karl clauser proteomics and biomarker discovery sample experiment summary 1 spider 1 ug venom...
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Karl ClauserProteomics and Biomarker Discovery 1
Sample Experiment SummarySpider
1 ug venom
Reduce/alkylate
De-salt
LC-MS/MS 300ngC18 300A Microsorb 3um
Orbitrap Elite CID/HCD/ETD
Spectrum Mill
Cysiodoacetamide
CysMeAziridine
DigestLys-C
Database• Venom gland mRNA library• Ion Torrent sequencing
• 75-150 bp reads• Transcriptome assembly (Trinity)• Translation (extractORFS.pl)
Database• Venom gland mRNA library• 454 sequencing
• 300-400 bp reads• Transcriptome assembly (MIRA)• Translation (extractORFS.pl)
Centipede 1 ug venom
Reduce/alkylateCys-ethanolyl-gp
Digest500ng & 500 ng Trypsin Glu-C
De-salt
LC-MS/MS 150 ng300A Microsorb 3um C18
C18 120A Reprosil 3umOrbitrap Elite CID/HCD/ETD
Spectrum Mill
Scorpion 1 ug venom
Reduce/alkylate
De-salt
LC-MS/MS 300,600 ngC18 300A Microsorb 3um
Orbitrap Elite CID/HCD/ETD
Spectrum Mill
Cys-ethanolyl-solCys-iodoacetamide
C18 200A Magic 3um
Cys-MeAziridine
DigestLys-C
Database• Venom gland mRNA library• 454 sequencing
• 300-400 bp reads• Transcriptome assembly (Trinity)• Translation (extractORFS.pl)
Karl ClauserProteomics and Biomarker Discovery 2
K121115_Hi_dsrak#Spectra
K121115_Hi_dsrakIntens i ty
K121115_Hi_dsrak#peps
K120223_Hi#Spectra
K120223_HiIntens i ty
K120223_Hi#peps
both#peps
scoreUnique subgroupNum entry_name BLAST2GO-Protein-Name
mature#Cys
cyspattern accession_number
SpiderPSignal peptide:
SpiderPPropeptide - MS/MS covered
80 2.75E+10 17 169 1.31E+10 38 53 775.68 2.1 unknown omega-hexatoxin-hi2a 6 37_1680.3d.comp485_c0_seq1_F3_R4 MKFSKLSLTLALILTQALFVLC GKINGGELLLHFPDFMENGLESHALHDEIRKPIDTEKADAER
61 4.03E+09 16 141 7.25E+09 35 49 706.92 2.2 omega-hexatoxin-Hi2b omega-hexatoxin-hi2b 6 AS58251 2.02E+09 15 124 3.90E+09 29 42 588.62 2.3 omega-hexatoxin-Hv2i omega-hexatoxin-hi2b 6 AS126353 2.06E+09 15 120 4.23E+09 26 39 553.17 2.4 omega-hexatoxin-Hv2n omega-hexatoxin-hi2b 6 AS127146 1.59E+09 13 120 4.38E+09 26 37 540.07 2.5 omega-hexatoxin-Hv2h_1 omega-hexatoxin-hi2b 6 AS126229 1.62E+09 9 112 2.56E+09 25 33 459.03 2.6 omega-hexatoxin-Hv2l omega-hexatoxin-hi2a 6 AS126938 1.41E+09 10 100 1.73E+09 21 29 435.72 2.7 omega-hexatoxin-Hv2o omega-hexatoxin-hi2a 6 AS1272
3 1.08E+07 1 66 2.03E+09 9 9 129.68 2.8 omega-hexatoxin-Hv2f omega-hexatoxin-hi2b 6 AS125812 1.02E+09 3 107 9.33E+09 25 28 431.57 6.1 unknown hntx- precursor 6 37_1680.3d.comp492_c0_seq1_F1_R2 MNTLIAFAVLLLLSTTLG DTDDKVSHEEIQERKELSGISEELLLQQLEAVEAALMEKERLEEMEEDGNSREKR
