SupplementaryFiguresCharacterisationofurinaryWFDC12insmallnocturnalbasalprimates,mouse

lemurs(Microcebusspp.)

JenniferUnsworth,GraceLoxley, AmandaDavidson,JaneLHurst,GuadalupeGömez-Baena,NicholasIMundy,RobertJBeynon,ElkeZimmermannandUte

Radespiel

SupplementaryFigureS1

Evidencefor‘long’and‘short’formsofWFDC12inmouselemurmaleurine

SupplementaryFigureS2

Evidencefor‘longform’ofWFDC12in Lemurcatta (ring-tailedlemur) urine(PEAKSDB&SPIDER)

SupplementaryFigureS3

EvidenceforanalternativespliceformofWFDC12inLcatta.

SupplementaryFigureS1Evidencefor‘long’and‘short’formsofWFDC12inmouselemurmaleurine(PEAKSDB)

a)Evidencefor‘long’and‘short’formsofWFDC12inmouselemurmaleurineUrinefrommouselemurmaleswasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘longform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingthePEAKSDBtoolofthesoftware.Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).Bluebarsaredatabasematches,greybarsaredenovo sequencetags

Dataarefromallmalesofbothspecies,irrespectiveofbreedingstatusorseason

b)Evidencefor‘shortform’ofWFDC12inmouselemurmaleurine (PEAKSDB)UrinefrommouselemurmaleswasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘shortform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingthePEAKSDBtoolofthesoftware.Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).

Dataarefromallmalesofbothspecies,irrespectiveofbreedingstatusorseason

Regionpredictedin‘longform’Nopeptide-levelevidence

Fullcoverage

c)Evidencefor‘longform’ofWFDC12inMmurinusfemaleurine(PEAKSDB)

UrinefrommouselemurfemaleswasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘longform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingthePEAKSDBtoolofthesoftware.Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).

Dataarefromallfemales.Thelowdegreeofcoverage,comparedtomales,reflectsthesubstantiallylowerlevelofproteinexpression.

d)Evidencefor‘shortform’ofWFDC12inMmurinusfemaleurine(PEAKSDB)

UrinefrommouselemurfemaleswasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘shortform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingthePEAKSDBtoolofthesoftware.Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).

Dataarefromallfemales.Thelowdegreeofcoverage,comparedtomales,reflectsthesubstantiallylowerlevelofproteinexpression. TheN-terminalmatchesarecommontobothshortformandlongformsearches.

e)Evidencefor‘longform’ofWFDC12inmalemouselemur urine(SPIDER)

UrinefrommouselemurmaleswasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘longform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingtheSPIDERtoolofthesoftware,whichperformsexhaustivesearchesforMS/MStagsequences..Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).

Dataarefromallmalesofbothspecies.Thelowdegreeofcoverage,comparedtomales,reflectsthesubstantiallylowerlevelofproteinexpression.

Evidencefor‘shortform’ofWFDC12inmalemouselemur inbreedingseason(SPIDER)

Urinefrommouselemurmales(bothspecies)wasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘shortform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingtheSPIDERtoolofthesoftware,whichperformsexhaustivesearchesforMS/MStagsequences..Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).

Dataarefromallbreedingseasonmales.Thehighdegreeofcoveragereflectsthesubstantiallyhigherlevelofproteinexpression.

Evidencefor‘longform’ofWFDC12inmalemouselemururineoutofbreedingseason(SPIDER)

Urinefrommouselemurmales(bothspecies)wasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘longform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingtheSPIDERtoolofthesoftware,whichperformsexhaustivesearchesforMS/MStagsequences..Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).

Dataarefromallmalesoutofbreedingseason.Thelowdegreeofcoveragereflectsthesubstantiallylowerlevelofproteinexpression.

Evidencefor‘shortform’ofWFDC12inmalemouselemururineoutofbreedingseason(SPIDER)

Urinefrommouselemurmales(bothspecies)wasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘longform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingtheSPIDERtoolofthesoftware,whichperformsexhaustivesearchesforMS/MStagsequences..Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).

Dataarefromallmalesoutofbreedingseason.Thelowdegreeofcoveragereflectsthesubstantiallylowerlevelofproteinexpression.

Evidencefor‘longform’ofWFDC12infemalemouselemururine(SPIDER)

Urinefrommouselemurfemales(Mmurinusonly)wasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘longform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingtheSPIDERtoolofthesoftware,whichperformsexhaustivesearchesforMS/MStagsequences..Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).

Dataarefromallmalesoutofbreedingseason.Thelowdegreeofcoveragereflectsthesubstantiallylowerlevelofproteinexpression.

