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High Sensitivity Quantitative Proteomics Using Automated Multidimensional Nano-flow Chromatography and Accumulated Ion Monitoring on Quadrupole-Orbitrap-Linear Ion Trap Mass Spectrometer

2017; Elsevier BV; Volume: 16; Issue: 11 Linguagem: Inglês

10.1074/mcp.ra117.000023

1535-9484

Paolo Cifani, Alex Kentsis,

Metabolomics and Mass Spectrometry Studies

Quantitative proteomics using high-resolution and accuracy mass spectrometry promises to transform our understanding of biological systems and disease. Recent development of parallel reaction monitoring (PRM) using hybrid instruments substantially improved the specificity of targeted mass spectrometry. Combined with high-efficiency ion trapping, this approach also provided significant improvements in sensitivity. Here, we investigated the effects of ion isolation and accumulation on the sensitivity and quantitative accuracy of targeted proteomics using the recently developed hybrid quadrupole-Orbitrap-linear ion trap mass spectrometer. We leveraged ultrahigh efficiency nano-electrospray ionization under optimized conditions to achieve yoctomolar sensitivity with more than seven orders of linear quantitative accuracy. To enable sensitive and specific targeted mass spectrometry, we implemented an automated, two-dimensional (2D) ion exchange-reversed phase nanoscale chromatography system. We found that automated 2D chromatography improved the sensitivity and accuracy of both PRM and an intact precursor scanning mass spectrometry method, termed accumulated ion monitoring (AIM), by more than 100-fold. Combined with automated 2D nano-scale chromatography, AIM achieved subattomolar limits of detection of endogenous proteins in complex biological proteomes. This allowed quantitation of absolute abundance of the human transcription factor MEF2C at ∼100 molecules/cell, and determination of its phosphorylation stoichiometry from as little as 1 μg of extracts isolated from 10,000 human cells. The combination of automated multidimensional nano-scale chromatography and targeted mass spectrometry should enable ultrasensitive high-accuracy quantitative proteomics of complex biological systems and diseases. Quantitative proteomics using high-resolution and accuracy mass spectrometry promises to transform our understanding of biological systems and disease. Recent development of parallel reaction monitoring (PRM) using hybrid instruments substantially improved the specificity of targeted mass spectrometry. Combined with high-efficiency ion trapping, this approach also provided significant improvements in sensitivity. Here, we investigated the effects of ion isolation and accumulation on the sensitivity and quantitative accuracy of targeted proteomics using the recently developed hybrid quadrupole-Orbitrap-linear ion trap mass spectrometer. We leveraged ultrahigh efficiency nano-electrospray ionization under optimized conditions to achieve yoctomolar sensitivity with more than seven orders of linear quantitative accuracy. To enable sensitive and specific targeted mass spectrometry, we implemented an automated, two-dimensional (2D) ion exchange-reversed phase nanoscale chromatography system. We found that automated 2D chromatography improved the sensitivity and accuracy of both PRM and an intact precursor scanning mass spectrometry method, termed accumulated ion monitoring (AIM), by more than 100-fold. Combined with automated 2D nano-scale chromatography, AIM achieved subattomolar limits of detection of endogenous proteins in complex biological proteomes. This allowed quantitation of absolute abundance of the human transcription factor MEF2C at ∼100 molecules/cell, and determination of its phosphorylation stoichiometry from as little as 1 μg of extracts isolated from 10,000 human cells. The combination of automated multidimensional nano-scale chromatography and targeted mass spectrometry should enable ultrasensitive high-accuracy quantitative proteomics of complex biological systems and diseases. The emerging ability to measure cellular and physiological states accurately and quantitatively promises to transform our understanding of biology and disease (1.Steen H. Pandey A. Proteomics goes quantitative: measuring protein abundance.Trends Biotechnol. 