Beliaev, Alexander S.

Elias, Dwayne A.

Goddard, Carrie D.

Lipton, Mary S.

Mottaz, Heather M.

UNT Digital Library

Pacific Northwest National LaboratoryU.S. Department of EnergyParallel Proteomic Identification ofMetal Reductases and Determinationof their Relative Abundance in aSeries of Metal Reducing BacteriaMary  S. LiptonDwayne A. Elias, Heather M. Mottaz, Carrie D. Goddard andAlexander S. BeliaevBiological Separations and Mass SpectrometryBiological Sciences DivisionPacific Northwest National Laboratory2MotivationHistorically, identification of proteins with metal reduction activity hasbeen challengingNecessary to purify proteins to homogeneityLost metal reduction activityNew advances in by mass spectrometry allowed for the identification ofmultiple proteins from global lysates and mixturesThis project has applied these mass spectrometry techniques withorthogonal protein purification and enzymology techniques for thepreliminary identification of proteins with these activities.3Concept of ExperimentsChemostat grown cellsFrench press lysis, centrifugation and ultracentrifugationSoluble fraction Insoluble fractionAnion, Cation, and Ni-NTAcolumn enrichmentAnion, Cation, Ni-NTA, andHydrophobic column enrichmentLTQ-FT MS analysisFractions with Fe(III)-NTA reductionactivity analyzed• Protein detection was valid if 2 peptides unique to the parent protein were present in all3 MS analyses and passed strict filters.• Proteins from all column fractions of each matrix were then pooled and queried againstother matrix results to find proteins in common for insoluble or soluble subcellular fractions.4 Accurate mass observed: 1830.1985 DA AMT is validated Separation, analytical conditionsestablished500 750 1,000 1,250 1,500m/z916.0 920.5m/z200 600 1,000 1,400 1,800m/zy9y8b10y7b9b8y6y12y10y11b12b6y5b7b11y13b14b13y4 Sequence determined from MS- MSfragmentation pattern Potential mass tag (PMT) identifiedfrom D. radiodurans proteome  (APEEKAR) (GITINTAHVEYQTETR) (HYSHVDCPGHADYVK)confirm sequence as GITINTAHVEYQTETRcalculated mass = 1830.1987Separate & measure usingCapillary LC- Ion Trap MS/MSSeparate & measure usingCapillary LC-FTICR MSAccurate Mass Tag (AMT) Concept5• Peptide generated from proteolytic digest of a protein• Identified from sequenced genome• High accuracy mass measurements by FTICR• Combination of conventional mass spectrometry andFTICR techniques– Higher confidence in peptide identification– High throughput analyses enabledAMT tags provide uniquebiomarkers for nearly allproteins in a microbe.PNNL Accurate Mass and Time (AMT) Tag ApproachFTICR mass spectrometry allows quantitation•   Stable isotope labeling provides relative abundance changes•   Direct peak intensities provides absolute quantitation6Fe(III) reduction activity measurements• Activity of eachfraction wastested by amethod modifiedfrom thatdescribed(Magnuson et al.,(2001) Biochem.J. 359:147-152.)as shown usingpurified MtrC andOmcA from S.oneidensis MR-1.• Proteinconcentration wasmeasured usingthe BCA microplate method.pH 6.8pH 7.0pH 7.5blankNo Fe(III)-NTA addedMtrC/OmcA pi,pH 6.8, 7.0, 7.5MtrA MtrC OmcA MtrC/OmcAMtrA/MtrC MtrA/OmcA MtrC/OmcA700.511.522.5protein (mg/ml)050100150200250Activity (mM/min, mM/min/mg protein)Insoluble fractionAnion exchangeProtein concentrationTotal activitySpecific ActivityShewanella oneidensis8Insoluble fractionMatrix Number of ProteinsCell Extract 972Hydrophobic 515Anion Exchange 360Cation Exchange 392Ni(II)-NTA 845Common to all Matrices 143Likely Candidates   41Shewanella oneidensisSoluble fractionMatrix Number of ProteinsCell Extract 275Anion Exchange 188Cation Exchange 212Ni(II)-NTA 196Common to all Matrices 120Likely Candidates     129Shewanella oneidensis candidates“Insoluble proteins”SO1778 decaheme cytochrome c MtrCSO1779 decaheme cytochrome c OmcA OM protein, functional purified protein shows Fe(III)-reduction; mutants deficient in Fe(III)-reductionSO4591 tetraheme cytochrome chigh homology to tetraheme