additional requirements need met top those research alluded above (e.g., coverage, sensitivity). mostly pertains analyze (possibly hundreds) un-interrupted fashion order achieve sufficient statistical power patient cohorts, simplify workflow, removing personnel specific technical skills proteomics, achieving acceptable turn-around time receiving generation complete profile analysis, (iv) cost-effectiveness Most these bottlenecks resolved simultaneously automation, avoiding handling, eliminating risk error while at same standardization irrespective number Although nowadays sufficiently excellent performance hundreds (Bache 2018), preceding highly cumbersome, involving steps extract, purify, digest before subsequent LCMS. ideal scenario, procedure should accepts any type, facilitating universal applicability. Despite existing methods (Rappsilber 2003; Huynh 2009; Wiśniewski Kulak 2014; Wu Guo 2015; HaileMariam 2018; Ludwig very few satisfy demands universally accommodate different imposed types. For instance, blood lysed under gentle than formalin-fixed paraffin-embedded (FFPE) requires harsh detergent-based efficiently extract (Wiśniewski 2013). Many currently available demonstrated great utility application proteomics; however, they also come some drawbacks. stage tips 2003), its derivative iST (Kulak 2014), do tolerate detergents, restricting generic use. Other approaches involve extensive procedures such as filtration centrifugation precipitation (Wu electrophoresis (Huynh 2009) difficult standardize scale up, lead undesirable especially solve several issues, recently introduced retrieval (Hughes 2014, 2019). method utilizes paramagnetic beads presence organic solvent (> 50% ACN EtOH) promote binding beads, washing eliminate contaminants, detergents SDS Triton X-100. After release off aqueous buffer, proteolytic digestion produces clean injected Another feature efficient recovery, maintaining coverage. combined characteristics scalability, tolerance ease operation, qualify methodology enabled “difficult” types diverse 2016; Erich 2018) historical bones (Cleland, 2018). well (Sielaff 2017), single human oocytes (Virant-Klun 2016), micro-dissected (Dilillo 2017; Pellegrini A property fully exploited yet nature which automation robotic platform. genomics, magnetic already years ago (Fisher 2011) now commonly used library through kits vendors. far less common restricted segments enrichment sub-proteomes AssayMap purify phosphorylated peptides; Murillo peptide clean-up (Kuras 2019), cases avoided (iST, system plasma cell lysates; Geyer 2016). study, Bravo (Agilent Technologies, USA), build scalable starting lysate delivering digests verified stability over period weeks handle down ng containing less. realistic successfully associating pathological tumor patterns strong prognostic implications (Warth 2012) distinct signatures. extraction downstream SP3, generating without steps. provides attractive solution routine, studies, easing introduction translational consumption. Results Establishing fast simple types, foundation bead render it adaptable automation. Here, Agilent system, widely laboratories. Doing so required optimization steps, positioning consumables, reagent, waste volumes, accessories, magnet, shaker, heating block ensure components each consecutive task, tips-on, aspiration dispensing, tips-off, volumes reagents, buffers, waste. overview deck setup shown Fig 1A. As result, space motion pipetting head were automate ready MS simultaneously. Figure 1. schematic shows protocol input enzymatic digestion. core protocol. acidification recovery ends injection-ready versions. provided three options reduction alkylation post-digestion described Appendix Protocols (A D). Protocol A: one-step TCEP/CAA mixture 5 min 95°C, followed autoSP3. B: two-step DTT CAA consecutively 30 incubation 60 23°C, respectively, C: omitting user flexibly pre-treat manually prepared D: new plate. Download figure PowerPoint evaluate autoSP3, HeLa execute (Protocol C; 1A C, C). was preceded off-deck TCEP 95°C PCR thermocycler. Alternatively, performed integral part (Fig 1C), begins aliquoting suspension sample, spotting μl droplet wall then gently moved agitation orbital shaking accessory. binding, 2014) rather EtOH 2019) properties releasing droplets pipette tips. Continuous switching between slow maintains distribution preventing sedimentation formation protein-bead aggregates. At pre-empts mixing associated losing tend stick inner conditions. rinsing Hughes al (2019), two times 