2002; 20: 361-364Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). For example, time-resolved and multiparametric quantitative analyses of cellular signaling are enabling the elucidation of fundamental paradigms of cell development and homeostasis (2.Olsen J.V. Vermeulen M. Santamaria A. Kumar C. Miller M.L. Jensen L.J. Gnad F. Cox J. Jensen T.S. Nigg E.A. Brunak S. Mann M. Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis.Sci. Signal. 2010; 3: ra3Crossref PubMed Scopus (1150) Google Scholar, 3.Dephoure N. Gygi S.P. Hyperplexing: a method for higher-order multiplexed quantitative proteomics provides a map of the dynamic response to rapamycin in yeast.Sci. 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For example, selected reaction monitoring (SRM) 1The abbreviations used are: SRM, selected reaction monitoring; ABC, ammonium bicarbonate; ACN, acetonitrile; AIM, accumulated ion monitoring; AGC, automatic gain control; CID, collision induced dissociation; FA, formic acid; HCD, higher-energy collisional dissociation; HPLC, high pressure liquid chromatography; IRM, ion routing multipole; LOD, limit of detection; PRM, parallel reaction monitoring; PTM, post-translational modification; Q1, (first) quadrupolar mass filter; SCX, strong cation exchange; SIC, specific ion current; SIM, selected ion monitoring; TSQ, triple stage quadrupole (also QQQ); XIC, extracted ion chromatogram. 1The abbreviations used are: SRM, selected reaction monitoring; ABC, ammonium bicarbonate; ACN, acetonitrile; AIM, accumulated ion monitoring; AGC, automatic gain control; CID, collision induced dissociation; FA, formic acid; HCD, higher-energy collisional dissociation; HPLC, high pressure liquid chromatography; IRM, ion routing multipole; LOD, limit of detection; PRM, parallel reaction monitoring; PTM, post-translational modification; Q1, (first) quadrupolar mass filter; SCX, strong cation exchange; SIC, specific ion current; SIM, selected ion monitoring; TSQ, triple stage quadrupole (also QQQ); XIC, extracted ion chromatogram. uses quadrupole mass analyzers to filter specific precursor and fragment ions produced by collision-induced dissociation (CID) (6.Lange V. 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Here, we investigated the effects of ion selection and accumulation on the sensitivity and quantitative accuracy of targeted proteomics using the recently developed hybrid Fusion quadrupole-Orbitrap-linear ion trap mass spectrometer. We leveraged high efficiency low μm-scale electrospray ionization to analyze synthetic peptides in neat solvent and thus determine the absolute limits of sensitivity, achieving yoctomolar absolute limits of quantitation with more than seven orders of linear quantitative accuracy. We observed that ion coisolation led to significant reduction in sensitivity in analyses of complex human cellular proteomes. To partially overcome this limitation, we implemented an automated, scalable two-dimensional ion exchange-reversed phase nano-scale chromatography system, suitable for robust, high-resolution, high-capacity separations necessary for quantitative targeted mass spectrometry. By quantifying the endogenous transcription factor MEF2C and its phosphorylation stoichiometry in 1 μg of extracts from as few as 10,000 human cells, we achieved significant improvements in the sensitivity and quantitative accuracy of both PRM and accumulation monitoring (AIM) methods, permitting the detection and quantitation of ∼100 molecules/cell. Mass spectrometry grade (Optima LC/MS) water, acetonitrile (ACN), and methanol were from Fisher Scientific (Fair Lawn, NJ). Formic acid of 99%+ purity (FA) was obtained from Thermo Scientific. Ammonium formate and all other reagents at MS-grade purities were obtained from Sigma-Aldrich (St. Louis, MO). MRFA peptide was obtained from Sigma-Aldrich. Based on consensus protein sequences in the UniProt database (as of January 30th, 2015), N-terminally isotopically