MassIVEapplication/xmlftp://massive-ftp.ucsd.edu/v01/MSV000082296/primary200OK0000Laurence Florenshttps://massive.ucsd.edu/ProteoSAFe/dataset.jsp?task=c7b07c867e7543c69c574ad5309f3864laf@stowers.orgMassIVE403MOD:00719 - "A protein modification that oxygenates an L-methionine residue to one of the diastereomeric L-methionine sulfoxide residues."ProteomicsThermo Fusion LumosQ ExactiveSaccharomyces Cerevisiae (ncbitaxon:4932)Stowers Institute for Medical ResearchThe use of mass spectrometry as a tool to detect proteins of biological interest has become a cornerstone of proteomics. The popularity of mass spectrometry-based methods has increased along with instrument improvements in detection and speed. The Orbitrap Fusion Lumos mass spectrometer has recently been shown to have better fragmentation and detection than its predecessors. Here, we determined the sensitivity of the Lumos using the NIST monoclonal antibody reference material at various concentrations to detect its peptides in a background of S. cerevisiae whole cell lysate, which was kept at a constant concentration. The data collected by data-dependent acquisition showed that the spiked protein could be detected at 10 pg by an average of 4 peptides in 250 ng of whole cell lysate when the instrument was operated by detecting the peptide masses in the Orbitrap and the fragment masses in the ion trap (FTIT mode). In contrast, when the peptides and fragments were both detected in the Orbitrap on either the Lumos or Q-Exactive Plus (FTFT mode), the lowest concentration of NIST monoclonal antibody detected was 50 pg. The Lumos can detect a single protein at a level 2500 times lower than the whole cell background and the combination of detecting ions in the Orbitrap and ion trap can improve the identification of low abundance proteins. Furthermore, the total number of proteins identified from decreasing starting amounts of whole cell extracts was determined. The Lumos, when operated in FTIT mode, was able to identify twice as many proteins compared to the Q-Exactive+ at 5 ng of whole cell lysate. Similar numbers of proteins were identified on both platforms at higher concentrations of starting material. Therefore, the Lumos mass spectrometer is especially useful for detecting proteins of low abundance in complex backgrounds or samples that have limited starting material.Probing the Sensitivity of the Orbitrap Lumos Mass Spectrometer Using a Standard Reference Protein in a Complex Background.Levy Michaella J MJ, Washburn Michael P MP, Florens Laurence Ld230, Meth, Procedures, ion, Extract, Peptidomics, instrument configuration, number, Brewer's, Gene, baker's yeast, dTAFII250, protein, Spectrum Analyses, protein-containing complex, Chalk, EfW1, supernumerary, Saccharomyces italicus, hardware, peptide, dmTAF[[II]]230, Polypeptides, Techniques, protein polypeptide chains, Antibody, peptido, polypeptide chain, dmTAF1, Method, sensitive, IT, Taf230, yeast, Tina, Mass, Gene Products, Studies, Extracts, Low, Analysis, protein aggregate, Technique, sensitivity, Mass Spectroscopy, lager beer yeast, TAF250, Mass Spectrum Analysis, average, increased, Taf200, me75, Glass, dTAF[[II]]250, peptides, TFIID TAF250, Analyses, cel, cell, iones, HardwareType, Saccharomyes