3 2.69E+06 1 82 4.05E+10 19 20 266.31 9.1 omega-hexatoxin-Hi1a_2 omega-hexatoxin-ar1a 6 AS192.310 2.01E+08 2 54 1.35E+10 13 15 204.53 9.2 omega-hexatoxin-Hi1g_1 omega-hexatoxin-ar1a 6 AS747
6 1.00E+08 2 59 2.06E+10 13 15 202.12 9.3 unknown omega-hexatoxin-ar1a 6 37_1680.3d.comp499_c1_seq1_F4_R1 MNTATGFIVLLVLATVIGCISA DFQGAFEPHEEEEAERIFRR
3 2.69E+06 1 48 7.22E+09 11 12 147.39 9.4 unknown omega-hexatoxin-ar1a 6 spider.q17.comp1689_c0_seq1_F1_R1 MNTATGFIVLLVLATVIGCISA DFQGAFEPHEEEEAERIFRR
3 2.69E+06 1 50 1.47E+10 10 11 150.20 9.5 omega-hexatoxin-Hi1b_8 omega-hexatoxin-ar1d 6 AS195.43 2.69E+06 1 40 1.20E+08 9 10 119.99 9.6 omega-hexatoxin-Hi1c_2 omega-hexatoxin-ar1e 6 AS197.2
26 2.40E+08 6 6 80.01 17.1 unknown mu-hexatoxin-mg1a 6 spider.q17.comp522_c0_seq2_F5_R2 MRVTLVFLVLSALAVTHG DTNLDMERKEAEESSLSEMKEMLLLQELQAVEAALFGKMNAENDENRDFREKR
58 7.92E+09 2 2 47.56 18.1 unknown 6 37_1680.3d.comp125_c0_seq1_F2_R1 MDVKTVLIFTVLFGVISNNAA None predicted
5 8.62E+07 2 2 31.60 22.1 unknown 6 37_1680.3d.comp466_c0_seq1_F4_R3 MNTMIGFIVLLVSATVLG DPELDALRKELDERFDMEFKDGPMSEIQEKLFLQELEALDSELLEDDPQTDTYENSNFREKR
2 5.66E+06 1 1 20.87 25.1 unknown 6 spider.q17.comp522_c0_seq1_F5_R2 MRVTLVFLVLSALAVTHG DTNLDMERKEAEESSLSEMKEMLLLQELQAVEAALFGKMNAENDENRDFREKR
6 3.77E+07 1 1 18.55 27.1 unknown 6 37_1680.3d.comp183_c0_seq1_F1_R19 1.27E+08 2 2 17.69 28.1 unknown 6 37_1680.3d.comp496_c0_seq1_F6_R2 MKITIFCLTILTLSITFG ETNEDVKDGSLKEMDEMSLLQELMSVEAALVEKEMEAEAEENRDSREKR
117 8.55E+09 20 381 4.84E+10 87 107 1647.02 1.1 unknown hntx-xviii precursor 8 CECCG 37_1680.3d.comp348_c0_seq1_F5_R3 MKLLYFFVVITVLVAVAAA LPAKTEEQIAAEENQLVEDLVQYAGTRLTQKRATR
30 1.22E+09 5 17 8.68E+08 7 12 174.31 11.1 unknown hntx-xviii precursor 8 CECCG 37_1680.3d.comp159_c0_seq1_F1_R13 8.62E+07 1 1 15.22 29.1 unknown 8 CECCG 37_1680.3d.comp5507_c0_seq1_F5_R1
32 4.96E+09 12 12 173.17 12.1 unknown hntx- precursor 8 CECCG spider.q17.comp2075_c0_seq1_F2_R2 MKLYLVILVTSVALAAA SPTRTKEEPIEDELLEALLSVEKSLFNEETTVMEKR
79 3.20E+09 14 22 1.53E+09 7 21 315.67 8.1 unknown u1-cyrtautoxin-as1a 8 CC,CYIC 37_1680.3d.comp410_c0_seq1_F2_R3 MKTILSMLIFVALFAAIVG None predicted
4 4.10E+07 2 2 25.46 24.1 unknown 8 CC,CYIC 37_1680.3d.comp429_c0_seq1_F3_R3 MKLILLIAIFSALAVVNLG None predicted
13 7.13E+08 5 41 4.80E+09 9 14 163.06 13.1 unknown 8 CC,CXC,CXC 37_1680.3d.comp454_c0_seq1_F4_R5 MRIHYLLILSFLLLASG VLINSEEMPRSEMSPSR
16 5.68E+08 4 7 3.99E+08 3 7 109.57 15.1 unknown 8 CC,CXC,CXC 37_1680.3d.comp454_c0_seq2_F4_R5 MRIHYLLILSFLLLASG VLINSEEMPRSEMSPSR
