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ParkerMass SpectrometryShotgun proteomicsNot availableNano-liquid chromatography-tandem mass spectrometryElectrospray ionizationProteomicsContaminant ionshttp://www.ebi.ac.uk/pride/archive/projects/PXD013073BrainWhole mouse brain was lysed in 4% sodium deoxycholate (SDC) in 100 mM Tris, pH 8.5 by tip-probe sonication. The lysate was centrifuged at 20,000 x g for 10 min and quantified by BCA. Two milligrams of protein was reduced/alkylated with 10 mM tris(2-carboxyethyl)phosphine and 40 mM 2-chloroacetamide at 95°C for 5 min. The mixture was diluted to a final concentration of 1% SDC and digested with 40 μg of sequencing grade trypsin (Sigma Aldrich, St. Louis, MO, USA) at 37°C for 16 h. The digestion was acidified to a final concentration of 1% trifluoroacetic acid (TFA) and centrifuged at 20,000 x g for 10 min. The peptide containing supernatant was loaded onto a hydrophilic-lipophilic balanced solid phase extraction (HLB-SPE) cartridge (Waters, Milford, MA, USA) that was pre-equilibrated with 100% methanol, 100% acetonitrile and then 5% acetonitrile containing 0.1% TFA. Peptides bound to the column were washed with 5% acetonitrile containing 0.1% TFA and eluted with 50% acetonitrile containing 0.1% TFA prior to evaporation to dryness by vacuum concentration. Peptides were resuspended in 2% acetonitrile containing 0.1% TFA and quantified by Qubit (Thermo Fisher, Waltham, MA, USA). Liquid chromatography-tandem mass spectrometry analysis Peptides were analysed with an Easy-nLC 1000 (Thermo Fisher) coupled to a Q Exactive Orbitrap. Peptides were separated on 7 cm x 100 μm inner diameter column with an integrated laser pulled ESI emitter packed in-house with 3 μm C18-AQ particles (Dr. Maisch GmbH, Germany). Separation was achieved by loading the peptides directly onto the column in 2% buffer B (A=0.1% formic acid; B=80% acetonitrile, 0.1% formic acid) and peptides eluted over a linear gradient of buffer B for either 12 or 120 min at 500 nl/min. The gradient was then ramped to 80% buffer B and held for 2 min to wash the column and then re-equilibrated with 2% buffer B. The MS was operated in positive polarity with an ionization of 2.4 kV and a capillary temperature of 300°C. A full-MS scan was recorded at 300-1600 m/z, 35,000 resolution and an automatic gain control (AGC) target of 3x106. Data-dependent acquisition of the top 15 most intense ions were selected for MS/MS by HCD at 25 normalized collision energy with a 17,500 resolution, AGC target of 1e5 and a maximum injection time of 60 ms. Only analytes with charge 2-5 were selected with an isolation window of 2 m/z and then excluded for 40 s. The NanoBlow was operated with a sweep gas setting of 10 and was switched on only during re-equilibration of the column, peptide loading and during washing of the column at 80% buffer B.PrideNot availableiodoacetamide derivatized residuemonohydroxylated residueacetylated residueRaw data were analysed with MaxQuant/Andromeda (v1.6.1.0) and searched against the mouse Uniprot database containing both SwissProt and TrEMBL (March 2018, 55,608 entries) [PMID: 19029910]. Spectra were search with protein N-terminus acetylation and methionine oxidation set as variable modifications, and cysteine carbamidomethylation set as a fixed modification. A maximum of 2 miss-cleavages were allowed and following mass recalibration, 4.5 ppm MS and 20 ppm MS/MS mass tolerance was applied. Peptide spectral matches and protein false discovery rates were set to 1%. Minimum peptide length was set to seven amino acids. All raw data has been deposited into the ProteomeXchange Consortium via the PRIDE repository with identifier X.ProteomicsBenjamin ParkerQ ExactiveThe University of SydeyPARTIALMus