cerevisiae, Taf1p, Baker, proteins, procedures, Saccharomyces uvarum var. melibiosus, ions, D17Mit170, T1, S. cerevisiae, Study, allergic reaction, dTAF250, S cerevisiae, lysate, Candida robusta, Methodological Studies, Ice, Saccharomyces capensis, T1., Baker's, Sensitivity, peptidos, Mycoderma cerevisiae, 1883, brewer's yeast, TAF, dTAF[[II]]230, TAF[[II]]250, cou, Saccharomyces cerevisiae 'var. diastaticus', protein complex, Monoclonal Antibody, Proteins, TAF200, l(3)84Ab, BG:DS00004.13, Procedure, TAFII-250, TAF250/230, Tl3, Monoclonal, Tl2, Spectrum Analysis, Peptide, Saccaromyces cerevisiae, Cell, dTAF230, Spectroscopy, Baker's Yeasts, Sccharomyces cerevisiae, polypeptide, TAFII250, MS, Lr, native protein, natural protein, Ionen, Monoclonal Antibodies, Saccharomyces diastaticus, p230, Protein, TAF[[II]]250/230, TFIID, techniques, background, Mass Spectrum Analyses, Crystal, Saccharomyces oviformis, Taf[[II]]250, Mass Spectrum, Yeast, TAF[[II]]230, instrument, increased number, Spectrometry, Specificity, TAF[II]250, Methodological, CG17603, Methodological Study, TAF[[II]], introduction, Protein Gene Products, Speed, Gene Proteins, present in greater numbers in organism, Saccharomyces cerevisiae (Desm.) Meyen ex E.C. Hansen, DmelCG17603, Cell Extract, Specificity and Sensitivity, Taf250, Ion, concentration, SR3-5, cardinality, Bra, Peptid, Polypeptide, Baker's Yeast, Brewer's Yeast, accessory, Baker Yeast, TAF230, methodology, TAF1allergic reaction, Extracts, Specificity, Sensitivity, Cell., Cell Extract, Specificity and Sensitivity, Extract, sensitivity, sensitive, CellProtein Gene Products, allergic reaction, Gene Proteins, protein polypeptide chains, native protein, Specificity and Sensitivity, natural protein, polypeptide chain, sensitive, protein complex, Protein, Gene Products, Proteins, Specificity, Sensitivity, introduction., Gene, proteins, background, protein, protein aggregate, protein-containing complex, sensitivitysodium salt, Water, l(4)17, FLN-B, l(4)13, SCT, ammonium formate, Gli, 13C-labeled, cadmium salt, MeCN, odd(Oz), YPD, Cid[Mel], Ci[D], Abcb2, H(2)O, Heparinic acid, BOUND WATER, Basodexan, magnesium formate, Apaf-1, oxidane, zinc salt, heavy chain disease, CENP-A, Ham1, WATER, CENP-C, HOH, Bca, 5730420M11Rik, heparina, dmTAF[[II]]230, lenses, Polypeptides, protein polypeptide chains, Personal, heparine, hydrogen chloride, Ly6, cobalt(II) formate dihydrate, CH3-C#N, ionic compounds, ten-m, 1, 2, Msal-1, AGM4, 2-amino-3-mercaptopropanoic acid, Software Engineering, Analysis, 3-Propanetriol, bca, Heparinic Acid, C, SET, Oelsuess, BcDNA:RE21270, F, C79325, l(3)rP126, hac-1, TFIID TAF250, Analyses, cel, N, yeast extract peptone dextrose, Salt, ocular lens, Saccharomyes cerevisiae, Msal, composition, proteins, T6G21.3, Saccharomyces uvarum var. melibiosus, BcDNA:AT25108, sel, Salz, D17Mit170, salts, AI047805, Social, DmelCG4299, salt, set, Wasserstoffchlorid, decreased, Reviparin, CI, column, anatomical tube, ACN, sample, Acn, RCB0007, Mycoderma cerevisiae, Half Cystine, Social Power, nickel salt, Power, Ten-mc, CiD, Glyceritol, Ce, dTAF[[II]]230, Degradations, Stars, Ci, Ly-6A.2, Unfractionated Heparin, Zinc Cysteinate, cobalt (+2) salt, bHLHc9, Trypsin/K, CG5723, anon-53Fa, Liquid Chromatography, TAF200, l(2)SH0173, Tl3, Tl2, Spectrum