LC-MS/MS Analysis of Apical and Basolateral Plasma Membranes of Rat Renal Collecting Duct ...

15 Pages·2006·0.98 MB·English

Research
LC-MS/MS Analysis of Apical and Basolateral
Plasma Membranes of Rat Renal Collecting
Duct Cells*
□
S
Ming-Jiun Yu‡, Trairak Pisitkun‡, Guanghui Wang§, Rong-Fong Shen§,
and Mark A. Knepper‡¶
Weusedbiotinylationandstreptavidinaffinitychromatog- lates several transport proteins including aquaporin 2 (1, 2),
raphy to label and enrich proteins from apical and baso- aquaporin 3 (3), the epithelial sodium channel ENaC1 (4, 5),
lateralmembranesofratkidneyinnermedullarycollecting andtheureatransporterUT-A(6,7).Abnormalitiesofregula-
ducts (IMCDs) prior to LC-MS/MS protein identification. tory processes in the collecting ducts are responsible for a
Toenrichapicalmembraneproteinsandboundperipheral
large number of clinically important disorders of salt and
membrane proteins, IMCDs were perfusion-labeled with
waterbalance(8,9).
primary amine-reactive biotinylation reagents at 2°C us-
The terminal portion of the collecting duct is the IMCD.
ing a double barreled pipette. The perfusion-biotinylated
Investigationofthemechanismsofvasopressinactioninthe
proteins and proteins bound to them were isolated with
IMCDisbenefitingfromanalysisoftheproteomeoftheIMCD
CaptAvidin-agarose beads, separated with SDS-PAGE,
andslicedintocontinuousgelpiecesforLC-MS/MSpro- cells. The work so far has identified a large number of IMCD
tein identification (LTQ, Thermo Electron Corp.). 17 inte- proteins that have been included in a publicly accessible
gral and glycosylphosphatidylinositol (GPI)-linked mem- database, the IMCD Proteome Database (dir.nhlbi.nih.gov/
brane proteins and 44 non-integral membrane proteins papers/lkem/imcd/index.htm).Becausemostoftheproteom-
were identified. Immunofluorescence confocal micros- ics methods used so far (10–13) to construct this database
copy confirmed ACVRL1, H(cid:1)/K(cid:1)-ATPase (cid:2)1, NHE2, and arebiasedagainstintegralmembraneandglycosylphospha-
TauTexpressionintheIMCDs.Basementmembraneand
tidylinositol(GPI)-linkedmembraneproteins,theseclassesof
basolateral membrane proteins were biotinylated via in-
proteinsappearunderrepresented.Thedifficultyindetecting
cubationofIMCDsuspensionswithbiotinylationreagents
membraneproteinshasarisenlargelybecauseofthedifficulty
onice.23integralandGPI-linkedmembraneproteinsand
of solubilizing them in detergents that are compatible with
134 non-integral membrane proteins were identified.
two-dimensional electrophoresis. However, shotgun pro-
Analyses of non-integral membrane proteins preferen-
tiallyidentifiedintheperfusion-biotinylatedandnotinthe teomicsusingLC-MS/MSoffersgreaterefficiencyinidentifi-
incubation-biotinylated IMCDs revealed protein kinases, cation of integral membrane proteins because they can be
scaffold proteins, SNARE proteins, motor proteins, small solubilized using the strong ionic detergent SDS and then
GTP-binding proteins, and related proteins that may be separated on one-dimensional gels prior to trypsinization.
involved in vasopressin-stimulated AQP2, UT-A1, and However,biochemicalapproachesareneededtoisolatespe-
ENaCregulation.AWorldWideWeb-accessibledatabase cificmembranefractionspriortoidentification.Oneobjective
wasconstructedof222membraneproteins(integraland
ofthepresentstudywastodeviseanapproachthatincreases
GPI-linked)fromthisstudyandpriorstudies. Molecular
identification of integral membrane proteins and GPI-linked
&CellularProteomics5:2131–2145,2006.
proteins in plasma membrane domains as well as proteins
thatareboundtointegralmembraneproteins.
Plasma membrane segregation into apical and basolateral
The renal collecting duct is the terminal part of the renal
tubule.Itsmajorfunctionistotransportwaterandsolutesin
aregulatedmanner.Althoughtherearemanyregulatoryfac- 1The abbreviations used are: ENaC, epithelial sodium channel;
torsthataffectcollectingducttransportfunctions,oneofthe ACVRL1, activin A receptor type-II like I; AQP, aquaporin; DAPI,
most important factors is vasopressin, a peptide hormone 4(cid:1),6-diamidino-2-phenylindole; GPI, glycosylphosphatidylinositol;
H(cid:2)/K(cid:2)-ATPase,potassium-transportingATPase;IMCD,innermedul-
secreted by the posterior pituitary gland. Vasopressin regu-
larycollectingduct;Na(cid:2)/K(cid:2)-ATPase,sodium/potassium-transporting
ATPase; NHE2, sodium/hydrogen exchanger 2; SPA-1, signal-in-
From the ‡Laboratory of Kidney and Electrolyte Metabolism and duced proliferation-associated 1-like protein 1; TauT, sodium- and
§Proteomics Core Facility, NHLBI, National Institutes of Health, chloride-dependent taurine transporter; UT-A, urea transporter A;
Bethesda,Maryland20892 Wnt4, wingless-type mouse mammary tumor virus integration site
Received,May15,2006,andinrevisedform,July26,2006 familymember4;SNARE,solubleN-ethylmaleimide-sensitivefactor
Published, MCP Papers in Press, August 9, 2006, DOI 10.1074/ attachment protein receptor; NHS, N-hydroxysuccinimide; FD&C,
mcp.M600177-MCP200 UnitedStatesFederalFood,Drug,andCosmeticAct.
Thispaperisavailableonlineathttp://www.mcponline.org Molecular & Cellular Proteomics 5.11 2131
This is an Open Access article under the CC BY license.
IMCD Plasma Membrane Proteome
domainsatthetightjunctionsprovidesthekeyfunctionalityof brieffixationofthecellmembranelipidswith4%paraformaldehyde
epithelialcells.Discreteproteomesareexpectedintheapical (16)priortobiotinylationpreventedintracellularbiotinylationat2°C
(Fig. 1, C and D). The optimized labeling process took place in the
andthebasolateralmembranestoaccountforstructuraland
following sequence. One barrel delivered the fixative to the IMCD
functionaldifferencesincludinghormoneresponsesandvec-
lumenfor5minfollowedbytheotherbarreldeliveringthebiotinyla-
torial transport across the epithelia. In the kidney, Cutillas et tionreagent(sulfo-NHS-LC-biotin)foranother5min.Afterthebioti-
al. (14) were the first to profile the apical and basolateral nylation step, the solution was switched back to the fixative that
proteomesofrenalcortextissueusingsamplespreparedfrom flushedoutthebiotinylationreagentsandstayedinthelumenwhile
otherductsofBelliniwerebeingperfusion-biotinylated.Onaverage,
differential centrifugation and free flow electrophoresis. Be-
betweensixandeightductsofBelliniofasingleinnermedullawere
cause of the relative abundance of proximal tubules in the
perfusion-biotinylated. Two non-toxic food dyes (FD&C Blue No. 1
renal cortex, the findings from this study are probably appli-
andFD&CRedNo.3)wereusedintheperfusatestovisualizesolution
cable to the proximal tubules but not the collecting ducts. change. Another pipette (single barreled) was situated at the top of
Here we devised methods combining surface biotinylation theperfusionpipettetodripTris-bufferedisolationsolutionontothe
and streptavidin affinity chromatography to label and enrich tissuetomoisturizethetissueandtoquenchthereactiveNHSgroup
of the biotinylation reagents if backflow occurred. After perfusion-
proteins from apical and basolateral membranes of IMCDs
biotinylation,theinnermedullaewereimmediatelyfrozenondryice.
