Supporting Information for Proteomics DOI 10.1002/pmic.200700142 Karl Skld, Marcus Svensson, Mathias Norrman, Benita Sjgren, Per Svenningsson and Per E. Andren´ The significance of biochemical and molecular sample integrity in brain proteomics and peptidomics: Stathmin 2-20 and peptides as sample quality indicators ª 2007 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.proteomics-journal.com SUPPORTING INFORMATION Supporting Information Table 1. Degraded protein identities and peptide sequences in the striatum after 1, 3, and 10 min post-mortem. UniProtKBa. Protein name Sequenceb Scorec P60710/P63260 Actin, cytoplasmic 1,2 A.LVVDNGSGMCK.A 56 E.MATAASSSSLEKS.Y 55 W.IGGSILASLSTFQQ.M 64 W.ISKQEYDESGPSIVHRK.C 93 M.WISKQEYDESGPSIVHRK.C 56 Q8K021 Secretory carrier-associated F.ATGVMSNKTVQTAAANAASTAATSAAQNAFKGNQM.- 124 membrane protein 1 Q9D164 FXYD domain-containing ion L.ITTNAAEPQK.A 58 transport regulator 6 precursor L.ITTNAAEPQKA.E 57 L.ITTNAAEPQKAE.N 89 L.ITTNAAEPQKAEN.- 54 P99029 Peroxiredoxin 5, mitochondrial M.APIKVGDAIPSVEVF.E 57 precursor P01942 Hemoglobin alpha subunit F.LASVSTVLTSKYR.- 106 M.FASFPTTKTYFPHF.D 72 L.ASHHPADFTPAVHASLDK.F 76 T.LASHHPADFTPAVHASLDK.F 59 L.LVTLASHHPADFTPAVHAS.L 56 L.LVTLASHHPADFTPAVHASLDK.F 71 L.LVTLASHHPADFTPAVHASLDKFLASVST.V 66 T.LASHHPADFTPAVHASLDKFLAS.V 55 L.VTLASHHPADFTPAVHASLDKFLAS.V 68 -.VLSGEDKSNIKAAWGKIGGHGAEYGAEALER.M 97 -.VLSGEDKSNIKAAWGKIGGHGAEYGAEALERM.F 58 P02088/P02089 Hemoglobin beta-1,2 subunit L.LVVYPWTQRY.F 53 L.LVVYPWTQRYF.D 52 Q00623 Apolipoprotein A-I precursor Y.VDAVKDSGRDYVSQFESSSLGQQLN.L 100 P10637 Microtubule-associated protein D.MVDSPQLATLADEVSASLAKQ.G 148 tau M.VDSPQLATLADEVSASLAKQ.G 111 P20357 Microtubule-associated protein L.LESPQLATLAEDVTAALAKQ.G 99 2 L.LESPQLATLAEDVTAALAKQG.L 98 P68369/Q922F4 Tubulin alpha-1,6 chain F.HSFGGGTGSGFTS.L 63 F.FSETGAGKHVPRA.V 62 F.LVFHSFGGGTGSGFTS.L 68 L.THSLGGGTGSGMGTLLI.S 85 F.SVMPSPKVSDTVVEPYNA.T 61 Q9D6F9 Tubulin beta-4 chain R.SGPFGQIFRPDNF.V 53 Q62361 Thyroliberin precursor A.LLEAAQEEGAVTPDLPGLEKVQVRPE.R 89 Q9Z0P4 Paralemmin F.MGYQNVEDEAETKKVLGLQDTIKA.E 81 F.MGYQNVEDEAETKKVLGLQDT.I 58 P05201 Aspartate aminotransferase, -.APPSVFAQVPQAPPVLVFK.L 85 cytoplasmic -.APPSVFAQVPQAPPVLVFKL.T 81 Q60771 Claudin-11 F.YYSSGSSSPTHAKSAHV.