cytochrome c3; classically involved in metal reductionSO3286 cytochrome d ubiquinol oxidase SO0610 ubiquinol-cytochrome c reductase cytochrome c1 SO4666 cytochrome c classically involved in mediating electron transferSO4321 OmpA family proteinSO3545 OmpA family protein OM protein, likely redox reactive SO2361 Cytochrome c oxidase cbb3 type[low O2] expressed terminal reductase and so may also be reactive with oxidized metals.SO1429 anaerobic dimethyl sulfoxide reductase A subunitterminal reductase for anaerobic respiration of DMSO; likely redox reactive “Soluble Proteins”SO1776  outer membrane protein MtrB loosely OM associated protein; mutants deficient in Fe(III)-reduction10Dsv. desulfuricans G2000.20.40.60.811.21.41.61.8protein (mg/ml)05101520253035Activity (mM/min,mM/min/mg protein)Insoluble fractionAnion exchange11Dsv. desulfuricans G20Soluble fractionMatrix Number of ProteinsCell Extract 836Anion Exchange  NDCation Exchange  NDNi(II)-NTA 657Common to all Matrices N/ALikely Candidates   71Insoluble fractionMatrix Number of ProteinsCell Extract 326Hydrophobic 122Anion Exchange 164Cation Exchange 198Ni(II)-NTA 175Common to all Matrices  26Likely Candidates     1112“Insoluble proteins”Bacterioferritin (cytochrome b1)   *detected in both fractionslikely involved in electron transfer pathwayNi,Fe-hydrogenase I large and small subunit  *detected in both fractionsredox reactive protein and known to be involved in Tc(VII)-reductionCoenzyme F420-reducing hydrogenase   *detected in both fractionsredox reactive protein, may be involved in Tc(VII)-reduction(?) similar to the NiFe-Hydrogenase.“Soluble proteins”Predicted NADH:ubiquinone oxidoreductase, RnfC and RnfG s.u. Putative NADPH-quinone reductaseNADH:ubiquinone oxidoreductaseCytochrome bd-type quinol oxidase, subunit 1classically involved in mediating electron transferPredicted Fe-S oxidoreductases (X3)Fe-S oxidoreductases (X2)redox reactive proteins apparently in the periplasmOuter membrane protein (X4)OM protein, possibly redox reactiveDsv. desulfuricans G20 candidates1300.511.522.50 5 10 15 20 25 30 35Elution time (m051015202530354045Geobacter sulfurreducensInsoluble fractionNi(II)-NTA14Geobacter sulfurreducensInsoluble fractionMatrix Number of ProteinsCell Extract 414Hydrophobic 228Anion Exchange 207Cation Exchange 234Ni(II)-NTA 227Common to all Matrices   87Likely Candidates   38Soluble fractionMatrix Number of ProteinsCell Extract 398Anion Exchange 119Cation Exchange 137Ni(II)-NTA 360Common to all Matrices   91Likely Candidates    2015“Insoluble proteins”GSU0509/ GSU0510 Fe(III) reductase, a and b subunit (sfrA, sfrB)annotated as Fe(III) reductaseGSU3259/ GSU0274 cytochrome c family proteinGSU2811* cytochrome c Hsc (hsc)GSU2813 cytochrome c551 peroxidase (ccpA-2)heme containing; classic electron transfer proteins and may function as terminal reductase.GSU1468/69/70* keto/oxoacid ferredoxin oxidoreductase, a,b,g subunitsGSU0343/44/46/50 NADH dehydrogenase I; F,G,I,M subunits (nouF,l-1,M-1)classically involved in mediating electron transferGSU0024/0360 OmpA domain protein (X2)GSU0073/2267/2268 outer membrane protein, putative (X3)OM proteins, possibly redox reactive“Soluble proteins”GSU2504/1397 cytochrome c family proteinheme containing; classic electron transfer proteins and may function as teminal reductase.GSU2731 polyheme membrane-associated cytochrome c (ferA)fer system well-known to be involved in Fe(III)-reductionGSU1177/1178 fumarate reductase, flavoprotein iron-sulfur subunits(frdA,B)redox reactive proteinGeobacter sulfurreducens candidates16Charge state of Fe in gas phase - critical to determine the hememodification massFragmentation pattern of heme-containing peptides-C-X-Y-C-H- Under-represented2D gel analysis:  Giometti, C. S. et. al. Proteomics 2003, 3, 777-785.    Fumarate reductase   both abundance and pI changes    pI: 7.6 under aerobic condition    pI: ~6.5 under low oxygen conditionpotential modification changesIdentification of c-type heme-containingpeptides in Shewanella oneidenis17Purification of heme-containingpeptides from Horse Cyt.C-C-X-Y-C-H-Soret band absorptionAliAlieUV detection395 nm detection0. 