priortoLC-MS/MSproteinidentification.62integralandGPI-
A total of 12 inner medullae from six rats were collected. Some
linked membrane proteins were identified. Subtractive com- non-fixed inner medullae were perfused with sulfo-NHS-SS-biotin.
parison of non-integral membrane proteins identified in the Both fixed and non-fixed perfusion-biotinylated IMCDs were pre-
apicalandnotthebasolateralmembranerevealed25poten- paredforLC-MS/MSanalysisindifferentexperiments.
BasolateralBiotinylationofIMCDs—Tolabelthebasementmem-
tialsignalingandtraffickingproteinsinvolvedinvasopressin-
braneandbasolateralmembraneproteinswithbiotin,anIMCDsus-
regulatedAQP2,UT-A,andENaCregulation.
pensionwaspreparedfromtheexcisedinnermedullaeasdescribed
previously(17).Brieflytheinnermedullaeweremincedanddigested
EXPERIMENTALPROCEDURES with 2 mg/ml hyaluronidase and 3 mg/ml collagenase B. A 60 (cid:3) g
centrifugation was then carried out to precipitate the heavier IMCD
Animals—Pathogen-free male Sprague-Dawley rats (Taconic
segments from the non-IMCD components of the inner medulla
FarmsInc.,Germantown,NY)weremaintainedonadlibitumratchow
(loops of Henle, interstitial cells, vasa recta, and capillaries). The
(NIH-07;Zeigler,Gardners,PA)anddrinkingwaterintheSmallAnimal
isolatedIMCDsuspensionwasfixedwith4%paraformaldehydefor5
Facility, NHLBI, National Institutes of Health. Animal experiments
minonicebeforeincubationwith1.5mg/mlsulfo-NHS-LC-biotinfor
were conducted under the auspices of the animal protocol H-0110
5minonicetolabelselectivelythebasementmembraneandbaso-
approvedbytheAnimalCareandUseCommittee,NHLBI,National
lateral membrane proteins as described previously (18). Another
InstitutesofHealth.Adultanimalsweighingbetween200and250g
IMCD suspension that was not fixed was labeled with 1.5 mg/ml
were injected intraperitoneally with furosemide (5 mg/rat) 20 min
sulfo-NHS-SS-biotinonicefor5min.Bothfixedandnon-fixedincu-
before decapitation and removal of kidneys. Furosemide dissipates
bation-biotinylatedIMCDsuspensionswereusedforLC-MS/MSpro-
the medullary osmolality, thereby preventing osmotic shock to the
teinidentificationindifferentexperiments.
cellsuponisolationoftheinnermedullae(15).Immediatelyafterthe
PlasmaMembrane-enrichedFraction—Toenrichforplasmamem-
innermedullaewereexcisedfromthekidneys,theyweretransferred
brane components of the IMCD cells, a high density membrane
inice-coldisolationsolution(250mMsucrose,10mMTris,pH7.4)to
acoldroom(2°C)forapicalsurfacebiotinylation.Someexcisedinner fraction was prepared using differential centrifugation as described
medullae were used to prepare IMCD suspensions for basolateral previously(3,19).Perfusion-biotinylatedinnermedullaewerehomog-
surfacebiotinylation. enized in liquid nitrogen using a mortar and a pestle. The inner
Perfusion Biotinylation of IMCDs—In the cold room, each inner medullahomogenatewassuspendedinice-coldisolationbuffercon-
medulla was placed on a porous support that allows drainage of tainingproteaseinhibitors(0.1mg/mlPMSFand1(cid:1)g/mlleupeptin)
excessfluidandinbetweentwostacksoffilterpapersthatmoisturize and centrifuged at 1,000 (cid:3) g for 10 min at 4°C to remove incom-
thetissue(Fig.1A).Tointroducebiotinylationreagentstothelumens pletely homogenized fragments and nuclei. The supernatant was
of IMCDs, a double barreled pipette was made from a theta glass collected and centrifuged again at 17,000 (cid:3) g for 20 min. The
capillary(TST150-6;WorldPrecisionInstruments,Inc.,Sarasota,FL). 17,000 (cid:3) g pellet is a high density membrane fraction that was
Thetipofthepipettewasbentcloseto90°anddrawntoaspindle reportedpreviouslytobeenrichedforplasmamembrane(19).
shapewithadiameterof100(cid:1)mtaperingto30(cid:1)matitsopeningto Incubation-biotinylated IMCD suspensions were homogenized in
fittheopeningsoftheIMCDs(ductsofBellini)attheinnermedullary theice-coldisolationbuffercontainingtheproteaseinhibitorsusinga
tip.Thegeometryofthepipettetipwasmadesuchthatthebodyof tissuehomogenizer(TH;OmniInternational,Marietta,GA).Thehigh
thespindlesealstheductofBellinitopreventbackflowoftheper- densitymembranefractionwaspreparedasdescribedabove.
fusedfluid.Twosurfacebiotinylationreagents(thiol-cleavablesulfo- IsolationofBiotinylatedProteins—Whenthenon-cleavablebiotiny-
NHS-SS-biotin or non-cleavable sulfo-NHS-LC-biotin, Pierce) were lationreagentwasused,highdensitymembranefractionswerepre-
usedindifferentexperimentsataconcentrationof1.5mg/mlinPBS pared,andthemembranesweresolubilizedwith1mloflysissolution
(5.1 mM Na2HPO4, 1.2 mM KH2PO4, 154 mM NaCl, pH 7.4). These (150 mM NaCl, 5 mM EDTA, 50 mM Tris, pH 7.4) containing 1%
reagents covalently link biotin to surface proteins through the N- NonidetP-40plusproteaseinhibitors(0.1mg/mlPMSFand1(cid:1)g/ml
hydroxysuccinimide(NHS)groupthatreactswithprimaryaminesat leupeptin). The solubilized membrane fraction was centrifuged at
theN(cid:1)terminiandonsidechainsoflysineresidues.Theyarebelieved 10,000(cid:3)gfor10minat4°Ctoremoveinsolublecomponents.100
tobeexcludedfromthecellinteriorduetotheirnegativelycharged (cid:1)loftheresultingsupernatantwassavedaspreisolationcontrol,and
sulfonategroups.However,ourpreliminaryexperimentsshowedthat 900(cid:1)lofitwasmixedwith200(cid:1)lofsedimentCaptAvidin-agarose
two of these nominally “membrane-impermeant” reagents readily beads(Invitrogen)thatbindbiotinylatedproteins(20).Afterremovalof
enteredtheIMCDcells.Preliminaryexperimentsalsoshowedthata unboundproteins,theCaptAvidin-agarosebeadswerewashedwith
2132 Molecular & Cellular Proteomics 5.11
IMCD Plasma Membrane Proteome
thefollowingsolutions(900(cid:1)lforeachwash)toremovenonspecifi- nofluorescencestainingorRT-PCR.However,allsingle-peptideiden-
cally bound proteins: 1) lysis solution containing 1% Nonidet P-40 tifications that passed manual inspection are presented in Supple-
threetimes,2)highsaltsolution(500mMNaCl,5mMEDTA,50mM mentalTablesS-2,S-4,andS-6.