- 63 Q68ED7 Mucoepidermoid carcinoma L.STEARRQQAQQVSPTLSPLSPITQAV.A 55 translocated protein 1 homolog P80313 T-complex protein 1, eta subunit F.VWEPAMVRINALTAASEAA.C 69 Q99KB8 Hydroxyacylglutathione -.MKVELLPAL.T 53 hydrolase P70699 Lysosomal alpha-glucosidase T.PVLEPGKTEVTGYFPKG.T 60 precursor P17751 Triosephosphate isomerase L.NAANVPAGTEVVCAPPTAYID.F 75 L.NAANVPAGTEVVCAPPTAYIDFARQK.L 66 P32037 Solute carrier family 2, L.NSMQPVKETPGNA.- 56 facilitated glucose transporter member 3 P09411 Phosphoglycerate kinase 1 L.LEGKVLPGVDA.L 58 F.LKDCVGPEVENACANPAAGTVI.L 124 F.LKDCVGPEVENACANPAAGTVIL.L 92 L.FLKDCVGPEVENACANPAAGTVI.L 96 L.FLKDCVGPEVENACANPAAGTVIL.L 70 P16858 Glyceraldehyde-3-phosphate L.ISWYDNEYGYSNR.V 76 dehydrogenase P97450 ATP synthase coupling factor 6, F.KFDDPKFEVIDKPQS.- 52 mitochondrial precursor Q9DB20 ATP synthase O subunit, P.FAKLVRPPVQVYGIEGRYAT.A 63 mitochondrial precursor Q9D3D9 ATP synthase delta chain, F.FDSANVKQVDVPTLTGAFG.I 52 mitochondrial precursor Y.AEAAAAPAPAAGPGQMSFTFASPTQ.V 126 Q61548 Clathrin coat assembly protein F.DSAPEVAAAPKPDAAPS.I 57 AP180 D.AFAAPSPASTASPAKAESSGVIDL.F 91 P17742 Peptidyl-prolyl cis-trans T.ADDEPLGRVSF.E 66 isomerase A F.FDITADDEPLGR.V 71 F.DITADDEPLGRVSF.E 61 F.DITADDEPLGRVS.F 67 M.ANAGPNTNGSQFFICT.A 60 M.ANAGPNTNGSQFFICTA.K 71 Q9R0P9 Ubiquitin carboxyl-terminal L.LLLFPLTAQHENF.R 68 hydrolase isozyme L1 Q9DCT8 Cysteine-rich protein 2 Y.IYDKDPEGTVQP.- 62 O08553 Dihydropyrimidinase-related A.FVTSPPLSPDPTTPDFLNS.L 52 protein 2 Q99LX0 Protein DJ-1 L.AIVEALVGKDMANQVKAPLVLKD.- 56 Q66JT5 Protein C18orf10 homolog S.KVFKSKNKILI.P 60 P56564 Excitatory amino acid L.IAQDNEPEKPVADSETKM.- 51 transporter 1 Q9QZM0 Ubiquilin-2 L.ATEAPGLIPSFAPGVGMGVLGT.A 59 P70296 Phosphatidylethanolamine- Y.AGVTVDELGKVLTPTQV.M 61 binding protein W.AGPLCLQEVDEPPQHAL.R 63 P34884 Macrophage migration Y.DMNAANVGWNGSTFA.- 64 inhibitory factor O70251 Elongation factor 1-beta -.GFGDLKTPAGLQVL.N 66 P35455 Vasopressin-neurophysin 2- Q.LAGTRESVDSAKPR.V 68 copeptin precursor L.VQLAGTRESVDSAKPR.V 64 L.VQLAGTRESVDSAKPRVY.- 62 R.LVQLAGTRESVDSAKPRVY.- 73 P60761 Neurogranin G.RKGPGPGGPGGAGGARGGAGGGPSGD.- 97 P57774 Neuropeptide Y precursor R.SSPETLISDLLMKESTENAPRTRLEDPS.M 56 P60041 Somatostatin precursor R.SANSNPAMAPRE.R 52 P22005 Proenkephalin A precursor Q.LEDEAKELQ.K 54 R.SPQLEDEAKE.L 61 R.SPQLEDEAKEL.Q 51 R.SPQLEDEAKELQ.K 72 K.DADEGDTLANSSDLLK.E 102 Q8BME9 Cerebellin-4 precursor N.SKVAFSAVRSTN.H 69 R.AANSKVAFSAVRSTN.H 78 Q9R171 Cerebellin precursor R.SGSAKVAFSAIRSTN.H 61 R.SGSAKVAFSAIRSTNH.E 58 P01193 Corticotropin-lipotropin