Analysis, collisionally activated dissociation, Cid, CID, ciD, Software Tools, RING4, Baker's Yeasts, Computer Applications, H2O, common salt, sodium (4:1:1) salt, Stickstoff, soluble, Dm1, magnesium salt, Tap-1, Saccharomyces diastaticus, Computer Applications Software, Chlorwasserstoff, ci-D, (2R)-2-amino-3-sulfanylpropanoic acid, CG6829, Hydrogen Oxide, dark/hac-1/dapaf-1, DmelCG7659, Gro, Software Applications, HLA-DR-associated protein II, glycerine, CENP-A/Cid, CENP-A/CID, DI-2, FLN3, ionic compound, LY57, lens, CENPA, Source Software, content, I-2Dm, Protein Digestions, formate, Spectrometry, pooled, cromium (+3), study protocol, beta Trypsin, MEX67, I-2PP1, Saccharomyces cerevisiae (Desm.) Meyen ex E.C. Hansen, Applications, nitrogen, TAF-IBETA, Taf250, Mex67, duct, ammonium (4:1) salt, 2017, TAF-Ibeta, methyl cyanide, Sodium Heparin, chlorane, lead (+2) salt, STARS, l(3)01658, T-cell-activating protein, dipeptide chemoreceptor protein, Ci-155, Brewer's Yeast, TAF230, Computer Software Applications, Psychological Power, 5301, nickel formate dihydrate, lead formate, False, chlorure d'hydrogene, Protein USO1 homolog, aluminum salt, number, Pierce, L-Zystein, Lens <eudicots>, Carbamide, Stem cell antigen 1, protein-containing complex, thomson, 17alpha)-isomer, Saccharomyces italicus, CG7826, lens crystallina, agua, mass-to-charge ratio, CenpA, Odz, Kingdom of Norway, glycyl alcohol, Certoparin, TAP1, Ly57, IT, Mex67h, yeast, Gene Products, CG7835, CG42273, Half-Cystine, Carmol, Siah, lager beer yeast, Application, Open Source Softwares, odz, ABP-280-like protein, anatomical systems, dTAF[[II]]250, Acinus, 2310061K05Rik, Hydrogen chloride, thallium (+1) salt, cell, L Cysteine, Software Application, E-920, Glycerin, Monosodium Salt, beta-Trypsin, rubidium salt, Open Source Software, 2pp2a, Hcys, methanoic acid, MCP-IV, l(3)rJ307, CG10574, S. cerevisiae, Computer Software Application, acinusL, Injectable, l(3)05301, dTAF250, Melting, S cerevisiae, glycerolum, 2PP2A, Tools, CYSTEINE, acinusS, alpha-Heparin, Kochsalz, dSET, dSet, FREE CYSTEINE, CG2125, peptidos, Ly-6A.2|Ly-6E.1, (2R)-2-amino-3-mercaptopropanoic acid, Squalene transfer protein, 4-(2-hydroxyethyl)-, Ly6a, C22orf6, dark/dapaf-1/hac-1, lens capsule, cou, Spf, SPF, 1-(14)C-labeled, dApaf-1, Proteins, Ly-6E.1, Sca-1, chloridohydrogen, potassium formate, BG:DS00004.13, copper (+2) salt, compositionality, acetonitrile, buffer, Digestions, Saccaromyces cerevisiae, Cell, mKIAA0670, Engine, Ten79E, Tool, dTAF230, Sccharomyces cerevisiae, polypeptide, APAF1, Lr, MS, native protein, Temperatures, Natriumchlorid, Proteolyses, carbonyldiamide, Hac-1, ci[D], I-2PP2A, cupric formate, Dm I-2, TAF[[II]]250/230, 5alpha, Liquaemin, Sodium, NaCl, aluminum formate, Unfractionated, dJ142L7.2, Mass Spectrum Analyses, Saccharomyces oviformis, AU020952, Taf[[II]]250, lithium formate, CT16449, DmelCG2125, CenH3, ci155, Alpha-tocopherol-associated protein, underdeveloped, ensemble, PSF1, increased number, Striated muscle activator of Rho-dependent signaling, Dark/Dapaf-1/HAC1, B130022O04Rik, (R)-2-amino-3-mercaptopropanoic acid, azote, ethanenitrile, AOI, Supernatant protein factor, 2-Amino-3-mercaptopropionic acid, primary structure of sequence macromolecule, ME-IV, plan