Musculus (mouse)ben.parker@unimelb.edu.au31268342 Humphrey SJ, Crossett B, Parker BL. NanoBlow: A Simple Device To Limit Contaminants during NanoLC-MS. J Proteome Res. 2019 10.1021/acs.jproteome.9b00175The University of MelbourneAustraliaLimiting contamination of liquid chromatography/tandem mass spectrometry (LC-MS) systems and reducing the downtime associated with maintenance and cleaning is essential for productivity. We developed a simple device that creates a gas curtain barrier to prevent ions from entering the MS inlet. The gas can be quickly and easily applied when certain contaminant ions are known to elute. We show that the device can prevent the buildup of contaminants on the heated transfer capillary following >100 injections of a crude tissue lysate and improves peptide identifications. The device may provide a promising approach toward improving instrument robustness.NanoBlow: A Simple Device To Limit Contaminants during NanoLC-MS.Humphrey Sean J SJ, Crossett Ben B, Parker Benjamin L BLsodium salt, Nup32D, CPD photolyase activity, ammonium formate, PLIP, 13C-labeled, MeCN, cadmium salt, PhrB photolyase activity, NCMe, ion, 1-bis(diphenylmethoxy-2'-ethyldimethylammoniumacetoxy)decane dichloride, zinc formate, lead salt, protein, magnesium formate, pigmented epithelium, zinc salt, Type 2A-interacting protein, P270, Long Term, Bca, Trifluoroacetate, peptide, actin-dependent regulator of chromatin subfamily F member 1, Polypeptides, BAF250a, protein polypeptide chains, ammonium tetraformate, ClvPrd, peptido, cobalt(II) formate dihydrate, dJ69E11.3, CH3-C#N, B1, SWI|SNF-related, 1, Min, AGM4, 3, synganglion, NUP96, germacrene A synthase activity, outer pigmented layer of retina, protein aggregate, present in fewer numbers in organism, epithelium, Injectables, Effect, HTATIP, DmelCG42273, nup154, wood alcohol, bca, Divorced, peptides, reference sample, DmelCG13176, hypoplasia, pigmented retina, min, ORF19, mAPC, proteins, Divorces, purification, suprasegmental levels of nervous system, copper, SCAN, DNA cyclobutane dipyrimidine photolyase activity, SUPPRESSOR OF AUXIN RESISTANCE 3, AI047805, isolation and purification, PA1, pigment epithelium of retina, decreased, lithium salt, cPLA2, column, trifluoro-, Solid Phase Extractions, D1, C16orf53, pigmented retina epithelium, Lyw-57, blood capillary, house mouse, Sodium Methoxide, buffer., TIP, ammonium (2:1) salt, Long-Term Effects, ELD, nickel salt, (+)-germacrene A synthase activity, PRE, hOSA1, suprasegmental structures, Alcohol, cobalt (+2) salt, ZC2HC5, deoxyribonucleic cyclobutane dipyrimidine photolyase activity, mouse, Osa homolog 1, Dmel_CG7826, Longterm Effect, Th, HTATIP1, pigmented retinal epithelium, tip, GAST, ANAC091, Putative MAPK-activating protein PM10, SLP65, Ly-57, sodium (4:1:1) salt, HCD, Dm1, retinal pigment, magnesium salt, Ionen, dipyrimidine photolyase (photosensitive), B120, 1110049F14Rik, TIP60, WASH, GAS, nickel (+2) salt, Carbinol, 6-trans-farnesyl-diphosphate diphosphate-lyase (germacrene-A-forming) activity, retinal pigment layer, Dmel_CG7835, Acetic acid, carbinol, Mnb, MNB, Acid, SLP-65, ammonium salt, BASH, LY57, mice, retinal pigmented epithelium, Sonications, Encephalon, Trifluoroacetic, formate, OSA1, cobaltous formate, cromium (+3), gas, AW124434, lysed material, beta Trypsin, Solid Phase, chloracetamide, METHANOL, phr A photolyase activity, Wood, DNA-photoreactivating enzyme, Cesium, spirit of wood, copper salt, ammonium (4:1) salt, Mouse, Polypeptide, methyl cyanide, tlp, lead (+2) salt, TIP41, time, Methyl Alcohol, the brain, nickel formate dihydrate, lead formate, Effects, aluminum salt, cesium salt, 2-trans, Methyl, CH3OH, photoreactivating enzyme activity, protein-containing