Advisorycommi,eeP.Smith(SeniorAssociateDeanforResearch)A.Fenton,Scien8ficdirector(BiochemistryandMolecularBiology)K.Peterson(vice-chair,BiochemistryandMolecularBiology)L.Swint-Kruse(BiochemistryandMolecularBiology)S.Weinman(InternalMedicine)K.McCarson(Pharmacology,ToxicologyandTherapeu8cs)M.Winter(RodentBehaviorFacility)

Mass Spectrometry/ Proteomics M. T. Villar and A. Artigues

Dept of Biochemistry and Molecular Biology, KU Medical Center ThemissionoftheMassSpectrometryLaboratoryoftheUniversityofKansasMedicalCenter istoprovideaccesstomassspectrometrybasedproteomics applica;ons toKU researchers. The technologyandexper;seofferedby theKUMCMassSpectrometryCorelaboratoryareintendedtoenhancetheextramuralfundingofinves;gatorsofKUMedicalCenter.TheMSPCLaboratoryispartoftheKDRIKCandislocatedinroom1058,HemenwayLifeSciencesInnova;onCenter(HLSIC).

For more information please visit our web page:hNp://www.kumc.edu/mspc.html

Proteomicsisthestudyofallproteinsexpressedinapar;cularcelltype,;ssueororganism,andthechangesintheirrela;veabundanceduringdevelopment,agingor as a result of an injury or trauma. This discipline has acquired significance inbiologicalandhealthsciencesduetoadvancesinotherrelatedareassuchas:•  Thepossibilityofgenera;ngionsinthegasphaseofverylargemoleculessuch

asproteins/pep;des.•  Theusenewmass spectrometers than canmeasure theirmasseswith ahigh

degreeofaccuracy,resolu;onandreproducibility•  Theavailabilityof largedatabasescontainingthecompletegenome/proteome

ofmanyspecies•  Theavailabilityofpowerfulcomputersandcomputeralgorithmstointerrogate

thosedatabaseswithMSinforma;on

•  StandardMassspectrometryproteomics:Proteiniden;fica;onMudPITtechnologyProteinquan;fica;on:freelabel,SILACandusingchemicallylabeledpep;deswithisobaricTags(TMT,iCAT,andothers)

•  SRMandMRM:methoddevelopmentandquan;fica;on•  AnalysisofproteinposNransla;onalmodifica;ons•  Denovopep;deandproteinsequencing•  Analysisofprotein-proteininterac;onsandchemicalcross-linking•  Deuterium-exchangemassspectrometryfortheanalysisof

ProteindynamicsProtein-ligandinterac;onsProteinconforma;on

•  GelimagingandspotpickingonaProPicII.•  Note: Inaddi;ontothestandardandspecializedmassspectrometrymethods

above, theMSPCLaboratoryhas thecapability to introduce/adaptordevelopmethodsforspecificstudiesasneeded.

DirectspotpickingandImaging

TheProPicIIisafullyintegratedrobo:cpla;ormforproteingelimagingandpicking(spotexcision).

Directspotpickingandgelimaging

TheProPicIIisafullyintegratedrobo8cplaMormforproteingelimagingandspotexcision.

LTQFTHybridiontrap–ICRFTmassspectrometerSAIDEAnin-housebuildinterfaceforHDXMS

Highresolving-highsensi;vehybridmassspectrometry

OrbitrapFusionLumosThemostadvancedmassspectrometerEASYnLC1200anultrahighresolu8onHPLC

Tota

l de

uter

ium

co

nten

t

-10 -5 0 5

10

Residue number 0 20 40 60 80

Diff

eren

ce in

de

uter

ium

co

nten

t

0 2 4

Lower H/D rate for mutant, EX2 kinetics for WT; EX1 kinetics for mutant No differences

No H/D exchange

Slower H/D rate for the mutant

Histidine tag

An#MinCDdomain Topologicalspecificitydomain

MTS β1 β2 β3α1

PathwayforconversionofMinEtotheac:veformKTPark,AAr#guesandJLutkenhaus,inpress

Hydrogenexchange–MSanalysisofproteindynamicsinMinE

m/z

200 400 600 800 1000 1200

Inte

nsi

ty (

co

un

ts)

