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)