Tris,pH7.4)twotimes,and3)nosaltsolution(10mMTris,pH7.4)one Immunofluorescence Confocal Microscopy—Adult rats were
time. The biotinylated proteins were eluted from the CaptAvidin- treatedwithfurosemidebeforedecapitationandremovalofthekid-
agarosebeadstwice,eachtimewith45(cid:1)lofalkalinesolution(50mM neys as described above. Kidney slices containing cortex and me-
Na2CO3,pH10.1)plus10mMD-biotin(Invitrogen)thatcompetesthe dullawerefixedwith4%paraformaldehydeovernightandprocessed
biotinbindingsitesontheCaptAvidinmoleculestherebyenhancing using an automated tissue-embedding console system (Tissue-Tek
elutionofbiotinylatedproteins(20). VIPTM5andTissue-TekTECTM5;SakuraFinetek,Torrance,CA)using
When the thiol-cleavable biotinylation reagent was used, the iso- paraffin as the embedding agent. Tissue sections (4 (cid:1)m) were ob-
lation procedures for the biotinylated proteins were similar to those tained using a microtome (RM2125; Leica Microsystems Inc., Ban-
for the non-cleavable biotinylated proteins except 1) radioimmune nockburn, IL). The sections were rehydrated, and antigen retrieval
precipitationassaydetergentsolution(0.1%SDS,0.5%sodiumde- wascarriedoutwithmicrowaveheatfor15mininTEGbuffer(10mM
oxycholate,and1%NonidetP-40)wasusedinthelysisbufferand Trisand0.5mMEGTA,pH9.0).AfterneutralizationwithNH4Clbuffer,
thewashingbuffer,2)streptavidin-agarosebeads(Pierce)wereused, the sections were blocked with 1% BSA, 0.2% gelatin, and 0.05%
and3)theelutionwasdoneviacleavingthedisulfidebondwithlysis saponin in PBS before incubation overnight with primary antibody
buffercontaining100mMDTTfor30minatroomtemperature. dilutedin0.1%BSAand0.3%TritonX-100inPBS.Therabbitand
PreparationofProteinsforMassSpectrometricIdentification—The chicken primary antibody against AQP2 (LL127 and LL265) were
purifiedbiotinylatedproteinswereconcentratedwith10,000-Dacut- generated in our laboratory (1, 22). Other primary antibodies were
off Microcon centrifugal filter devices (YM-10; Millipore Corp., Bed- gifts or commercial products: H(cid:2)/K(cid:2)-ATPase (cid:2)1 (Adam Smolka,
ford, MA) and separated by SDS-PAGE using 10% polyacrylamide MedicalUniversityofSouthCarolina,Charleston,SC),TauT(Russell
minigels (Bio-Rad) to reduce sample complexity. Gels were silver- Chesney,UniversityofTennesseeHealthScienceCenter,Memphis,
stainedusingSilverQuestTM(Invitrogen)orGelCode(Pierce)tovisu- TN),ACVRL1(AbgentInc.,SanDiego,CA),andNHE2(AlphaDiag-
alizetheproteins.Theentiresamplelanewascutinto15–16sequen- nostics International Inc., San Antonio, TX). After rinsing with 0.1%
tial slices of about 2-mm thickness. Proteins in each gel slice were BSA, 0.2% gelatin, and 0.05% saponin in PBS, the sections were
destained,reduced,alkylated,andtrypsin-digestedusingaprotocol incubatedfor1hwithsecondaryantibodydilutedin0.1%BSAand
describedpreviously(21). 0.3% Triton X-100 in PBS. The secondary antibodies used were
LC-MS/MSProteinIdentification—Trypticpeptidesextractedfrom FITC-,Alexa488-,orAlexa568-conjugated(Invitrogen).Afterwashing
eachgelslicewereinjectedusinganAgilent1100nanoflowsystem withPBS,thesectionsweremountedinVectashieldsolutioncontain-
(Agilent Technologies, Palo Alto, CA) into a reversed-phase liquid ing DAPI to stain nuclei (H-1500; Vector Laboratories, Burlingame,
chromatographic column (PicoFritTM, Biobasic C ; New Objective, CA).ConfocalfluorescenceimagesweretakenusingaZeissLSM510
18
Woodburn, MA) to further reduce sample complexity before mass microscopeandsoftware(CarlZeissMicroImaging,Inc.,Thornwood,
analysesusinganLTQmassspectrometer(ThermoElectronCorp., NY).Immunostainingwithoutprimaryantibodyservedasanegative
SanJose,CA)equippedwithananoelectrosprayionsource.Them/z controlandyieldednostaining.
ratiosofpeptidesandtheirfragmentedionswererecordedasspectra Sites of biotinylation in the perfusion- or incubation-biotinylated
bythemassspectrometer.Thespectrawithatotalioncurrentgreater IMCDswereexaminedwithstreptavidin-FITCstainingandconfocal
than 10,000 were used to search for matches to peptides from rat microscopy. Perfusion-biotinylated inner medullae were fixed with
proteins (4,296 entries) in the Swiss-Prot database using the Bio- 4%paraformaldehyde,processed,embedded,andsectionedasde-
works software (Version 3.1; Thermo Electron Corp.) based on the scribed above. After rehydration, the sections were stained with
Sequestalgorithm.Thesearchparametersincluded:1)precursorion streptavidin-FITCfor1h,washedwithPBStwice,andmountedinthe
masstolerancelessthan2amu,2)fragmentionmasstoleranceless Vectashield medium for observation. Incubation-biotinylated IMCDs
than1amu,3)uptothreemissedtrypticcleavagesallowed,and4) werefixedwith4%paraformaldehydefor10min.Afterremovalofthe
amino acid modifications cysteine carboxyamidomethylation (plus fixative, the IMCDs were suspended in 50 (cid:1)l of OCT compound
57.05amu),methionineoxidation(plus15.99amu),andlysinebioti- (Sakura Finetek) and frozen at the bottom of the Cryomold (Sakura
nylation (plus 89.00 amu for residual thiol-cleavable biotin mass or Finetek).AdditionalOCTcompoundwasgraduallyaddedtothefro-
plus 339.00 amu for non-cleavable biotin mass). Matched peptide zen IMCD suspension to make the IMCD suspension block. 4-(cid:1)m
sequences must pass the following filters for provisional identifica- sections were obtained from the block using a Leica CM3050 S
tion:1)thecross-correlationscores(Xcorr)ofmatchesweregreater cryostat. The sections were fixed with (cid:6)20°C methanol for 5 min,
than 1.5, 2.0, and 2.5 for charged state 1, 2, and 3 peptide ions, dried, washed with PBS twice for 5 min each, and stained with
respectively,2)theuniquenessscoresofmatches((cid:4)Cn)werehigher streptavidin-FITCfor1h.AfterawashwithPBS,thesectionswere
than0.08,and3)theranksoftheprimaryscores(Rsp)werelessthan mountedintheVectashieldmediumforobservation.