complex, Methylalkohol, thomson, ESA1, CG7826, deoxyribodipyrimidine photolyase activity, stratum pigmentosum retinae, Buffer, formic acid, mass-to-charge ratio, period, hELD, potassium salt, polypeptide chain, reduced, isolation, Ly57, subnumerary, Injection, 14C-labeled, SWI-like protein, p63, A-17, Arabidopsis NAC domain containing protein 91, p65, CG7835, zk, CG42273, Wood Alcohol, stratum pigmentosa retinae, tiny, Separated, Cesium Trifluoroacetate, l(2)10432, MOS3, deoxyribocyclobutadipyrimidine pyrimidine-lyase activity, FAM39E, PRECOCIOUS, thallium (+1) salt, Longterm, iones, beta-Trypsin, rubidium salt, BAF250, F23A5.3, Extractions, methanol, Long-Term, decreased number, calcium formate, methanoic acid, ESI, ions, Injectable, DmelCG4579, ms(2)zk, lysate, wood naphtha, BRG1-associated factor 250, peptidos, cromium (+3) salt, Long-Term Effect, l(2)01501, F23A5_3, sodium formate, TCEP, PTHB1, BLNK-S, Controlled, small, DYRK1, Controlling, data, SMARCF1, 1-(14)C-labeled, nickel formate, BM029, 3H-labeled, protein complex, BcDNA:LD21772, 6-trans-farnesyl-diphosphate diphosphate-lyase [(+)-germacrene-A-forming] activity, strontium formate, Methyl alcohol, potassium formate, Mischung, copper (+2) salt, Extraction, acetonitrile, strontium salt, buffer, (+)-(10R)-germacrene A synthase activity, encephalon, Peptide, light amplification by the stimulated emission of radiation, Methanol, polypeptide, stratum pigmentosum (retina), Wash2, MODIFIER OF SNC1, Wash1, capillary vessel, native protein, Temperatures, natural protein, Mus, C18, cupric formate, Protein, Long Term Effects, deoxyribonucleic photolyase activity, Sodium, Dyrk1, MeOH, Vacuums, aluminum formate, ACETONITRILE, cyanomethane, CG13176, HLB, AU020952, MRD14, Separation, BAF250A, C1orf4, nup32D, RPE, lithium formate, underdeveloped, photolyase activity, Separations, CC1, CG4579, Tripcellim, ethanenitrile, retinal pigment epithelium, SWI|SNF complex protein p270, chromic formate, Longterm Effects, ME-IV, p. pigmentosa retinae, Trypure, matrix-associated, ARID domain-containing protein 1A, BRG1-associated factor 250a, Ion, concentration, wood spirit, TCV-interacting protein, calcium salt, Peptid, deoxyribonucleate pyrimidine dimer lyase (photosensitive), Methoxideinstrument., SIMPLE, PIG7, TP53I7AU043776, Bru, IPP2A2, 2-amino-4-(methylsulfanyl)butanoic acid, Cysteine Hydrochloride, Raw, False, Aminosaeure, Aminocarbonsaeure, L Isomer, FBN, Amino acid, protein, alpha-amino acid, protein-containing complex, PHAPII, 5730420M11Rik, peptide, Polypeptides, protein polypeptide chains, L-Isomer Methionine, Methionine, Drug Tolerance, ClvPrd, peptido, polypeptide chain, template-activating factor I, ECTOL1, L-Methionine, Del(8)44H, Half-Cystine, protein aggregate, Pedameth, WMS, Svc, SET, F, amino acids, peptides, TAF-I, c14_5346, M, L Cysteine, phosphatase 2A inhibitor I2PP2A, ipp2a2, Acetylations, 2pp2a, proteins, CG10574, DmelCG4299, OCTD, set, IGAAD, 2-amino-4-(methylthio)butanoic acid, 2PP2A, 10^[-6], DmelCG10574, taf-ibeta, data., ppm, dSET, dSet, peptidos, house mouse, Half Cystine, metionina, GPHYSD2, Racemethionine, drug tolerance, SGS, phapii, data, DL-Methionine, Zinc Cysteinate, protein complex, immune system tolerance, Aminokarbonsaeure, acetylation, mouse, 2-Amino-4-(methylthio)butyric acid, igaad, C14orf32, StF-IT-1, 2310009E07Rik, alpha-amino acids, L-Isomer, Peptide, alpha-amino carboxylic acids, ACMICD, polypeptide, native protein, Immune Tolerance, natural protein, Mus, I-2PP2A, Protein, Dm I-2, I2PP2A, Immunologic Tolerance, Methionin, MFS1, methionine, Hmet, Data Base, Met, WMS2, Amino Acid, Acid, Col4a-1, HLA-DR-associated protein II, Amino acids, DI-2, mice, I-2Dm, L-Cysteine, CG4299, MASS, inhibitor of granzyme A-activated DNase, MISS, Amino, I-2PP1, dSET/TAF-Ibeta, 