80% one 100% ACN. effective removal wash solvents within task achieved dividing latter aspirates air plug avoid hanging droplets. classes optimized aspirating dispensing velocities defined optimal movements. Thus, clearance residual achieved, example, taking account propensity drain side well. Proteins trapped resuspended trypsin (or other enzyme choice) incubated 16 h 1A). Following acidified recovered on-deck plate after supplying 1B, D, manually. further possibility re-using tasks explored throughput reduce cost. Therefore, adjusted heights every never touch surface possible liquid-adding aspirate volume only once successively dispense row-by-row entire Subsequently, during wash-disposal inevitably dip solution, again transfer sticking tip re-used specifically disposal thus excluding cross-contamination. Having place, first confirmed equally established respect obtained ion intensities identified processed (Appendix S1). Next, cross-contamination negligible, 10 μg alternating empty controls half subset seven peptide-containing eleven randomly selected subjected direct acquisition, S2A B. Compared sample-containing injections, most injections had intensity < 0.03%, 1% S2B). could attributed autolytic (which added ones), (non-peptidic) contaminants +1 charge state, sharply contrasting rich chromatograms protein-containing S2C). summary, care producing estimated cost reduction, alkylation, 92.39 euros (< 1 euro per sample), (600 50 μg/μl), (576 0.05 reagent plates, boxes, buffers/solvents. Precision strict leading precise workflows. evaluated assessing precision [defined EMEA observed laboratory (EMEA, 2009)], both span day (intra-day precision) longitudinally month (inter-day (Grant Andrew, 2014). end, lysed, DNA RNA digested, alkylated above. Intra-day assessed morning afternoon days (days 1, 13, 27), i.e., roughly month, resulting total six plates 576 individual 2A). Inter-day inferred correlating 3 days. Specifically, respective immediately completing selecting five (i.e., samples). exact batch, distinguish potential variance fluctuation performance. allowed us plate, day, days, variation Intensities consistent average Pearson correlation 0.9 2B). 2. Evaluation intra-day inter-day representation experimental design. batch (Plate A) B) (day 27) month. From (red dots). (ten day) measured judge influence Box-whisker plots log2-transformed raw files. central line represents median. Boxes extend 25th 75th percentile whiskers smallest highest value. color coding highlights origin. Cumulative frequency curve [%] coefficient (CV) quantified minimum valid values day. ten files individually. median CV shown. heatmap sixteen displayed filtered 75% completeness (Table 1). Please note narrow scaling (1–0.94). online figure. Data 2 [msb199111-sup-0002-SDataFig2.zip] assess level, generated least values, 2,672, 2,537, 2,663 LFQ value 27, respectively 2C). protein, calculated, demonstrating 91% 30%, 12.9, 10.9 12.2% reflecting replicates sequentially determined datasets compared SP3. 14,140 spectrum matches 3,191 S3A) CVs 7.1 2.1% respectively. maximize included assessment, match-between-runs functionality MaxQuant, proportion missing 33.62 58.37%. list (n = 3,750) 18.1 20.5%. evaluation, calculated out 3,688) 45 2,964) corresponds measurements corresponding 2,964 (79.04%) S3B) proteins, comparison preparation, daily (median 14.3%; Table 1) similar collection 13.3%; 1), indicating differences minimal, acquisition extended periods. (14.7 17.4%, respectively) showing marginal but noticeable improvement 16.3% 18.6% illustrated S4A B, consistently improved versus per-protein basis. Summary variations (CVs) intra- No. Minimum Median (%) Average Within 11.9 14.6 13.3 16.5 13 9.8 12.2 11.3 14 27 10.8 13.5 12.3 15.5 Across 15.7 18.6 w/o 14.3 17.3 17.2 20.1 Overall 14.7 17.4 20.6 Manual 16.3 20 2, table summarizes either requirement (~80% proteins) 3. lower-limit capabilities Schematic design 1:2 dilution series ng. four replicates, lowest concentration. concentrated material injected, whereas sub-microgram used. sum iBAQ standard deviation bars 4 replicates. numbers Series decreasing 10,000 cells, eight [msb199111-sup-0003-SDataFig3.zip] coefficients showed 0.97) among automatically robust 2D). no observable extremely precision. Only slightly lower 0.94) robustness itself, likely subtle protocols volumes). sectioned experiments abundance bins (A: 1–500; 501–1,251; 1,252–2,001; 2,002–2,964) investigated S5A). B), > 97.5% 10% bin (D), 39.1% which, comprises group ~1,000 rec