10.Toassesstheoverallqualityofpeptidematches,thesamespec- RT-PCR—IMCDsuspensionswerepreparedasdescribedabove.
trawereusedtosearchforrandommatchesusingthesamesearch- Total RNA of the IMCD suspensions was extracted using TRIzol(cid:2)
ingparameters,filtersettings,anddatabaseexceptthesequencesin reagent(Invitrogen).FirststrandcDNAwasproducedfromthetotal
the database were reversed. The random match results served as RNA extract (5 (cid:1)g) using an oligo(dT) primer and SuperScriptTM
references for accepting spectra after manual examinations. For reagents(Invitrogen)inareactionvolumeof20(cid:1)l.PCRproductwas
those peptides that passed the filters, the spectra with the highest generated from 0.5 (cid:1)l of the first strand cDNA mixture using gene-
Xcorr were manually examined to confirm protein identification. 38 specificprimers(SupplementalTableS-3)andtheImmoMix(Bioline
examples of the accepted spectra are listed in Supplemental Table USA Inc., Randolph, MA). The PCR was performed on a Peltier
S-1.Mostofthesespectraareselectedfromthefixedperfusion-bio- PCT-200thermalcycler(MJResearch,Waltham,MA)withthefollow-
tinylatedIMCDs(n(cid:5)30).Regardlessofthismanualspectruminspec- ingconditions:activationoftheDNApolymeraseat95°Cfor7min;
tion,theonlyproteinsthatarepresentedunder“Results”arethose 30cyclesof94°Cfor30s,58°Cfor30s,and72°Cfor30s;anda
with high quality spectra for two or more distinct peptide ions or final extension at 72°C for 10 min. The RT-PCR products were
single-peptideidentificationsthatwereconfirmedwitheitherimmu- resolved with 1.6% agarose gel electrophoresis, stained with
Molecular & Cellular Proteomics 5.11 2133
IMCD Plasma Membrane Proteome
FIG.1. Apical surface biotinylation
viaretrogradeperfusionofIMCDs.A,
a rat kidney medulla was excised and
placedonaporoussupportthatallows
fluid drainage and in between filter pa-
pers that moisturize the tissue. Apical
membraneproteinswerelabeledwithbi-
otinviaperfusingtheIMCDlumensusing
acustom-madedoublebarreledpipette
in a cold room (2°C) to inhibit endocy-
tosis. One barrel delivered paraformal-
dehydetofixthemembranelipidsbefore
theotherbarreldeliveredsulfo-NHS-LC-
biotin or sulfo-NHS-SS-biotin to label
theapicalmembraneproteins.Thefixa-
tionoflipidspriortobiotinylationofpro-
teins was necessary to ensure apical
protein biotinylation. Blue and red food
dyes were used in the perfusates to vi-
sualizefluidchangeintheIMCDs.Asin-
gle barreled pipette at the top of the
perfusionpipettesuppliedTrisbufferto
moisturizethetissueandtoquenchthe
biotinylation reagent if backflow oc-
curred.B,apictureshowstheperfusion
setup in the cold room. C, a fluores-
cence micrograph shows biotinylation
occurringthroughouttheperfusedIMCD
cells without prior fixation revealed by
streptavidin-FITC that stains biotin in
green.TheIMCDmarkerAQP2staining
andtheDAPInuclearstainingareshown
in red and blue, respectively. D, a fluo-
rescence micrograph shows restricted
apicalmembranebiotinylationintheper-
fusedIMCDswithpriorfixation.
ethidium bromide, and visualized using the Gel Logic 100 imaging vidin-FITC revealed that the reagent entered the IMCD cells,
system(EastmanKodakCo.).
resulting in diffuse biotinylation distributed throughout the
cells (Fig. 1C, green). Another similar biotinylation reagent
RESULTS
sulfo-NHS-LC-biotin yielded the same results (data not
EntryofBiotinylationReagentsintoIMCDCells—Thestrat-
shown), indicating a common problem with these reagents.
egy used in this study was to use surface biotinylation and
The biotinylation, however, was restricted to the perfused
streptavidinchromatographytolabelandenrichproteinsin
IMCDcells(comparewithdistributionofacollectingductcell
the apical or basolateral plasma membrane and then to
proteinAQP2,red).
identifythemusingcombinedLC-MS/MS.Thestrategywas
As shown in Fig. 1D, when the IMCDs were first perfused
designed to identify integral membrane proteins and GPI-
with a fixative (4% paraformaldehyde) to fix the lipid and
linked proteins as well as peripheral membrane proteins
protein components of the cells (16), the biotinylation was
bound to the labeled proteins. To enrich proteins of the
limited to the apical plasma membrane. Thus, the technique
apical plasma membrane, we perfused rat IMCDs from the
can be used to label all proteins in the IMCD cells if used
ducts of Bellini at the tip of isolated renal medullae with
without prior fixation or to selectively label apical plasma
biotinylation reagents (Fig. 1, A and B). Perfusion from a
singleductofBelliniresultedinlabelingofalargenumberof membrane proteins if used with prior fixation. Because the
IMCDs because of the merging organization of the collect- non-ionic detergents used in the affinity isolation of biotiny-
ing ducts in the inner medulla. Although the biotinylation lated proteins do not in general disrupt protein-protein bind-
reagent sulfo-NHS-SS-biotin chosen for this task is classi- inginteractions,thistechniqueallowsisolationofbothbiotin-
fied as a membrane-impermeant form and the tissue proc- ylated proteins and other proteins that are bound in
essingandperfusionwasdoneat2°C,stainingwithstrepta- complexeswiththebiotinylatedproteins.
2134 Molecular & Cellular Proteomics 5.11
IMCD Plasma Membrane Proteome
FIG.2.A,aflowchartsummarizingthe
isolation, preparation, and identification
ofproteinsinthefixedperfusion-biotin-
ylatedIMCDs.B,asilver-stainedimage
showing the isolation processes of bio-
tinylated proteins and their associated
proteins in the fixed perfusion-biotiny-
lated IMCDs. The total membrane frac-
tion(laneT)waspreparedandboundto
theCaptAvidin-agarosebeads.Afterre-
moval of the unbound (U) and the non-
specifically bound proteins in three
washing buffers (W ), the biotinylated
1–3
proteins and their associated proteins
wereeluted(E)fromtheCaptAvidin-aga-
rose beads. C, a silver-stained image
showing how the biotinylated proteins
andtheirassociatedproteinsweresep-
arated with SDS-PAGE and sliced into
16pieces(numbersontheleft)toreduce
sample complexity prior to preparation
for LC-MS/MS protein identification.