labeled (13C615N2 lysine and 13C615N4 arginine) MEF2C and MARK4 peptides (Table I) were synthesized using solid phase chemistry by New England Peptides (Gardner, MA), and purified by reversed phase chromatography. Extinction coefficients were calculated as described by Kuipers and Gruppen (24.Kuipers B.J.H. Gruppen H. Prediction of molar extinction coefficients of proteins and peptides using UV absorption of the constituent amino acids at 214 nm to enable quantitative reverse phase high-performance liquid chromatography-mass spectrometry analysis.J. Agric. Food Chem. 2007; 55: 5445-5451Crossref PubMed Scopus (278) Google Scholar), and listed in supplemental Table S1. Peptides were quantified using UV absorbance spectroscopy at 214 nm using 3 mm QS quartz cuvettes (Hellma, Plainview, NY) and the SpectraMax M5 analytical spectrophotometer (Molecular Devices, Sunnyvale, CA). A standard peptide mixture was created by mixing individual peptides at final concentration 10 pmol/μl in a clean glass vial. For direct infusion experiments, the peptide mixture was serially diluted 1:10 in 30% ACN, 0.1% FA containing 1 μg/ml MRFA peptide (Sigma-Aldrich).Table IPeptide propertiesIDSequencem/zM1SEPVSPPR439.7339M2SEPV(pS)PPR479.7171M3NSPGLLVSPGNLNK709.3981M4N(pS)PGLLVSPGNLNK749.3813M5YTEYNEPHESR717.8116M6LFEVIETEK558.3073M9VLIPPGSK409.7649 Open table in a new tab Human OCI-AML2 cells were obtained from the German Collection of Microorganisms and Cell Cultures (Brunswick, Germany). Cells were cultured as described (25.Kentsis A. Reed C. Rice K.L. Sanda T. Rodig S.J. Tholouli E. Christie A. Valk P.J.M. Delwel R. Ngo V. Kutok J.L. Dahlberg S.E. Moreau L.A. Byers R.J. Christensen J.G. Vande Woude G. Licht J.D. Kung A.L. Staudt L.M. Look A.T. Autocrine activation of the MET receptor tyrosine kinase in acute myeloid leukemia.Nat. Med. 2012; 18: 1118-1122Crossref PubMed Scopus (140) Google Scholar), collected while in exponential growth phase, washed twice in ice-cold PBS, snap frozen and stored at −80°C. Protein extraction and proteolysis was performed as previously described (26.Dhabaria A. Cifani P. Reed C. Steen H. Kentsis A. A high-efficiency cellular extraction system for biological proteomics.J. Proteome Res. 2015; 14: 3403-3408Crossref PubMed Scopus (17) Google Scholar). Briefly, frozen pellets of 5 million cells were thawed on ice, resuspended in 100 μl of 6 m guanidinium hydrochloride, 100 mm ammonium bicarbonate at pH 7.6 (ABC) containing PhoStop phosphatase inhibitors (Roche Diagnostics GbmH, Mannheim, Germany), and lysed using the E210 adaptive focused sonicator (Covaris, Woburn, CA). The protein content in cell lysate was determined using the BCA assay, according to the manufacturer's instructions (Pierce, Rockford, IL). On reduction and alkylation, proteomes were digested using 1:100 w/w (protease/proteome) LysC endopeptidase (Wako Chemical, Richmond, VA) and 1:50 w/w MS sequencing-grade modified trypsin (Promega, Madison WI). Digestion was stopped by acidifying the reactions to pH 3 using formic acid (Thermo Scientific), and peptides were subsequently desalted using solid phase extraction using C18 Macro Spin columns (Nest Group, Southborough, MA). Peptides were eluted in 60% acetonitrile, 1% formate in water, lyophilized using vacuum centrifugation, and stored at −20°C. Tryptic peptides were reconstituted in 0.1% formate, 3% acetonitrile to a final concentration of 0.5 μg/μl. For experiments in cellular proteomes, the synthetic peptide mixture was initially diluted to 5 pmol/μl in a tryptic digest of whole OCI-AML2 cell proteome at 0.5 μg/μl, and subsequently serially diluted 1:10 in the same solution. Detailed description of the instrumental and operational parameters, as well as step-by-step protocol for system construction and operation are provided in supplementary Materials. Both direct infusion sample delivery and liquid chromatography experiments were performed using the Ekspert NanoLC 425 chromatograph (Eksigent, Redwood City, CA), equipped with an autosampler module, two 10-port and one 6-port rotary valves, and one isocratic and two binary pumps. Polyimide-coated