0

200

400

600

800

y2+

y6+

y7+

b72+-NH3

b112+

b152+

z8+

a7+

c62+

x143+ x13

2+

x73+

y93+ -NH3

y5+

y153+

x102+

b102+ +H2O

y142+ +H2O

YYKYILtRnFEALNAR

Abu

ndan

ce (%

)

m/z600 800 1000 1200 1400 1600 1800 2000

0

20

40

60

80

100

+4b

+316y

+5y

+211b

+318b

+212b

+−+ 343 ]3[ POHHM

+7y

+221y

+12y

+230y

+321b

+323y

+216y

+8y

+7b

+8b

+219y

+9y

+330y

+220y

+321b

+10y

+222y

+332b

+223y

+11y

+13y

+228y

+14y

+230b

+232y

+16b

+19y

x5 x5

H Q Q Q pS P V S Q S oM Q T L S D S L S G S S L Y S T S A N L P V oM G H E K

H Q Q Q S P V S Q S M Q T L S D S L S G S S L Y S T S A N L P V oM G H E K

Abu

ndan

ce (%

)

m/z

+12y

+7y

+221y

+230y

+232y

+17a

+13y

+5y

+9y

+8y +

10y

+14y

+19y

+4b

+5b

+213y

+214y

+7b

+325y

+216y+8b

+218y

+221b

+223y

+11y+224y

x5 x5

+336y

+224b

+228y

+230b

+15y +2

33y+16y

+16b

+235y

+236b

600 800 1000 1200 1400 1600 1800 20000

20

40

60

80

100

+219y

+222y

A

B

Identification of a phospho-Serine-574 site on FOXO3. A. The figure shows the CID spectrum of the triple charged ion with m/z of 1353.6, containing a phosphoSer (pS) in position 574 and two oxidized Met (oM) in position 580 and 602, in comparison with (B) the CID spectrum of the non-phosphorylated counterpart peptide with m/z of 1321.6 containing only one oxidized M at position 602. Fragmentation of the phosphorylated peptide results in the loss of 98 Dalton and in the formation of a prominent neutral loss ion ([M+3H-HPO4]3+). The modified residues are indicated by the presence of an unmodified b4

+ for both forms of the peptide, and on the differentially modified y322+ and b11

+ ions, which together identify S574 as a phosphorylation site.

I.  Tikanovich et al Hepatology (2013)

Analysis of protein post-translational modifications

Proteinquan;fica;onviaSILACAlteringO-GlcNAccyclingdisruptsmitochondrialfunc8on

Analysisofproteinpost-transla;onalmodifica;onsIden8fica8onofpSer574onFOXO3

H/Dexchange-MSAnalysisofproteindynamicsinMinE

A)ThefigureshowstheCIDspectrumofthetriplechargedionwith m/z of 1353.6, containing a pS two oxidized, incomparisonwithB) the CID spectrum of the non-phosphorylated counterpartpep;decontainingonlyoneoxidizedM.Fragmenta;on of the phosphorylated pep;de results in thelossof98Daltonandintheforma;onofaprominentneutrallossion([M+3H-HPO4]3+).The arrows point to the differen;ally modified y322+ whichiden;fiesS574asaphosphoryla;onsite.

Proteiniden;fica;onIden8fica8onofCHD4humanprotein

Mitochondrial proteins were immmuniprecipitated using an;-OGlcNAc IgG.Protein iden;fica;onwasperformedfollowingHPLCsepara;ononlinetandemMSontheLTQFT.A)Pep;demassmapofhumanCHD4,pep;desingreenandyellowareiden;fiedataFDRof<0.1%or<0.5%,respec;vely.B) the CID spectrum of a O-Glc_Nacylated pep;de. Color codingindicatesthetypeoffragmention

A. Schemeofsampleprocessing.B. Quan;fica;on of SILAC-labeled total (above) ormitochondrial(below) proteins from OGT (green) - and OGA (light) -overexpressingSY5Ycells.

C. Heatmapshowsproteinsdemonstra;nga>1.5-foldexpressionchangeintheOGT/OGA-overexpressingcellsnormalizedtoGFPcontrolcells.

D. ComponentsoftheelectrontransportchainthatarealteredinOGT-orOGA-overexpressingcells. JTPark,M.Villar,A.Ar8gues,J.Lutkenhaus.(submi`ed)

Z.Zang,M.Villar,A.Ar8guesandCSlawson

E.Tanetal,JBC,2013

I.TikanovichetalHepatology(2013)

m/z

;me

ChromatographicSepara8on(reversed-phase)

Tandemmassspectraof50-150,000pep8des

ProteasedigestionPep,deextrac,on

Nano-HPLC MS/MS

ProteinsequenceDatabase

DatabasesearchingSoBware(SEQUEST)

Output:

• Proteiniden,fica,oninsimple/complexmixtures

• Extensivesequencecoverageandpep,demapping

• Analysisofmodifiedpep,despossible

• Proteinquan,fica,on

Sample

Solware/Searchengines

•  OliverC,HernándezMA,TandbergJI,ValenzuelaKN,LagosLX,HaroRE,SánchezP,RuizPA,Sanhueza-OyarzúnC,CortésMA,VillarMT,Ar;guesA,Winther-LarsenHC,Avendaño-HerreraR,YáñezA.TheProteomeofBiologicallyAc;veMembraneVesiclesfromPisciricke`siasalmonis LF-89 Type Strain Iden;fies Plasmid-Encoded Puta;ve Toxins.. J Front Cell InfectMicrobiol. 2017 Sep 28; 7:420. doi: 10.3389/fcimb.2017.00420.eCollec;on2017.PMID:29034215