LaneM,molecularmassmarkers.
Proteomics Analyses of Fixed Perfusion-biotinylated Prot database and Bioworks software with default settings
IMCDs—Fig. 2A shows the purification process of biotiny- (see “Experimental Procedures”). As shown, most identifica-
latedproteinsfromthefixedperfusion-biotinylatedIMCDs.In tions with these settings would be based on matches of a
general,weperfusedbetweensixandeightductsofBelliniin singletrypticpeptide.Animportantobjectiveinproteinmass
each inner medulla of a total between 10 and 12. Fig. 2B spectrometry is to devise approaches that eliminate or mini-
shows a silver-stained gel image of the proteins prepared mizefalsepositiveidentifications.Towardthisend,wecarried
from the fixed perfusion-biotinylated IMCDs, indicating that out computational control experiments in which the same
the final eluate (lane E) from the CaptAvidin-agarose beads spectra were analyzed in exactly the same way except that
containedbutaverysmallfractionofthetotalproteinpresent thedatabaseelementsconsistedofreversedsequencesfrom
intheoriginalsample(laneT).Fig.2Cshowsasilver-stained the Swiss-Prot database. The number of proteins putatively
gelimageoftheconcentratedeluate(laneE),indicatinghow identifiedinthiscontrolexperimentissummarizedinthethird
the gel was cut into 16 slices (slice numbers are on the left) column of Table I labeled “Reversed database search.” As
prior to in-gel trypsinization and protein identification using canbeseen,therewasahighrateofputative“identifications”
LC-MS/MS. derived from randomly matched sequences. The fourth col-
Table I summarizes the number of proteins identified by umnofTableIlabeled“Netdifference”showsthenumbersin
LC-MS/MSfromallslicesaddedtogether.Thefirstcolumnof thesecondcolumnsubtractedfromthenumbersinthethird
Table I indicates the number of peptides that were identified column. The comparison indicates that, for identifications
forparticularproteins.ThesecondcolumnofTableI(labeled based on one- or two-peptide matches, a relatively small
“Originaldatabasesearch”)indicatesthenumberofproteins fraction is likely to be valid based only on computerized
identified by a standard computer search using the Swiss- identification. Therefore, to efficiently eliminate false positive
Molecular & Cellular Proteomics 5.11 2135
IMCD Plasma Membrane Proteome
TABLEI
Number of proteins identified from the fixed perfusion-biotinylated
innermedullarycollectingducts
Original Reversed Manually
Peptide Net
database database accepted
numbera differenced
searchb searchc identificatione
1 378 263 115 75
2 39 24 15 21
3 12 3 9 8
4 5 1 4 4
(cid:7)4 4 0 4 3
Total 438 291 147 111
aNumberofpeptidesidentifiedforparticularproteins.
bNumberofproteinspassingthedefaultcriteriaoftheBioworks/
SequestsearchalgorithmusingtheSwiss-Protdatabase.
cSameasFootnotebexceptthesequencesinthedatabasewere
reversed.
dEstimatednumberofproteinslikelytobevalididentifications.
eNumberofproteinspassingmanualspectruminspection.
FIG.4.RT-PCRconfirmationofmRNAexpressionofone-pep-
tideidentificationsintheIMCDs.PCRwithpriorRTreaction(lane
(cid:2))usingAQP2,anIMCDmarkerprotein,specificprimersservedasa
positivecontrol.PCRwithoutreversetranscriptionreaction(lane(cid:6))
servedasthenegativecontrols.Parenthesesindicatesamplesourc-
es: FPB, fixed perfusion-biotinylated IMCDs; FIB, fixed incubation-
biotinylated IMCDs; NPB, non-fixed perfusion-biotinylated IMCDs;
NIB,non-fixedincubation-biotinylatedIMCDs.ACE,angiotensin-con-
vertingenzyme;APC,adenomatouspolyposiscoliprotein;CA4,car-
bonicanhydrase4;CD40L,CD40ligand;CLIC4,chlorideintracellular
channelprotein4;ERC2,ERCprotein2;FZD1,frizzled1precursor;
GPR64, G-protein coupled receptor 64 precursor; GRIN2D, gluta-
mate (N-methyl-D-aspartate) receptor subunit (cid:3)4 precursor; HCN1,
potassium/sodium hyperpolarization-activated cyclic nucleotide-
gatedchannel1;Kv4.3,potassiumvoltage-gatedchannelsubfamily
D member 3; LPL, lipoprotein lipase; LRP4, low density lipoprotein
receptor-related protein 4; MAPK12, mitogen-activated protein ki-
nase 12; MCT2, monocarboxylate transporter 2; PalmT, palmitoyl-
transferase ZDHHC7; PIK3-C2(cid:4), phosphatidylinositol-4-phosphate
3-kinase C2 domain-containing (cid:4)polypeptide; PIK3-C(cid:5), phospha-
FIG.3. Confocal immunofluorescence microscopy confirming tidylinositol-4,5-bisphosphate 3-kinase catalytic subunit (cid:5)isoform;
theexpressionofproteinsidentifiedfromIMCDs.ActivinArecep- PKA-(cid:5)C, cAMP-dependent protein kinase, (cid:5)-catalytic subunit;
tor type II-like I, potassium-transporting ATPase (cid:2)1, sodium/hydro- PODXL, podocalyxin; ROMK, ATP-sensitive inward rectifier potas-
genexchanger2,andsodium-andchloride-dependenttaurinetrans- sium channel 1; S1P-R, sphingosine-1-phosphate receptor Edg-8;
porter in the IMCDs are visualized with secondary antibodies SCN5A, sodium channel protein type 5 (cid:2)subunit; SNAP29, synap-
conjugated to FITC or Alexa488 (green). These proteins co-localize tosomal-associatedprotein29;THIK-1,potassiumchannelsubfamily
withIMCDmarkerproteinaquaporin2(red,Alexa568),indicatingtheir K member 13; TYRO3, tyrosine-protein kinase receptor TYRO3;
expressionintheIMCDs.ThenucleiarestainedwithDAPI(blue). VAPA, vesicle-associated membrane protein-associated protein A;
VK(cid:4)C,vitaminK-dependentgamma-carboxylase;WASPIP,Wiskott-
identifications, we manually examined all spectra to be in- Aldrichsyndromeproteininteractingprotein.