fused silica capillaries (365 μm outer diameter, variable inner diameters) were obtained from Polymicro Technologies (Phoenix, AZ). Unions and fittings were obtained from Valco (Houston, TX). For direct infusion, samples were initially aspirated into a 10 μl PEEK sample loop. On valve switching, the content of the loop was ejected using a gradient pump (30% ACN, 0.1% FA at 100 nl/min) into an empty silica capillary (20 μm inner diameter) in-line with the DPV-566 PicoView nano-electrospray ion source (New Objective, Woburn, MA). Chromatographic columns were fabricated by pressure filling the stationary phase into silica capillaries fritted with K-silicate, as previously described (27.Dhabaria A. Cifani P. Kentsis A. Fabrication of capillary columns with integrated frits for mass spectrometry.Protocol Exchange. 2015; Google Scholar). Strong-cation exchange columns were fabricated by packing Polysulfoethyl A 5 μm silica particles (PolyLC, Columbia, MD) into 150 μm × 10 cm fritted capillary. Reversed phase columns were fabricated by packing Reprosil 1.9 μm silica C18 particles (Dr. Meisch, Ammerbauch-Entrigen, Germany) into 75 μm x 40 cm fritted capillaries. Trap columns were fabricated by packing Poros R2 10 μm C18 particles (Life Technologies, Norwalk, CT) into 150 μm x 4 cm fritted capillaries. Vented trap-elute architecture was used for chromatography (28.Ficarro S.B. Zhang Y. Carrasco-Alfonso M.J. Garg B. Adelmant G. Webber J.T. Luckey C.J. Marto J.A. Online nanoflow multidimensional fractionation for high efficiency phosphopeptide analysis.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). One of the 10-port valves was set to include in the flow path either the SCX column or an empty capillary of equal inner volume. Samples were initially loaded into a 10 μl PEEK sample loop, and subsequently delivered at 1 μl/min by the isocratic pump using 0.1% FA in water into either the empty capillary (for one-dimensional chromatography) or the SCX column (for two-dimensional chromatography). A step gradient of 50, 100, 150, 300, and 1000 mm ammonium formate (AF) in water, pH 3 was delivered in 3.5 μl (0.5 column volume) increments from auto-sampler vials to elute peptides into the trap column, where peptides were desalted. Finally, peptides were resolved by reversed phase chromatography hyphenated to the nano-electrospray ion source. On valve switch to connect the trap column in line with the analytical reversed phase column and ion emitter, the pressure was equilibrated at a flow of 250 nl/min for 5 min in 5% buffer B (ACN, 0.1% FA) in buffer A (water, 0.1% FA). Subsequently, a 60-min linear gradient of 5–38% of buffer B was used to resolve peptides, followed by a 5 min 38–80% gradient before column wash at 80% buffer B for 30 min. Electrospray emitters with terminal opening diameter of 2–3 μm were fabricated from silica capillaries as previously described (29.Cifani P. Dhabaria A. Kentsis A. Fabrication of Nanoelectrospray Emitters for LC-MS.Protocol Exchange. 2015; Crossref Google Scholar). The emitter was connected to the outlet of the reversed phase column using a metal union that also served as the electrospray current electrode. Electrospray ionization was achieved using variable voltage, programmed from 1750 to 1450 V with 50 V steps over 60 min of the gradient elution. During column loading, the electrospray emitter was washed with 50% aqueous methanol using the DPV-565 PicoView ion source (New Objective). For all measurements, we used the Orbitrap Fusion mass spectrometer (Thermo Scientific, San Jose, CA). During AIM measurements, the mass spectrometer was programmed to iteratively perform precursor ion scans with 8 Th isolation windows targeting both endogenous light and synthetic heavy peptides using Q1 isolation and S-lens voltage of 60 V (22.Senko M.W. Remes P.M. Canterbury J.D. Mathur R. Song Q. Eliuk S.M. Mullen C. Earley L. Hardman M. Blethrow J.D. Bui H. Specht A. Lange O. Denisov E. Makarov A. Horning S. Zabrouskov V. Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates.Anal. Chem. 2013; 85: 11710-11714Crossref PubMed Scopus (177) Google Scholar). Unless otherwise specified, ions were accumulated for a maximum of 200 ms with automatic gain control of 105 ions, and scanned at 240,000 resolution. For PRM scans, precursor ions were isolated using 2 Th isolation windows, and fragmented by HCD with normalized collision energy set at 32% before analysis of the fragment ions in the Orbitrap at 30,000 resolution. Optimal fragmentation conditions were preliminarily established for each target peptide by manual inspection of MS2 spectra collected within the same analysis on fragmentation with HCD energy 30, 32, 36, and 38% (30.Zhang Y. Ficarro S.B. Li S. Marto J.A. Optimized Orbitrap HCD for quantitative analysis of phosphopeptides.J. Am. Soc. Mass Spectrom. 2009; 20: 1425-1434Crossref PubMed Scopus (90) Google Scholar). Ion signal intensities and total ion currents were analyzed using Xcalibur Qual Browser 3.0.63 (Thermo Fisher Scientific). Automated chromatographic peak area integration was performed using Skyline 3.5.0 (31.MacLean B. Tomazela D.M. Shulman N. Chambers M. Finney G.L. Frewen B. Kern R. Tabb D.L. Liebler D.C. MacCoss M.J. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments.Bioinformatics. 2010; 26: 966-968Crossref PubMed Scopus (2983) Google Scholar), with mass tolerance set at 0.0075 Da corresponding to 10 ppm for m/z of 750 Da, and integration boundaries were manually verified for all peaks. Numerical and statistical analyses were performed using Origin Pro 9.0 (OriginLab Corporation, Northampton, MA). All raw and processed mass spectrometry data as well as Skyline chromatogram documents are available via ProteomeXchange with the identifier PXD006236 (32.Vizcaíno J.A. Côté R.G. Csordas A. Dianes J.A. Fabregat A. Foster J.M. Griss J. Alpi E. Birim M. Contell J. O'Kelly G. Schoenegger A. Ovelleiro D. Pérez-Riverol Y. Reisinger F. Ríos D. Wang R. Hermjakob H. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013.Nucleic Acids Res. 2013; 41: D1063-D1069Crossref PubMed Scopus (1596) Google Scholar). Quantitative data from serially diluted synthetic peptides were linearly fitted to obtain a signal-response function for each peptide. These functions were subsequently used to calculate amounts of endogenous peptides. Phosphorylation stoichiometry was defined as the fraction of each peptide being chemically modified, as described (33.Cifani P. Shakiba M. Chhangawala S. Kentsis A. ProteoModlR for functional proteomic analysis.BMC Bioinformatics. 2017; 18: 153Crossref PubMed Scopus (3) Google Scholar). The protein content of OCI-AML2 cells was established by BCA assay quantification of total protein extracted from cells, which were manually counted using a Neubauer hemocytometer. The study evaluated the sensitivity and specificity of targeted detection using peptides delivered either by direct infusion or by chromatography with variable peak capacities. Technical variability was established under direct infusion regime by collecting seven measurements for each data point. For chromatographically resolved peptides, triplicate measurements of the intensity of synthetic peptides were performed at three experimental conditions. Endogenous peptide measurements were performed in parallel with isotopolog targeting, for a total of 18 replicate measurements. To compare AIM and PRM, assays were performed within the same experiment to control for possible variation in chromatography and ionization performance. Sensitivity of mass spectrometric detection is in principle determined by two factors: the minimum number of ions necessary to produce a measureable electronic signal, and the baseline instrumental noise. In the case of high-resolution mass analyzers coupled to external ion storage devices, the number of ions delivered to the mass analyzer can thus be increased by prolonged ion accumulation before detection. The incorporation of high-capacity ion routing multipoles (IRM) on the recently developed Q Exactive (Q-OT) and Fusion (Q-OT-IT) mass spectrometers permits ion accumulation and storage for as long as 5 s. An acquisition strategy was thus designed, combining quadrupole precursor ion filtering, with precursor qua