3 5.71E+07 2 3 7.23E+07 1 3 40.15 21.1 unknown 8 CC,CXC,CXC spider.q17.comp1562_c0_seq3_F5_R4 MRIYSLLILSFLLLASTVLINS AEMPRSEKSLLYSIMQGREDSEEGR
7 1.56E+08 2 2 19.15 26.1 unknown 8 CC,CXC,CXC spider.q17.comp479_c0_seq2_F6_R2 MRISTLIVLSGLLLTTSVLIRA EEIPGLEESLVPAGLVDEESSPEQGR
37 1.07E+10 9 78 1.45E+10 25 32 541.87 5.1 unknown u2-hexatoxin-hi1a 8 CC,CXC,CXC 37_1680.3d.comp516_c0_seq1_F2_R2 MRNTTFLVLNVMLLVSVALFCAA DPEMEKSSFAEILDTGNPEQER
6 1.39E+08 4 4 41.24 20.1 unknown 8 CC,CXC,CXC spider.q17.comp1557_c0_seq1_F3_R1 MRNTTFLVLSALVLVSVALLCES APQMSEVDLVQSLEENPEGER
41 6.11E+09 8 51 1.41E+09 18 26 340.73 7.1 unknown delta-hexatoxin-mg1b 8 CCC 37_1680.3d.comp616_c0_seq1_F1_R1 None predicted MEAIEDAFLEKGFLPHEEDRNARPKR
3 2.75E+07 1 20 1.11E+09 5 6 116.98 14.1 unknown delta-hexatoxin-mg1b 8 CCC spider.q17.comp6008_c0_seq1_F1_R15 2.31E+08 3 16 1.30E+08 5 8 80.81 16.1 unknown delta-hexatoxin-mg1b 8 CCC spider.q17.comp1543_c0_seq2_F2_R1 MKIIIALYVLFFLTIALG DITEGNEDDLVDNFRKELSEADIPLLKKLEAIEDALLEKDFLPYEEEDRNARPKR
4 4.55E+07 2 2 13.28 30.1 unknown delta-hexatoxin-mg1b 8 CCC spider.q17.comp1516_c0_seq1_F6_R1168 3.01E+10 21 79 1.58E+10 21 39 643.52 3.1 unknown u1-hexatoxin-hsp201a 10 spider.q17.comp1699_c0_seq2_F3_R3 MLKFVVVIFLVIMASTFA None predicted
41 2.47E+09 10 25 6.24E+08 9 17 274.67 3.2 unknown u1-hexatoxin-hsp201c 10 spider.q17.comp1699_c0_seq3_F3_R3 MLKFVVVIFLVIMASTFA None predicted
66 2.06E+10 14 63 1.26E+10 20 34 579.80 4.1 unknown u1-hexatoxin-hi1a 10 37_1680.3d.comp452_c0_seq2_F2_R3 MLKFVVVIFLVIMASTFA None predicted
22 3.27E+08 8 27 7.84E+09 10 16 227.62 10.1 unknown hntx-xiv- precursor 10 37_1680.3d.comp799_c0_seq1_F2_R4 MMKAVIILVILFNAVYVSV None predicted
6 3.37E+08 2 2 2.00E+08 1 3 45.22 19.1 P81803 U1-hexatoxin-Hv1a u1-hexatoxin-hv1a 10 AS2093 9.42E+07 2 2 9.86E+07 1 3 29.83 23.1 unknown ?6,5 CC,CC spider.q17.comp1681_c0_seq3_F5_R6 MKLRITLALTSVLAFCVFG DKENENLMENLLEDDLLDIFTDAIHMER
Spider Protein Group.subgroup ListMatches to Arachnoserver but not RNA-SeqSuggests need to improve paralog inclusion inRNA-Seq Transcript Assembly
Samples are fromDifferent animals
Nov 2012 Feb 2012MeAzCys IAAcysLys-C undigested
Karl ClauserProteomics and Biomarker Discovery 3
Group 2 omega-hexatoxin Hi2
6 Cys
SubGroup
11215346666
MTPSCTLGICAPSVGGLVGGLLG reads: 4382MTPSCTLGICAPSVGGIVGGLLG reads: 0251MTPSCTLGICAPNVGGLVGGLLG reads: 0201MTPSCTMGLCVPNVGGLVGGILG reads: 0000MTPSCTMGICVPNVGGLVGGILG reads: 0000MTPSCTMGICVPNVGGLVGGLLG reads: 0000 CAPNVGG reads: 0442 CVPNVGG reads: 0000
Evidence exists in spider.q17 reads for paralogs missing from the assembly.