cluded in the results reported in this study (last column la-
beled“Manuallyacceptedidentification”).Criteriafortheac- peptide identifications, we required positive results for either
ceptance are described under “Experimental Procedures.” immunofluorescence staining or RT-PCR to confirm the ex-
Because many of these potential identifications were single- pression of these proteins (Supplemental Table S-1). How-
2136 Molecular & Cellular Proteomics 5.11
IMCD Plasma Membrane Proteome
TABLEII
IntegralandGPI-linkedmembraneproteinsidentifiedfromthefixedperfusion-biotinylatedinnermedullarycollectingducts
Gene Accession Ion
Proteinname Confirmedb CDDBc Typed
name no. (n)a
AminopeptidaseN ANPEP P15684 2(4) Aminopeptidases II
Angiotensin-convertingenzyme,somaticisoform ACE P47820 1(2) RT-PCR Signalingproteins I
precursor
ATP-bindingcassettesubfamilyAmember2 ABCA2 Q9ESR9 2(3) Transportersandchannels III
Chlorideintracellularchannelprotein4 CLIC4 Q9Z0W7 1(1) RT-PCR Transportersandchannels III
G-protein-coupledreceptor64precursor GPR64 Q8CJ11 1(1) RT-PCR Membranereceptors III
Lipoproteinlipaseprecursor LPL Q06000 1(1) RT-PCR Metabolismproteins GPI
Lowdensitylipoproteinreceptor-relatedprotein4 LRP4 Q9QYP1 1(1) RT-PCR Membranereceptors I
PalmitoyltransferaseZDHHC7 ZDHHC7 Q923G5 1(1) RT-PCR Biosyntheticproteins III
PotassiumchannelsubfamilyKmember13,THIK-1 KCNK13 Q9ERS0 1(1) RT-PCR Transportersandchannels III
Potassium-transportingATPase(cid:2)chain1 ATP4A P09626 1(14) IF Ionpumps III
Serine/threonine-proteinkinasereceptorR3 ACVRL1 P80203 2(5) Membranereceptors I
precursor
Sodium-andchloride-dependenttaurine SLC6A6 P31643 1(3) IF Transportersandchannels III
transporter
Sodium/hydrogenexchanger2 SLC9A2 P48763 1(1) IF Transportersandchannels III
Syntaxin-8 STX8 Q9Z2Q7 2(2) Membranetraffickingproteins II
Transmembraneprotease,serine9 TMPRSS9 P69526 2(6) Metabolismproteins II
Tyrosine-proteinkinasereceptorTYRO3precursor TYRO3 P55146 1(1) RT-PCR Membranereceptors I
VitaminK-dependent(cid:4)-carboxylase GGCX O88496 1(1) RT-PCR Biosyntheticproteins III
aNumberofpeptidesidentifiedwiththeirtotalhitnumberinparentheses.
bProteinidentificationsconfirmedbyimmunofluorescence(IF)orRT-PCR.
cCollectingductdatabase(CDDB)familygroupnames.
dMembrane protein types: I, one membrane-spanning helix with an extracellular N(cid:1) terminus; II, one membrane-spanning helix with an
extracellularC(cid:1)terminus;III,multiplemembrane-spanninghelices;GPI,glycosylphosphatidylinositol-linked.
ever,forcompleteness,allmanuallyinspectedsingle-peptide sion-biotinylated IMCDs, consistent with the relative lack of
identificationsarereportedinSupplementalTableS-2. externallysinesinthisprotein(see“Discussion”).
Fig. 3 shows immunofluorescence localization in the renal Proteomics Analyses of Fixed Incubation-biotinylated
inner medulla of four of the proteins identified in the fixed IMCDs—We next performed biotinylation in isolated IMCD
perfusion-biotinylated IMCDs for which antibodies could be segments in suspension to selectively label proteins in the
obtained,viz.ACVRL1,H(cid:2)/K(cid:2)-ATPase(cid:2)1,NHE2,andTauT. basement membrane and the basolateral membrane prior to
ACVRL1wasidentifiedwithtwopeptides,andtheotherthree CaptAvidin affinity protein isolation and LC-MS/MS protein
proteinswereidentifiedwithonepeptide.Eachwasfoundto identification.TheIMCDswereseparatedfromthenon-IMCD
co-localizewithAQP2,acollectingductmarkerprotein,con- structures (loops of Henle and vasculature) of the inner me-
firmingtheirpresenceintheIMCD.Forproteinidentifications dullae via enzymatic digestion and low speed centrifugation
thatlackavailableantibodies,RT-PCRwasusedtoexamine thatprecipitatestheheavierIMCDs(3,19).Afterisolation,the
theirmRNAexpressionintheisolatedIMCDsuspensions.As IMCD cell integrity was confirmed by the lack of trypan blue
shown in Fig. 4, 27 primer pairs designed for the single- permeation(datanotshown).TheIMCDpuritywasassessed
peptide identifications from the fixed perfusion-biotinylated by immunoblotting (Fig. 5A). As seen, the IMCD suspension
IMCDs yielded 17 positive RT-PCR products from the IMCD (lane I) was enriched in the IMCD marker protein AQP2 and
suspension (lane (cid:2)) with 30 cycles of amplification. AQP2 depleted of the non-IMCD marker protein AQP1 compared
expressionservedasapositivecontrolfortheRT-PCR.PCR with the whole inner medulla homogenate (lane W) and the
without prior RT reaction serves as negative controls and non-IMCDfraction(laneN).WhentheisolatedIMCDsuspen-
yieldednoPCRproducts(lane(cid:6)).Togethertheimmunofluo- sionwasincubatedwiththebiotinylationreagentsulfo-NHS-
rescence staining and RT-PCR confirmed 20 identifications of SS-biotin without prior fixation, biotinylation occurred chiefly
the30selectedmanuallyacceptedone-peptideidentifications. at the basement membrane and the basolateral membrane
Table II lists 17 integral or GPI-linked membrane proteins (Fig. 5B, green). For comparison, another IMCD suspension
thatwereidentifiedwithtwoormorepeptidesorwithasingle was fixed with 4% paraformaldehyde and biotinylated with
peptide and confirmed by immunofluorescence staining or sulfo-NHS-LC-biotin. Again the biotinylation occurred at the
RT-PCR.TypeIIImultiplemembrane-spanninghelixproteins basement membrane and the basolateral membranes of the
dominate the list. Note that the hallmark IMCD apical mem- IMCDsuspension(Fig.5C,green).Themembranefractionof
brane protein AQP2 (1) was not identified in the fixed perfu- thefixedincubation-biotinylatedIMCDsuspensionwasused
Molecular & Cellular Proteomics 5.11 2137
IMCD Plasma Membrane Proteome
TABLEIII
Number of proteins identified from the fixed incubation-biotinylated
innermedullarycollectingducts
Original Reversed Manually
Peptide Net
database database accepted
numbera differenced
searchb searchc identificatione
1 121 25 96 66
2 53 0 53 44
3 26 0 26 26
4 25 0 25 25
(cid:7)4 50 0 50 49
Total 275 25 250 210
aNumberofpeptidesidentifiedforparticularproteins.
bNumberofproteinspassingthedefaultcriteriaoftheBioworks/
SequestsearchalgorithmusingtheSwiss-Protdatabase.
cSameasFootnotebexceptthesequencesinthedatabasewere
reversed.
dEstimatednumberofproteinslikelytobevalididentifications.
eNumberofproteinspassingmanualspectruminspection.