Evidencefor‘shortform’ofWFDC12infemalemouselemururine(SPIDER)

Urinefrommouselemurfemales(Mmurinusonly)wasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘shortform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingtheSPIDERtoolofthesoftware,whichperformsexhaustivesearchesforMS/MStagsequences..Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).

Dataarefromallfemales.Thelowdegreeofcoveragereflectsthesubstantiallylowerlevelofproteinexpression.

Regionpredictedin‘longform’Nopeptide-levelevidence

Fullcoverage

SupplementaryFigureS2Evidencefor‘longform’ofWFDC12in Lemurcatta (ring-tailedlemur) urine(PEAKSDB&SPIDER)

Urinefromring-tailedlemurswasdigestedwithtrypsinandanalysedbyLC-MS/MS.Thepeptideswerematchedtothe‘longform’WFDC12sequenceusingPEAKSsoftware,andmatchedpeptideswerehighlightedagainstthesequenceusingthePEAKSDBandSPIDERtoolofthesoftware.Themapisintendedtobeillustrative,toindicatetheextent(horizontal)andfrequencyofobservations(vertical).Bluebarsaredatabasematches,greybarsaredenovo sequencetags

Evidencefor‘shortform’ofWFDC12in Lemurcatta (ring-tailedlemur) urine(PEAKSDB&SPIDER)

SupplementaryFigureS3

EvidenceforanalternativespliceformofWFDC12inLcatta.BasedonthepredictedsequenceofLcatta,togetherwithMS/MSanalysisofLcatta urine(previouspage),thereisconsiderableevidencethatthe'shortform'alsoistheformexpressedinLcatta urine.Thereisclearpotentialforasplicevariant(asinMmurinus,seeFigure5,maintext).

a)

Lemurcatta [MRSYSFWFLTAFLVFATLALGEA]VKGGKEKWGNCPAEKGSCIKSGPSQCHADNDCPGDKKCCFLSCSFKCVSPDRIRKEMmurinuspred [MKTLCFLVLTAFLVIATLTPGEA]VKGGKEKWGNCPAEKGSCIKSGPSQCHSDNDCPGDKKCCFLHCSYKCVSPERNRKEMmurinusobs VKGGKEKWGNCPAEKGSCJKSGPSQCHSDNDCPGDKKCCFJHCSYKCVSPERNRKE

Lemurcatta -GNEDEDVSRSSPEPGGEPRPPGSSPSTSILSYYAVSFPPPGIGQMAPVPQGAESWN---VGQEASPQKEWSMmurinuspred GGNEDEDVSRSSLGPGWEPRPPGSSPSTSILSSYAVFFLPPGIGQMAPVPQGADTWNVGQVGQEASPQKEWSMmurinusobs GJGQMAPVPQGADTWNVGQVGQEASPQKEWS

exon1 exon2

exon3

Alternate splice junction, exon 3

Signal peptide

ATGAGGTCCTACAGCTTCTGGTTCCTTACAGCGTTCCTGGTTTTTGCGACCCTGGCGCTCGGGGAAGCAGTCAAAGGGGGTGAATGGATATGGTGGACCTGGGTGGGGCTGGGCTAGGGAGAGGCCCTGAGGGGCCGTCTGGGGCTGTAGGCAGTGCTGGA-CCCTCACCTCAACCCGTGGCTTCCTCCACCAGGTAAAGAGAAGTGGGGAAATTGCCCAGCCGAGAAAGGAAGCTGCATCAAGTCCGGTCCGTCCCAGTGTCATGCCGATAATGACTGTCCTGGGGACAAGAAATGCTGCTTCCTGAGTTGTAGCTTCAAATGTGTGAGTCCTGACAGGATCAGAAAGGAAGGTAAGGAGCCCCTGCATACCAGGCCTGGTGCTCTCACTTCCCCTGCCTCTGTCTGGCCCATGAAAGCACGGAGGAATTAGTGCTTTTAGGCGGTGTGGGGAGGGATGGTTAGAGTTGGCAGGGCCCTAAGAGATCAACAAGTCTGAACATCTCCATTGCACAAAGGGGGAAATGAGGATGAAGATGTGTCAAGATCATCCCCAGAGCCAGGAGGGGAGCCCAGGCCTCCAGGTTCGTCCCCTTCCACAAGTATCCTCAGTTATTATGCTGTGTCCTTTCCACCTCCAGGTATCGGTCAAATGGCACCTGTTCCTCAGGGTGCTGAATCTTGGAAT---------GTGGGACAAGAAGCGAGCCCCCAGAAGGAATGGTCCTGA

exon 1

exon 2

exon 3

b)