Язык: Английский

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Decoding Post-Translational Modification Crosstalk With Proteomics

Molecular & Cellular Proteomics, Год журнала: 2021, Номер 20, С. 100129 - 100129

Опубликована: Янв. 1, 2021

Post-translational modification (PTM) of proteins allows cells to regulate protein functions, transduce signals and respond perturbations. PTMs expand functionality diversity, which leads increased proteome complexity. PTM crosstalk describes the combinatorial action multiple on same or different for higher order regulation. Here we review how recent advances in proteomic technologies, mass spectrometry instrumentation, bioinformatics spurred proteome-wide identification through measurements sites. We provide an overview basic modes crosstalk, methods elucidate approaches that can inform about functional consequences crosstalk.

Язык: Английский

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R2‐P2 rapid‐robotic phosphoproteomics enables multidimensional cell signaling studies

Molecular Systems Biology, Год журнала: 2019, Номер 15(12)

Опубликована: Дек. 1, 2019

Recent developments in proteomics have enabled signaling studies where > 10,000 phosphosites can be routinely identified and quantified. Yet, current analyses are limited throughput, reproducibility, robustness, hampering experiments that involve multiple perturbations, such as those needed to map kinase-substrate relationships, capture pathway crosstalks, network inference analysis. To address these challenges, we introduce rapid-robotic phosphoproteomics (R2-P2), an end-to-end automated method uses magnetic particles process protein extracts deliver mass spectrometry-ready phosphopeptides. R2-P2 is rapid, robust, versatile, high-throughput. showcase the method, applied it, combination with data-independent acquisition spectrometry, study dynamics mitogen-activated kinase (MAPK) yeast. Our results reveal broad specific events along mating, high-osmolarity glycerol, invasive growth branches of MAPK pathway, robust phosphorylation downstream regulatory proteins transcription factors. facilitates large-scale involving hundreds perturbations opening door systems-level aiming complexity.

Язык: Английский

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Glycoproteomics

Nature Reviews Methods Primers, Год журнала: 2022, Номер 2(1)

Опубликована: Июнь 23, 2022

Язык: Английский

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Decoupling astrocytes in adult mice impairs synaptic plasticity and spatial learning

Cell Reports, Год журнала: 2022, Номер 38(10), С. 110484 - 110484

Опубликована: Март 1, 2022

The mechanisms by which astrocytes modulate neural homeostasis, synaptic plasticity, and memory are still poorly explored. Astrocytes form large intercellular networks gap junction coupling, mainly composed of two channel proteins, connexin 30 (Cx30) 43 (Cx43). To circumvent developmental perturbations to test whether astrocytic coupling is required for hippocampal circuit function behavior, we generate study inducible, astrocyte-specific Cx30 Cx43 double knockouts. Surprisingly, disrupting in adult mice results broad activation microglia, without obvious signs pathology. We show that CA1 neuron excitability, excitatory transmission, long-term potentiation significantly affected. Moreover, behavioral inspection reveals deficits sensorimotor performance a complete lack spatial learning memory. Together, our findings establish connexins an intact astroglial network the brain vital cognition.

Язык: Английский

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Mass spectrometry-based proteomics as an emerging tool in clinical laboratories

Clinical Proteomics, Год журнала: 2023, Номер 20(1)

Опубликована: Авг. 26, 2023

Abstract Mass spectrometry (MS)-based proteomics have been increasingly implemented in various disciplines of laboratory medicine to identify and quantify biomolecules a variety biological specimens. MS-based is continuously expanding widely applied biomarker discovery for early detection, prognosis markers treatment response prediction monitoring. Furthermore, making these advanced tests more accessible affordable will the greatest healthcare benefit. This review article highlights new paradigms clinical has created microbiology laboratories, cancer research diagnosis metabolic disorders. The technique preferred over conventional methods disease detection therapy monitoring its combined advantages multiplexing capacity, remarkable analytical specificity sensitivity low turnaround time. Despite achievements development adoption number practices, are expected undergo transition from bench bedside near future. provides insights trials recent progresses (mainly covering literature NCBI database) application laboratories.

Язык: Английский

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A synthetic methylotrophic Escherichia coli as a chassis for bioproduction from methanol

Nature Catalysis, Год журнала: 2024, Номер 7(5), С. 560 - 573

Опубликована: Апрель 23, 2024

Methanol synthesized from captured greenhouse gases is an emerging renewable feedstock with great potential for bioproduction. Recent research has raised the prospect of methanol bioconversion to value-added products using synthetic methylotrophic

Язык: Английский

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