teins, the discrete biotinylation of the apical (Fig. 1D) versus
thebasolateral(Fig.5C)membraneproteinsallowsidentifica-
FIG.5.BasolateralsurfacebiotinylationofisolatedIMCDsus- tion of the non-integral membrane proteins from respective
pensions.A,animmunoblotshowsthattheIMCDsuspensions(I)are
membraneproteindomains.Apicallyassociatednon-integral
enriched in the IMCD marker protein AQP2 and depleted of the
membraneproteinsarethoseidentifiedinthefixedperfusion-
non-IMCDmarkerproteinAQP1comparedwiththewholeinnerme-
dullahomogenate(W)andthenon-IMCDsuspensions(N).BandC, biotinylated IMCDs. Basolaterally associated non-integral
fluorescencemicrographsshowthatbiotinylationoccursatthebase- membrane proteins are those identified in the fixed incuba-
mentmembraneandthebasolateralmembraneoftheisolatedIMCD tion-biotinylated IMCD suspension. Fig. 6 shows a general
segmentsinsuspensionswithout(B)orwithfixation(C)asrevealed
comparison of proteins from the two samples identified with
by streptavidin-FITC that stains the sites of biotinylation in green.
twoormorepeptidesorwithasinglepeptideandconfirmed
AQP2antibodyidentifiesIMCDsegments(red,Alexa568)andDAPI
stainsnuclei(blue). withRT-PCR.Asseen(Fig.6),themajorityofthenon-integral
membrane proteins identified in the fixed perfusion-biotiny-
forLC-MS/MSproteinidentificationfollowingthesameprep- lated IMCDs (25 of 44 or 57%) do not overlap with those
aration procedures used for the fixed perfusion-biotinylated identified in the fixed incubation-biotinylated IMCD suspen-
IMCDsample(Fig.2A). sion.These25non-integralmembraneproteinsidentifiedex-
Table III summarizes the numbers of proteins identified in clusivelyinthefixedperfusion-biotinylatedIMCDsarelistedin
the fixed incubation-biotinylated IMCDs. The computational Table V and include a number of proteins involved in cell
controlexperimentindicatesthelikelihoodofalargenumber signaling, vesicular trafficking, and cytoskeletal organization.
of random protein identifications for low numbers of peptide They can be considered potential components of the vaso-
identificationsperprotein.Consequentlyweagaincarriedout pressin-regulated apical trafficking mechanisms. However,
manualinspectionofallspectratoascertainwhichidentifica- this subtractive comparison of the non-integral membrane
tionswerevalid.Atotalof210identifiedproteinsarelistedin proteins between the fixed perfusion- and incubation-biotin-
Supplemental Table S-4. Three one-peptide protein identifi- ylated IMCDs cannot be used to exclude the existence of a
cationswereexaminedwithRT-PCRforexpressionatmRNA particular protein in either apical or basolateral membrane
level(Fig.4).23integralmembraneproteinsthatwereidenti- domainsduetothelimitedsensitivityofthemethods.
fiedwithtwoormorepeptidesorwithonesinglepeptideand Proteomics Analyses of Non-fixed Perfusion- and Incuba-
confirmed with RT-PCR are presented in Table IV. Type III tion-biotinylated IMCDs—Without fixation, biotinylation oc-
multiple membrane-spanning helix proteins again dominate cursthroughouttheIMCDcellsbutneverthelessislimitedto
the membrane protein identifications. Note that the IMCD IMCD cells (Fig. 1C) and therefore provides a means for
basolateralmembraneproteinAQP4(23)wasnotidentifiedin general identification of IMCD proteins. Similarly the high
thefixedincubation-biotinylatedIMCDsuspensionduetothe purity of the isolated IMCD suspension also permits valid
lackofexternallysineresidues(see“Discussion”). IMCD protein identifications (Fig. 5A). Therefore, samples
Analyses of Non-integral Membrane Proteins Identified in fromthenon-fixedperfusion-andincubation-biotinylated(us-
theFixedPerfusion-biotinylatedandNotintheIncubation-bio- ingsulfo-NHS-SS-biotin)wereisolatedwithstreptavidin-aga-
tinylated IMCDs—Because our strategy allows identification rose affinity chromatography and analyzed with mass spec-
ofintegralaswellasassociatednon-integralmembranepro- trometry. The computational control experiments and the
2138 Molecular & Cellular Proteomics 5.11
IMCD Plasma Membrane Proteome
TABLEIV
Integralmembraneproteinsidentifiedfromthefixedincubation-biotinylatedinnermedullarycollectingducts
Gene Accession
Proteinname Ion(n)a Confirmedb CDDBc Typed
name no.
ADP/ATPtranslocase1 SLC25A4 Q05962 2(2) Transportersandchannels III
ADP/ATPtranslocase2 SLC25A5 Q09073 4(16) Transportersandchannels III
Agrinprecursor AGRN P25304 4(20) Adhesionproteinsandcollagens II
Calnexinprecursor CANX P35565 13(27) Chaperones I
CD166antigenprecursor ALCAM O35112 1(2) RT-PCR Adhesionproteinsandcollagens I
Cytochromecoxidasesubunit4isoform1, COX4I1 P10888 3(4) Metabolismproteins II
mitochondrialprecursor
Dolichyl-diphosphooligosaccharide-protein RPN2 P25235 5(8) Biosyntheticproteins III
glycosyltransferase63-kDasubunitprecursor
Dolichyl-diphosphooligosaccharide-protein RPN1 P07153 5(8) Biosyntheticproteins III
glycosyltransferase67-kDasubunitprecursor
Epithelialcadherinprecursor CDH1 Q9R0T4 8(16) Adhesionproteinsandcollagens I
Frizzled1precursor FZD1 Q08463 1(1) RT-PCR Membranereceptors III
Golgiapparatusprotein1precursor GLG1 Q62638 2(4) Adhesionproteinsandcollagens I
Insulin-likegrowthfactor1receptorprecursor IGF1R P24062 3(4) Membranereceptors I
Integrin(cid:2)1precursor ITGA1 P18614 2(2) Adhesionproteinsandcollagens I
Integrin(cid:5)1precursor ITGB1 P49134 24(54) Adhesionproteinsandcollagens I
NeuralcelladhesionmoleculeL1precursor L1CAM Q05695 2(2) Adhesionproteinsandcollagens I
Neuropilin-1precursor NRP1 Q9QWJ9 2(2) Membranereceptors I
Phosphatecarrierprotein,mitochondrialprecursor SLC25A3 P16036 2(5) Transportersandchannels III
Podocalyxinprecursor PODXL Q9WTQ2 1(1) RT-PCR Adhesionproteinsandcollagens I
ProbableG-proteincoupledreceptor116 GPR116 Q9WVT0 2(4) Membranereceptors III
precursor
Sodium/potassium-transportingATPase(cid:2)-1chain ATP1A1 P06685 26(134) Ionpumps III
precursor
Sodium/potassium-transportingATPase(cid:2)-3chain ATP1A3 P06687 15(63) Ionpumps III
Sodium/potassium-transportingATPase(cid:5)-1chain ATP1B1 P07340 6(14) Ionpumps II
Voltage-dependentanion-selectivechannel VDAC2 P81155 4(8) Transportersandchannels III
protein2
aNumberofpeptidesidentifiedwiththeirtotalhitnumberintheparentheses.
bProteinidentificationsconfirmedbyRT-PCR.
cCollectingDuctDatabase(CDDB)familygroupnames.
dMembrane protein types: I, one membrane-spanning helix with an extracellular N(cid:1) terminus; II, one membrane-spanning helix with an
extracellularC(cid:1)terminus;III,multiplemembrane-spanninghelices;GPI,glycosylphosphatidylinositol-linked.
resultsofmanualinspectionaresummarizedinSupplemental
Table S-5. Specific proteins from non-fixed perfusion- and
incubation-biotinylatedIMCDsarecombinedinSupplemental
TableS-6.34integralandGPI-linkedmembraneproteinsthat
were identified with two or more peptides or with a single
peptideandconfirmedwithRT-PCR(Fig.4)arepresentedin
TableVI.TypeIIImembraneproteinsareagainthedominant
membrane proteins. Note that six GPI-linked proteins were
identifiedinthenon-fixedperfusion-biotinylatedIMCDs.Also
the perfusion-biotinylation without prior fixation led to the
identifications of apical (AQP2) and basolateral membrane
proteins (AQP4, barttin, and E-cadherin), indicating the entry
ofthebiotinylationreagents.Likewisetheincubation-biotiny-
lation without prior fixation led to the identification of the
apical(AQP2)andbasolateral(AQP4)membraneproteins.