specification, Parnaparin, Computer Programs, Gene Proteins, bps, SCA-1, Applications Softwares, Dietary Sodium, MTP1, structure, Power (Psychology), Bra, E920, ten(m), Baker's Yeast, [OH2], Vesicle-docking protein, CenpA/CID, Heparin, hydrochloric acid, CG11452, Hac-1/Dark, IPP2A2, E 920, NCMe, DmelCG6829, classic hairy cell leukaemia, zinc formate, Ly-6A|E, HEPES Monosodium Salt, lead salt, protein, Hydrogenchlorid, composed of, extracted material, Uniandra, peptide, DmelCG5723, halite, ammonium tetraformate, peptido, BANF, hTAP, Sandoparin, Min, Cystein, Apaf1, protein aggregate, present in fewer numbers in organism, ARK, Injectables, Computer Program, DmelCG42273, Mass Spectrum Analysis, Psychological, CG13329, HEPES Monosodium, 3-hydroxy-5-estrane-17-carbonitrile, increased, me75, Sca1, peptides, l(3)rL201, TAF-I, cisteina, E927b, 2610036I19Rik, Open, 2610510L13Rik, hypoplasia, min, arc, crystalline lens, Computer Programs and Programming, ten-m/odz, mAPC, Baker, copper, ten[m], ABC17, ark, SCAN, Protein Degradation, T1, cloruro sodico, reaction, IGAAD, fSAP152, Cy 222, lithium salt, LRS1, 1-Piperazineethanesulfonic acid, DmelCG10574, Saccharomyces capensis, Cenp-A, CG7659, dihydridooxygen, Baker's, stem cell antigen 1, Lyw-57, dApaf1, brewer's yeast, dihydrogen oxide, 115kDa, ammonium (2:1) salt, Contradens, phapii, Tube, rock salt, D-Apaf-1, aqua, Dmel_CG7826, StF-IT-1, Th, Search, Psychological Powers, classic hairy cell leukemia, TAFII-250, l(3)05309, TAF250/230, Cys, Thermo Scientific, ur, Source Softwares, Bemiparin, Programs, Spectroscopy, SLP65, TUBE, Program, Ly-57, carbamide, TAFII250, HCD, Digestion, glycerol, Computer Applications Softwares, Softwares, nickel (+2) salt, Dmel_CG7835, cenpA, Mnb, MNB, Yeast, SLP-65, ammonium salt, CenH3/CID, Ci/Gli, Ci/GLI, Spalt, BASH, AI256582, Xylella fastidiosa str. Temecula1, HCl, APT1, cobaltous formate, hydrogen hydroxide, common, L-Cysteine, hac1, CG4299, AW124434, natrii chloridum, CG17603, TAF[[II]], L-Cystein, acqua, dapaf-1S, nanospray, data analysis, H2NC(O)NH2, apaf-1, dapaf-1L, SR3-5, Computer Software Applications., copper salt, Polypeptide, Wasser, HEPES, 7N, Powers, Y3, i2pp2a, 1728, accessory, Lens <bivalves>, d230, Cysteine Hydrochloride, Dapaf-1/HAC-1, Ly-6A.2/Ly-6E.1, Professional Power, Degradation, cesium salt, Brewer's, Gene, baker's yeast, dTAFII250, Ten[m], ACINUS, Spectrum Analyses, Computer, EfW1, supernumerary, 1300013M05Rik, PHAPII, Buffer, formic acid, Dark/Hac-1/dApaf1, Karbamid, l(4)102ABc, method, Glycerine, Hac1, potassium salt, Propanetriol, Dark/Hac-1/dApaf-1, Enoxaparin, polypeptide chain, reduced, dmTAF1, Ms1, subnumerary, Taf230, Injection, 14C-labeled, method used in an experiment, Trihydroxypropane, Harnstoff, Mass, AW536553, Low, camera-type eye lens, tiny, Disulfide, cloruro de hidrogeno, Ham-1, Mass Spectroscopy, TAF250, CenH3[Cid], CenH3[CID], Search Engines, Taf200, Ly-6A/E, MS1, HeLa, ipp2a2, dapaf-1, chlorure de sodium, DmelCG13329, Taf1p, dapaf, dark, eau, decreased number, calcium formate, heparinum, Protein Degradations, TABP, Xylella fastidiosa (strain Temecula1 / ATCC 700964), X-linked combined immunodeficiency, Vdp, VDP, Table, lysate, Candida robusta, data processing, DARK, taf-ibeta, odz/ten-m, sels, male sterility 