•  Park KT, Villar MT, Ar;gues A, Lutkenhaus J. MinE conforma;onal dynamics regulate membrane binding, MinD interac;on, and Minoscilla;on.ProcNatlAcadSciUSA.2017Jul18;114(29):7497-7504.doi:10.1073/pnas.1707385114.Epub2017Jun26.PMID:28652337

•  Jadalannagari S, Converse G, McFall C, Buse E, Filla M, Villar MT, Ar;gues A, Mellot AJ, Wang J, Detamore MS, Hopkins RA, AljitawiDecellularizedWharton'sJellyfromhumanumbilicalcordasanovel3Dscaffoldingmaterialfor;ssueengineeringapplica;ons.OS.PLoSOne. 2017 Feb 21;12(2):e0172098. doi: 10.1371/journal.pone.0172098. eCollec;on 2017. Erratum in: PLoS One. 2017 Mar 7;12(3):e0173827.PMID:2822216

•  NaikS.,iAkkaladeviN.,MachenA.,O’NeilP.,WendyA.DivyaL,Amin,NVillarMT,Ar;guesA,TischerA,AutonMT,BurnsJR,MichaelT.BaldwinMTandFisherMT.SeeingisBelieving:AnalyzingproteincomplexassemblyfromBiolayerInterferometrybiosensorsurfacesusingElectronMicroscopyandMassSpectrometry.JoVE(inpress)

•  RimmerMA,NadeauOW,Ar;guesA,CarlsonGM.Structuralcharacteriza;onofthecataly;cγandregulatoryβsubunitsofphosphorylasekinaseinthecontextofthehexadecamericenzymecomplex..ProteinSci.

•  RimmerMA,NadeauOW,YangJ,Ar;guesA,ZhangY,CarlsonGM.Thestructureofthelargeregulatoryαsubunitofphosphorylasekinaseexaminedbymodelingandhydrogen-deuteriumexchange.ProteinSci.

•  Machen AJ,O’NeilPT,PenteluteBL,VillarMT,Ar;guesA,FisherMT.AnalyzingDynamicProteinComplexesAssembledOnandReleasedFromBiolayerInterferometryBiosensorusingMassSpectrometryandElectronMicroscopy.JoVE(inpress)

Abu

ndan

ce (%

)

m/z600 800 1000 1200 1400 1600 1800 2000

0

20

40

60

80

100

+4b

+316y

+5y

+211b

+318b

+212b

+−+ 343 ]3[ POHHM

+7y

+221y

+12y

+230y

+321b

+323y

+216y

+8y

+7b

+8b

+219y

+9y

+330y

+220y

+321b

+10y

+222y

+332b

+223y

+11y

+13y

+228y

+14y

+230b

+232y

+16b

+19y

x5 x5

H Q Q Q pS P V S Q S oM Q T L S D S L S G S S L Y S T S A N L P V oM G H E K

H Q Q Q S P V S Q S M Q T L S D S L S G S S L Y S T S A N L P V oM G H E K

Abu

ndan

ce (%

)

m/z

+12y

+7y

+221y

+230y

+232y

+17a

+13y

+5y

+9y

+8y +

10y

+14y

+19y

+4b

+5b

+213y

+214y

+7b

+325y

+216y+8b

+218y

+221b

+223y

+11y+224y

x5 x5

+336y

+224b

+228y

+230b

+15y +2

33y+16y

+16b

+235y

+236b

600 800 1000 1200 1400 1600 1800 20000

20

40

60

80

100

+219y

+222y

A

B

Identification of a phospho-Serine-574 site on FOXO3. A. The figure shows the CID spectrum of the triple charged ion with m/z of 1353.6, containing a phosphoSer (pS) in position 574 and two oxidized Met (oM) in position 580 and 602, in comparison with (B) the CID spectrum of the non-phosphorylated counterpart peptide with m/z of 1321.6 containing only one oxidized M at position 602. Fragmentation of the phosphorylated peptide results in the loss of 98 Dalton and in the formation of a prominent neutral loss ion ([M+3H-HPO4]3+). The modified residues are indicated by the presence of an unmodified b4

+ for both forms of the peptide, and on the differentially modified y322+ and b11

+ ions, which together identify S574 as a phosphorylation site.

I.  Tikanovich et al Hepatology (2013)

Analysis of protein post-translational modifications