2610030F17Rik, TAF-IBETA, Self Tolerance, Tolerance, SSKS, alpha-amino-gamma-methylmercaptobutyric acid, 2018, Peptid, Mouse, TAF-Ibeta, Acids, Polypeptide, variable, AA407739, Liquimeth, i2pp2a, Immunological Tolerance(+)-germacrene A synthase activity, portion of tissue, ion, peptides, instrument, iones, 6-trans-farnesyl-diphosphate diphosphate-lyase [(+)-germacrene-A-forming] activity, instrument configuration, Tissue, 2-trans, gas, instrument., Maintenances, GAST, ions, lysed material, (+)-(10R)-germacrene A synthase activity, Peptide, Productivity, PA1, Injectable, polypeptide, peptide, Polypeptides, capillary vessel, lysate, ClvPrd, peptido, Ionen, Ion, Injection, PIG7, C16orf53, tissue portion, Peptid, peptidos, simple tissue, germacrene A synthase activity, blood capillary, Polypeptide, associated, GAS, SIMPLE, 6-trans-farnesyl-diphosphate diphosphate-lyase (germacrene-A-forming) activity, Injectables, TP53I7(+)-germacrene A synthase activity, ion, Tandem, 6-trans-farnesyl-diphosphate diphosphate-lyase [(+)-germacrene-A-forming] activity, instrument configuration, Liquid Chromatography, 2-trans, GAST, Mass Spectrometry Mass Spectrometry, (+)-(10R)-germacrene A synthase activity, Peptide, Productivity, polypeptide, peptide, Polypeptides, capillary vessel, ClvPrd, peptido, Ionen, Injection, PIG7, tissue portion, simple tissue, germacrene A synthase activity, GAS, 6-trans-farnesyl-diphosphate diphosphate-lyase (germacrene-A-forming) activity, Injectables, tandem MS, portion of tissue, peptides, instrument, MS/MS, MS2, iones, Tissue, Mass Spectrometry, Mass Spectrometry-Mass Spectrometry, gas, instrument., Maintenances, ions, lysed material, PA1, Injectable, lysate, Ion, C16orf53, Peptid, peptidos, blood capillary, Polypeptide, associated, SIMPLE, TP53I7instrument., SIMPLE, PIG7, TP53I7falseNanoBlow: A simple device to limit contaminants during nanoLC-MSLimiting contamination of LC-MS systems and reducing downtime associated with maintenance and cleaning is essential for productivity. We developed a simple device that creates a gas curtain barrier to prevent ions entering the MS inlet. The gas can be quickly and easily applied when certain contaminant ions are known to elute. We show the device can prevent the build up of contaminants on the heated transfer capillary following >100 injections of a crude tissue lysate and improves peptide identifications. The device may provide a promising approach towards improving instrument robustness.2019-06-192019-03-13PXD013073280655164609918049829535851114542249420245278728421880905811363336351524260119832758385NCBITaxon:128068888561896111430118726355557114526236493461364031952065126957274670738538836411354615821582310665688954762721123294422291180992323122586365458333460792519365914320597129386558195851149315242592637371250232NCBITaxon:127211195774818661488584310430024513586722175543740816918045426683445305861266835149822505814963674585545281132081479564100392375146369420494332089642089634459741379791283844006679352936715675932165994048321659721659545501315282693042165915627161696833218321729204277944762376454317203094980197525556978159509439843948840479NCBITaxon:2157525338551531051067439899037122857759515205968762262311210884555588845587077918178081736309431794571698936160488409859NCBITaxon:37162104782679401505907148364320421061320491381693630355543204663263331199456545351102541110321745921612928867641002642036945694101676341021076127735113165823925472793342814613584301417298174633457744491675476048932128735675684030191726518130194111929439367110411310239320255598632582788396591047593474951652998355256631758543980110243260866310245980875741755518320196447145268045796631367019083233115611415535510421932555217634981812359871069955529NCBITaxon:475