1, N-2-Hydroxyethylpiperazine-N'-2'-ethanesulfonic Acid, liquid, cromium (+3) salt, 1883, dArk, TAF, Dapaf-1, eye lens, sodium formate, FLN1L, TCEP, Heparin Sodium, BLNK-S, small, Applications Software, TAP, Tap, Open Source, ABP-278, DYRK1, TAF[[II]]250, Computer Software, nickel formate, 3H-labeled, Saccharomyces cerevisiae 'var. diastaticus', protein complex, Glyzerin, sales, igaad, strontium formate, l(3)84Ab, cenH3, apaf1, Protein Digestion, strontium salt, Peptide, group, tap, [HCl], Software Tool, CAD, natural protein, Ci155, p230, water, Protein, FH1, AI551343, I2PP2A, Hydrochloride, sequence, TFIID, table salt, Professional, Dyrk1, Dark, (3alpha, Software, Dark/Apaf-I, ACETONITRILE, Mvb1, Zystein, ABP-280, cyanomethane, Data Base, Sal, 3-Trihydroxypropane, Mass Spectrum, alpha Heparin, PRSS, TAF[[II]]230, uree, MALE STERILITY 1 PROTEIN, 2-amino-3-sulfanylpropanoic acid, CC1, Engineering, Tripcellim, N 2 Hydroxyethylpiperazine N' 2' ethanesulfonic Acid, L-2-Amino-3-mercaptopropionic acid, Salze, TAF[II]250, project description, dApaf-1/DARK/HAC-1, sample population, chromic formate, nitrogeno, sal, Protein Gene Products, P115, present in greater numbers in organism, Trypure, dSET/TAF-Ibeta, Personal Power, 2610030F17Rik, DmelCG17603, Transcytosis-associated protein, concentration, cardinality, calcium salt, Peptid, SCH, AA407739, l(3)00844, dAPAF-1, Fluxum, Baker Yeast, TAF1, 3-OHECN, CID/CENP-A0PXD009548trueProbing the Sensitivity of the Lumos Mass Spectrometer by analyzing varying amounts of whole cell extractsPROJECT DESCRIPTION The Orbitrap Fusion Lumos mass spectrometer has recently been shown to have better fragmentation and detection than its predecessors. Previous studies using a HeLa digest analyzed on the Lumos mass spectrometer have reported the number of peptides and proteins identified by reverse phase elution: Espadas et. al. (2017) observed an increase in detected proteins of approximately 26% when the digest concentration was increased from 100ng to 1000ng. We observed similar increases in protein identification when varying the concentration of yeast whole cell lysate. Furthermore, the concentration of starting whole cell lysate was decreased to determine the number of proteins that could be identified from as low as 5ng of whole cell lysate analyzed following a logarithmic trend. SAMPLE PROCESSING PROTOCOL Yeast (S288C) was grown overnight to an OD600=1.5 in YPD media. The cells were pelleted by centrifuging at 6000 x g for 6 minutes at 4oC. Cells were resuspended in TAP extraction buffer (40 mM HEPES-KOH, pH 7.5, 10% glycerol, 350 mM NaCl, 0.1% Tween-20, 1 mM PMSF, 0.5 mM DTT) and lysed by freezing in liquid nitrogen. Proteins were extracted by breaking the cells using a blender (Waring Commercial). Heparin (Sigma) and salt active nuclease (ArticZymes, Tromso, Norway) were added for 20 minutes at room temperature before proteins were separated from cellular material by centrifuging for 15 minutes at 4000 rpm at 4oC on a table top centrifuge (Eppendorf 5810-R). The supernatant was reserved, and the cell pellet resuspended in TAP extraction up to 6 times to extract all soluble proteins. The supernatants were pooled and further clarified by centrifuging at 14, 000 rpm for 30 minutes at 4oC using a tabletop centrifuge (Beckman Coulter Microfuge 22R). Protein content was quantitated using the Pierce BCA Protein Assay (Thermo). Protein Digestion Yeast whole cell lysate was denatured by adding 8M urea and disulfide bonds were reduced with 5 mM TCEP for 30 minutes before modifying the cysteine residues by carbamidomethylation (10 mM for 30 min in the dark). Endoproteinase Lys-C was added, and proteins digested overnight at 37oC. Urea was diluted to 2M with 100mM Tris-HCl, pH 8.5 before adding trypsin to digest overnight at 37oC. The digestion reaction was quenched by the addition of formic acid to 5%. Peptides were stored at -20oC until use. Peptide Separation Yeast whole cell extract digests were diluted to varying final concentration with buffer A (5% acetonitrile (ACN), 0.1% formic acid (FA) in water). The samples were injected in triplicate using the auto-sampler on an UltiMate 3000 liquid chromatography system (Thermo Scientific) connected inline to the Q Exactive Plus or Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Thermo Scientific) fitted with the Nanospray Flex NG ion source. Peptides were trapped on a 15cm reverse phase column (5um Aqua (Phenomenex), 100um i.d.) and equilibrated with 2% buffer B (80% ACN, 0.1% FA in water) for 15 minutes at a flow rate of 300 nL/min before increasing the concentration of buffer B to 7% over 5 minutes. A linear gradient to 32% buffer B over 122 minutes was used to elute the majority of the peptides before increasing buffer B to 95% over 15 min and holding for an additional 12 minutes. The column was re-equilibrated with 2% buffer B before the next injection. Mass Spectrometry The eluted peptides were ionized for detection by the Orbitrap Fusion Lumos Tribrid Mass Spectrometer (Lumos) or Q Exactive Plus (QE+) mass spectrometer. Source conditions for both instruments were as follows: voltage, 2.5 kV, transfer tube temperature, 275oC. Peptides (MS1) were detected in the Orbitrap with the following settings: Lumos resolving power 60,000, QE+ resolving power 70,000; scan range m/z 400-1600; S-Lens RF 55%; Lumos AGC target 4.0 x 105, QE+ AGC target 1.0 x 106; microscans, 1. The top 15 peptides were selected in a data dependent mode for MS/MS fragmentation by CID at 35% NCE (Lumos) or HCD at 27% NCE (QE+) to be detected in the Orbitrap (Lumos resolving power 15,000 or QE+ resolving power 17,500) or ion trap on the Lumos with dynamic exclusion for 30s with the Lumos AGC target set to 1.0 x 104 and QE+ AGC target at 1.0 x 105. DATA PROCESSING PROTOCOL All datasets were searched with the ProLuCID search engine (v. 1.3.3) against the S. cerevisiae database downloaded from NCBI updated on May 16, 2017 with the NIST mAb sequence added as well as 160 common contaminants. The database also contained the shuffled protein sequences to estimate false discovery rates (FDRs). The result files from ProLuCID were further processed with DTASelect (v 1.9) to correlate peptide level information into protein information. Using in-house developed software, swallow, the peptide spectrum matches were maintained at FDR less than 5% for peptide and protein level matches. The datasets were compared using Contrast16 and quantitated using our in-house software NSAF7 (v 0.0.1). Thu Apr 19 15:43:00 BST 2018MSV00008229630180573