FIG.6. A Venn diagram summarizing the non-integral mem- IMCD Membrane Protein Database—Overall we identified
braneproteins(non-MPs)identifiedinthefixedperfusion-biotin- 62 integral and GPI-linked IMCD membrane proteins in this
ylated (FPB) IMCD and those identified in the fixed incubation-
study. These proteins together with previously identified
biotinylated (FIB) IMCD suspension. Numbers indicate protein
membrane proteins (n (cid:5) 187) were used to populate a new
identifications. Percentages indicate non-overlapping identifications
inaparticularsamplepreparation. IMCD Membrane Protein (IMP) Database (dir.nhlbi.nih.gov/
Molecular & Cellular Proteomics 5.11 2139
IMCD Plasma Membrane Proteome
TABLEV
Non-integralmembraneproteinsidentifiedinthefixedperfusion-biotinylatedandnotinthe
fixedincubation-biotinylatedinnermedullarycollectingducts
Gene Accession Ion
Proteinnamea Confirmedc Functionalremark
name no. (n)b
Alcoholdehydrogenase AKR1A1 P51635 2(2) Metabolismproteins.
(cid:2) -Antiproteinaseprecursor SERPINA1 P17475 10(12) Secretedprotein.
1
Bassoonprotein BSN O88778 2(4) Scaffoldprotein;myristoylated;formscomplexwithERC2;
similartopiccolo;membrane-associated;organizationof
thecytomatrixatthenerveterminalactivezone.
Calcyclin S100A6 P05964 1(1) RT-PCR Signalingprotein;regulatedbyvasopressin(41);bindsto
prolactinreceptor;bindstoannexins.
cAMP-dependentproteinkinase, PRKACB P68182 1(3) RT-PCR Proteinkinase,serine/threonine;maybemembrane-
(cid:5)-catalyticsubunit associatedviaAkinaseanchoringproteins.
Creatinekinase,sarcomeric CKMT2 P09605 3(12) Metabolismprotein;thoughttobefoundonlyinheartand
mitochondrialprecursor skeletalmuscles.
Glandularkallikrein-7, KLK7 P36373 2(4) Proteindegradationpathway;predominantkallikrein
submandibular/renalprecursor proteininthekidney;membrane-associatedand
secretedprotein.
Glialfibrillaryacidicprotein, GFAP P47819 3(77) Cytoskeletalproteins.
astrocyte
Hermansky-Pudlaksyndrome6 HPS6 Q7M733 2(2) Miscellaneousproteins.
proteinhomolog
Myosin-5A MYO5A Q9QYF3 3(10) Motorprotein;processiveactin-basedmotor;membrane-
associated;involvedinvesicletransportalongactin
filaments.
Nitric-oxidesynthase1,brain NOS1 P29476 3(10) Signalingprotein;producesNOfromarginine;stimulated
bycalcium/calmodulin.
PDZdomain-containingRING PDZRN3 P68907 3(4) Miscellaneousproteins.
fingerprotein3
Peroxiredoxin-2 PRDX2 P35704 2(2) Stressprotein;redoxregulationincell;roleineliminating
peroxidesgeneratedduringmetabolism.
Phosphatidylinositol-4,5- PIK3CB Q9Z1L0 1(1) RT-PCR Phosphoinositide3-kinase;peripheralmembraneprotein.
bisphosphate3-kinase
catalyticsubunit(cid:5)isoform
Piccoloprotein PCLO Q9JKS6 2(8) Scaffoldprotein;similartobassoon;
membrane-associated;organizationofthecytomatrixat
thenerveterminalactivezone;bindsprofilin.
Propionyl-CoAcarboxylase(cid:2) PCCA P14882 6(11) Metabolismprotein;keyenzymeinthecatabolicpathway
chain,mitochondrialprecursor ofodd-chainfattyacids,isoleucine,threonine,
methionine,andvaline.
RabGTPase-bindingeffector RABEP1 O35550 1(2) RT-PCR SmallGTP-bindingprotein-relatedprotein;Rabeffector
protein1 proteinactingaslinkerbetween(cid:4)-adaptin,Rab4A,and
Rab5A;involvedinendocyticmembranefusionand
membranetraffickingofrecyclingendosomes;stimulates
RabGEF1-mediatednucleotideexchangeonRab5A.
Ras-relatedproteinRab-31 RAB31 Q6GQP4 1(4) RT-PCR SmallGTP-bindingproteins.
Septin-9 SEPT9 Q9QZR6 1(4) RT-PCR Scaffoldprotein;bindsGTP;associateswithcell
membranesandtheactinandmicrotubule
cytoskeletons;polymerizetoformfilamentousstructures
undertheinfluenceofRhoandCDC42;regulatedbyphos-
phorylation;associatewithSNAREcomplexes(43).
SPA-1 SIPA1L1 O35412 2(2) SmallGTP-bindingprotein-relatedprotein;Rap1GAP;
implicatedinregulationofAQP2trafficking(44).
Synaptosomal-associated SNAP29 Q9Z2P6 1(1) RT-PCR SNAREprotein.
protein29
Synaptotagmin-likeprotein4 SYTL4 Q8VHQ7 3(9) SmallGTP-bindingprotein-relatedprotein;peripheral
(granuphilin) membraneprotein;partofaternarycomplexcontaining
STX1AandRAB27A;interactswithsyntaxinsinmunc18-
depenedentmanner;mayplayaroleinvesicle
trafficking.
Timelesshomolog TIMELESS Q9Z2Y1 2(2) Nuclearprotein;playaroleinepithelialmorphogenesis
duringkidneydevelopmentandbranchingoftubules
(45).
Wiskott-Aldrichsyndrome WASPIP Q6IN36 1(1) RT-PCR Scaffoldprotein;inducesactinpolymerizationand
protein-interactingprotein redistribution;bindsWASP,profilin,actin,andGRB2.
aNon-integral membrane proteins identified in the fixed incubation-biotinylated IMCDs subtracted from those identified in the fixed
perfusion-biotinylatedIMCDs.
bNumberofpeptidesidentifiedwiththeirtotalhitnumberintheparentheses.
cIdentificationsconfirmedbyRT-PCR.
2140 Molecular & Cellular Proteomics 5.11