F.MTSEKSQTPLVTL.F 52 precursor R.RPVKVYPNVAENE.S 72 E.EEAVWGDGSPEPSPRE.G 67 A.EEEAVWGDGSPEPSPRE.G 60 R.AEEEAVWGDGSPEPSPRE.G 101 R.RAEEEAVWGDGSPEPSPRE.G 75 P06880 Somatotropin precursor A.FPAMPLSSLFSNA.V 72 P41539 Protachykinin 1 precursor R.DADSSVEKQVALLKALYGHGQISH.K 67 P20065 Thymosin beta-4 K.KTETQEKNPLPSKETIEQEKQAG.E 52 L.PSKETIEQEKQAG.E 58 P16014 Secretogranin-1 precursor R.LGALFNPYFDPLQWKN.S 70 Q03517 Secretogranin-2 precursor F.QELGKLTGPSNQ.K 61 R.IPVGSLKNEDTPN.R 61 L.VKYPELLNTNQL.K 57 R.IPVGSLKNEDTPNR.Q 90 E.SVFQELGKLTGPSNQ.K 54 L.ESVFQELGKLTGPSNQ.K 90 L.KAGEKPNGLVEPEQDLE.L 56 L.NQEQAEQGREHLA.K 65 R.ESKDQLSEDASKVITYL.R 70 R.VPSPVSSEDDLQEEEQLEQAIKEHLGPG.S 74 P47867 Secretogranin-3 precursor L.RAITEKETVEKERQ.S 54 R.AITEKETVEKERQS.I 66 R.ELSAERPLNEQIAEAEAD.K 81 aAccession number in the UniProtKB bAmino acid sequence of the identified peptide cMascot score is based on the absolute probability (P) that the observed match between the experimental data and the database sequence is a random 5 event. The score reported is -10 x log10(P). If 1.5 x 10 peptides during a search are within the mass tolerance window of the precursor mass, and the significance threshold is chosen to be 0.05 (5% chance of a false positive), this translates into a threshold score of 51. Supporting Information Table 2. Identified endogenous peptides in hypothalamic brain tissue irradiated in vivo with focused microwaves. UniProtKBa Precursor proteinb Peptide namec Peptide sequenced Precursor Charge mass (m/z)e state (z)f Q9D3P9 Neuromedin N Neurotensin 150pyroglutamic acid LYENKPRRPYIL162 558.3 3 P22005 / Proenk A precursor, Met-enk YGGFM, multivalent 574.3 1 P01193 POMC P22005 Proenk A precursor Met-enk-RSL 188YGGFMRSL195 465.7 2 Q9QXV0 ProSAAS Big LEN 245LENPSPQAPARRLLPP260 586.0 3 P01193 Pro-opiomelanocortin Melanotropin alpha 124SYSMEHFRWGKPV136-amidation 555.6 3 1ac Ser124 acetylation P01193 Pro-opiomelanocortin Melanotropin alpha 124SYSMEHFRWGKPV136-amidation 569.7 3 2 ac Ser124, Ser126 acetylation P56942 Pro-MCH precursor Neuropeptide EI 132EIGDEENSAKFPI144-amidation 724.4 2 P20065 Thymosin beta-4 Thymosin beta-4 acetylation- 709.7 7 isoform short 2SDKPDMAEIEKFDKSKLKKTETQEKNPLPSK ETIEQEKQAGES44 Q6ZWY8 Thymosin beta-10 Thymosin beta-10 acetylation- 705.8 7 2ADKPDMGEIASFDKAKLKKTETQEKNTLPT KETIEQEKRSEIS44 P41539 Protachykinin 1 Substance P 58RPKPQQFFGLM68-amidation 674.4 2 P26339 Chromogranin