185029833547611361574146547718006655648433767729976798251021699823897648407413735115571108972444474153NCBITaxon:50557899698441000364653493341471474935480194675129911435974127647313708632654787656983532215998381920111067757727103481985247946409860995861571871766859919157295137221171101292921076878349741986020280914884063342492342351523359859886559746499870392021110729433117113972159874190850371170827117082841823502414511391902197822474636046147123749402375939723287581722532566489050794526591972211915853101774208588596NCBITaxon:615750521028307989511051989972375059576131720002634196102641088878176179212190659407121906772275885815689964975151950587244NCBITaxon:22023717NCBITaxon:170361220199985705061747679274506272491596153335541840052464321368615059648784866629417866613475153632910181943010867310185NCBITaxon:1763573039635586331492862871321650851865368595105135553482260710185687294296110091416333NCBITaxon:619184023210007348824123368127187878645159812894555663577553470951451225786260704NCBITaxon:13356262607052607076584574054163677483167233150561007133705654195NCBITaxon:305550445791399878168119011928752829126806315327341478281055524NCBITaxon:9606135858469008111167815022830519302836728593008529005NCBITaxon:569382688306401794699151011849943525815159014482428381116041937047592723708281677285035158199011951953267386370249240536088385816224888175626158355837153337605726711935019980861193502167879108579137113712724071906503714759175712562745757270903113335503728142433747924910078711082NCBITaxon:961559463318586260799141262200298492670NCBITaxon:12637192812492125283038568385695911692661006581237561275045426662551519788375036009412934979054632245352472140125764990851515143899237453747445446415243235959202228870515805955365550881579327160293851578114978633892894535936479437NCBITaxon:68544127542251171590109013416738715895521012966103061296715851584515849376593934327159100226376733889904614234512876895911983329664670414647929365761642803411602216413315992803828035369091089176894621812141080348179392287889295027113133295244568617493468191126526997227321890382107601247190400727159736114311402064119810871355477436581424507229533796982042321242231291632180334708714537209164573321535815754699287770202060328114319338293550833291292211487252649997283035739087267271037908550717629970767892683704152140532941281607839563924654898227377596207322086595882735551693605330797255181135491597495350261159556143213840607258781414471509493349194963401614334728005936046316461046603815184547448500356709747751240174841855601335755547920427460769946881037620148871005941289093164354282688500712303831389464940838283594144242322197857742171598921409212205851157781154756929793244704749906604162103591905158114452749907312989913112273170187546991103589915NCBITaxon:37296103609833418710491130798133679531640729946710340157865826453033373306383011306413992530642363650591169654788546981121162034317103372994044586207340115357111952965261174553111471613386541313166343055213231201798360253237125523299351299385057316317642619794011513458445772678728623219334151346186208621160791392227983516811961255228149539322323116761757312108077213487999948861856962814084341033519963218496209913924882097115338209611533920953304393755699286266447NCBITaxon:2697049339862436941103121958832050920290610130685999758644864324995252972467411583438942786378942625673786391233435514199834931799868655405534352914865451471245466104298635712467915164135825763288401937334508771823803912903912952971974869317978911028688999513373105487712516321389850762554701193806256318865713034431121308516558658862238NCBITaxon:44664946454636493NCBITaxon:11071142800624212428835649137514151678458810874391459438543311167810778543144080439334527031NCBITaxo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