27.5 55. 82.5 110.Time0. 27.5 55. 82.5 110.Time18ecenecen992.31 996.31 1000.3 1004.3 1008.3C44 H94 N12 O13m/z992.89 996.89 1000.8 1004.8 1008.8C42 H63 N12 O13 Fem/z4983.8 4991.8 4999.8 5007.7 5015.7C222 H355 N61 O66 S2 m/z4984.4 4992.4 5000.4 5008.4 5016.4C220 H338 N60 O66 S2 Fe m/z54Fe: 5.845%56Fe: 91.754%57Fe: 2.119%58Fe: 0.282%Fem/zHeme signature isotopes due to Fenaturally occurring isotopes19Heme-containing peptides (one hememodification on cysteine in CXYCH motif)identified from MtrA0.07271.78094.63K.LWEHAPVTENCVTC*HNPHGSVNDGMLK.T0.00903.9667203R.APQL C*QQCH ASDGHASNA.Y0.11180.01872.70043.9437704023R.SC*LNCHSQVHGSNHPSGK.L0.07993.4852123R.MSWNGGHHDNADVACASC*HQVHVAK.D0.26830.21980.11951.80441.65462.68331007728233K.QNSVC*MSCHQDDKR.MK.QNSVCMSC*HQDDKR.MK.QSTLSADKQNSVC*MSCHQDDKR.M0.03240.25292.00531.71641004323K.GADSCLMC*HK.KK.GADSCLMC*HKK.S0.01603.4542123K.SPMAGLQCEAC*HGPLGQHNK.G0.18030.29862.33061.29921004223K.NTEMEVCTSC*HTK.QK.NTEMEVC*TSCHTK.QCnXcorrHeme %CSPeptide (heme motif in bold)20Heme-containing peptides identified fromSO0970 and SO2727 proteinsNoYes2.74YesYes3.1, 2.02,3K.DGTPSADGAFEFAQCQSCHGK.LNoNo3.5, 3.04,5LSEMDAVHKPHDGNLVCADCHAVHDMNVGQKPTCESCHDDGRNoNoa2.83PHDGNLVCADCHAVHDMNVGQKNoYes3.0, 2.23,4LSEMDAVHKPHDGNLVCADCHAVHDMNVGQKYesYes1.8, 1.12,3K.LSDFHAESGGCESCHK.DYesYes1.5, 0.52,3K.PTCESCHDDGR.TSO2727YesNoNo3A.APEVLADFHGEMGGCDSCHVSDK.GYesYes2.6, 1.62,3K.SVAYCDACHSFGFDMPFGGK.WYesYes1.5, 0.62,3K.SHLIGEIACTSCHK.GYesYes0.6, 0.42,3K.SHLIGEIACTSCHKGHEK.SNoNoa4.63VSPHKSHLIGEIACTSCHKYesYes0.63K.GGVTNDNLTHENGQCVSCHGDLK.ESO0970SignatureisotopesEr r o r(ppm)L C QMS /MSFTICR MSCharg estatePeptide (heme attachment motif in bold)ORFa Due to low intensity and S/NLSDFHAESGGCESCHKDGTPSADGAFEFAQCQSCHGK21Conclusions• Combination of protein purification, enzymology and advanced massspectrometry schemes has yielded a method for identification of metalreduction proteins• Eliminates the need to purify each protein to homogeniety• Enrichment of subcellular fractions of S. oneidensis MR-1, Dsv.desulfuricans G20, and G. sulfurreducens by achieved by multiplepurification schemes•  Proteins isolated in fractions displaying Fe(III)-reduction activity inall schemes are identified as putatively being involved in metal-reduction activities• The combination of reductase enrichment with high-throughput,comprehensive MS analysis yields more information without lengthypurification of each protein and the possible loss of activity duringpurification.• Further use of characterization of heme containing proteins allowmore comprehensive understanding of these proteins.22AcknowledgementsShewanella Federation Margie Romine Jim FredricksonYuri Gorby Matt MarshallSeparationsYufeng Shen Kostas PetritisRui Zhao David SimpsonAlex ShvartsburgMass spectrometryLjiljana Paa-Toli Keqi TangDavid Anderson Ron MooreHarold Udseth Tao LiuAleksey Tolmachev Rui ZhangWeijun Qian Andre VilkovJason Page Therese ClaussData processing, software developmentand statisticsGordon Anderson Matt MonroeSam Purvine Angela NorbeckDave Clark Eric StrittmatterGary Kiebel Nikola TolicKen AuberrySample processing, etc.Mary Lipton Kim HixsonHeather Motaz Carrie GoddardMarina Gritsenko Josh Adkins*David Camp Jon JacobsEric Livesay Biological Systems Analysis and Mass SpectrometryRichard D. SmithDwayne A. Elias, Heather M. Mottaz, Carrie D.Goddard and Alexander S. Beliaev$$ $$NABIR program:  Office of Biological andEnvironmental Research

Parallel Proteomic Identification of Metal Reductases and Determination of their Relative Abundance in a Series of Metal Reducing Bacteria

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Parallel Proteomic Identification of Metal Reductases and Determination of their Relative Abundance in a Series of Metal Reducing Bacteria

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