A Chromogranin A 392AYGFRDPGPQL402 610.9 2 precursor 394-402 P20156* Neurosecretory VGF VGF 491-507 491PPEPVPPPRAAPAPTHV507 577.4 3 protein P01193 Pro-opiomelanocortin Joining peptide 103AEEEAVWGDGSPEPSPRE120-amidation 970.8 2 P22005 Proenkephalin A Proenk A 198-209 198SPQLEDEAKELQ209 693.9 2 precursor P22005 / Proenk A / proenk B Leu-enk YGGFL, multivalent 556.4 1 P06300 Q9QXV0 ProSAAS Little SAAS 42SLSAASAPLVETSTPLRL59 907.0 2 P01193 Pro-opiomelanocortin CLIP 141RPVKVYPNVAENESAEAFPLEF162 836.0 3 P01193 Pro-opiomelanocortin CLIP 141RPVKVYPNVAENESAEAFPLEF162 862.8 3 Ser-154 Phosphate P01193 Pro-opiomelanocortin Lipotropin gamma 165ELEGERPLGLEQVLESDAEKDDGPYRVEH 740.4 6 FRWSNPPKD202 P01193 Pro-opiomelanocortin Beta endorphin acetylation- 760.2 5 205YGGFMTSEKSQTPLVTLFKNAIIKNAH231 P01193 Pro-opiomelanocortin POMC 205-221 acetylation-205YGGFMTSEKSQTPLVTL221 951.0 2 P54227 Stathmin Stathmin 2-20 acetylation-2ASSDIQVKELEKRASGQAF20 702.8 3 a Accession number in the UniProtKB. b Name of the precursor protein from which the peptide originates. c Name of peptide if previously reported, or else precursor protein name and location. d Peptide sequence, modification and location in precursor protein. e Observed peptide precursor m/z. f Peptide precursor charge state * UniProtKB accession number to rat since mouse accession is non-existent. MS/MS spectra Q9D3P9, Neurotensin, mono mass 1672.0 P22005/P01193, Met-enkephalin, mono mass 573.2 P22005, Met-enkephalin-RSL, mono mass 929.5 Q9QXVO, big Len, mono mass 1755.1 P01193, Melanotrophin alpha, acetylated on second S, mono mass 1663.9 P01193, Melanotrophin alpha, acetylated on first and second S, mono mass 1705.8 P56942, Neuropeptide EI, mono mass 1446.8 P20065, Thymosin beta-4, mono mass 4960.8 Q6ZWY8, Thymosin beta-10, mono mass 4933.7 P41539, Substance P, mono mass 1346.8 P26339, Chromogranin A 392-402, mono mass 1346.8 P20156, Neurosecretory VGF protein 491-507, mono mass 1729.2 P01193, Joining peptide, mono mass 1939.8 P22005, Proenkephalin A precursor 198-209, mono mass 1385.7 P22005/P06300, Leu-enkephalin, mono mass 555.3 Q9QXVO, littleSAAS, mono mass 1812.0 P01193, CLIP, mono mass 2505.3 P01193, CLIP phosphorylation on S, mono mass 2585.3 P01193, Lipotropin gamma, mono mass 4436.2 P01193, Beta endorphin, mono mass 3036.7 P01193, Beta endorphin 1-17, mono mass 1900.0 P54227, Stathmin 2-20, N-terminalacetylation, mono mass 2105.1 .