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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