ERGVLDCVVNTLGC reads: 635ERGVVDCVLNTLGC reads: 106ERGLVDCVLNTLGC reads: 553ERGLADCVLNTLGC reads: 21ER_QLDCVLNTLGC reads: 26ERGVVGCVLNTLGC reads: 8
VLDCVVNTLGCSSDKDCCGMTPSCTLGICAPSVGGL reads: 99VVDCVLNTLGCSSDKDCCGMTPSCTLGICAPSVGGL reads: 33VVDCVLNTLGCSSDKDCCGMTPSCTLGICAPSVGGL reads: 33LVDCVLNTLGCSSDKDCCGMTPSCTLGICAPSVGGL reads: 72LADCVLNTLGCSSDKDCCGMTPSCTLGICAPSVGGL reads: 4VLDCVVNTLGCSSDKDCCGMTPSCTLGICAPNVGGL reads: 2QLDCVLNTLGCSSDKDCCGMTPSCTLGICAPSVGGL reads: 1QLDCVLNTLGCSSDKDCCGMTPSCTLGICAPNVGGL reads: 2
Karl ClauserProteomics and Biomarker Discovery 4
Only 1 spectrum supports LCVPN
(G)L C V P n\V\G\G\L\V\G\G\I\L(G)
MTPSCTLGICAPSVGGLVGGLLG reads: 4382MTPSCTLGICAPSVGGIVGGLLG reads: 0251MTPSCTLGICAPNVGGLVGGLLG reads: 0201MTPSCTMGLCVPNVGGLVGGILG reads: 0000MTPSCTMGICVPNVGGLVGGILG reads: 0000MTPSCTMGICVPNVGGLVGGLLG reads: 0000 CAPNVGG reads: 0442 CVPNVGG reads: 0000
Karl ClauserProteomics and Biomarker Discovery 5
Group 23 Fusion Toxin or Transcript Misassembly
23
looks like a fusion toxin, but could be transcript misassembly?• 12 Cys, 2 pairs of adjacent CC’s
• maybe 11 Cys in mature form, active dimer?• perhaps 2 concatenated 6 Cys toxins• Present in 2 libraries
>as:pi-theraphotoxin-Pc1a|sp:P60514... Toxin from venom of the spider Psalmopoeus cambridgei that inhibits ASIC1a channels
Score = 64.7 bits (156), Expect = 1e-13 Identities = 26/39 (66%), Positives = 28/39 (71%)
Query: 52 QECIAKWKSCAGRKLDCCEGLECWKRRWGHEVCVPITQK 90 ++CI KWK C R DCCEGLECWKRR EVCVP T KSbjct: 1 EDCIPKWKGCVNRHGDCCEGLECWKRRRSFEVCVPKTPK 39
Top BLASTHit does notspan fusion junction
pro/mature
Karl ClauserProteomics and Biomarker Discovery 6
Alignment of CCC 8 Cys containing groups 7,14,16,30
ProteinGroup
16.116.116.17.17.1
30.114.114.1
WCAKNEDCCCPMKCIGAWYN reads:312WCGKEGDCCCPWKCIGQWYN reads: 25RCNANSDCCCPLKCVIRLVG reads: 3YCEKDKDCCCPMRCVKSYWK reads: 10
Group Proposed names16.1 delta-hexatoxin-Hi3 7.1 delta-hexatoxin-Hi114.1 delta-hexatoxin-Hi230.1 delta-hexatoxin-Hi4
Karl ClauserProteomics and Biomarker Discovery 7
Coverage of CCC 8 Cys containing groups 7,14,16,30
Karl ClauserProteomics and Biomarker Discovery 8
Alignment of noCCC 8 Cys containing groups
ProteinGroup12.112.1 1.1 1.1 5.121.1 8.1 8.113.113.121.115.1 5.1 5.120.1
1,5,8,12,13,15,20,21
5,13,15,20,21ProteinGroup13.113.115.121.121.120.1 5.1 5.1 5.1
CC, CXC, CXC
Karl ClauserProteomics and Biomarker Discovery 9
Group 1
topBLAST hit not in Arachnoservergi|294653183|gb|ADF28499.1|putative mature sequence toxin-like ACSKQ [Pelinobius muticus]
<Hsp_qseq>EQIAAEENQLVEDLVQYAGTRLTQKRATRCSKKLGEKCNYHCECCGATVACSTVYVGGKETNFCSDKTSNNGALNTVGQGLNVVSNGLSAFQCWG<Hsp_midline>+ A+E ++L+E L + + Q+ A CSK++GEKC + C+CCGATV C T+YVGG C KTSNN LNT+G G+N V N ++ CWG
<Hsp_hseq>RKTASETSKLLEKL-GVSREAIPQEMARACSKQIGEKCEHDCQCCGATVVCGTIYVGGNAVEQCMSKTSNNAVLNTMGHGMNAVQNAFTSVMCWG
8 Cys
CSDKTSNNGALNTVGQGLNVVSNGLSAFQC reads: 1023
CSKKLGEKCNYHCECCGATVACS reads: 371CSKKLGEKCDYHCECCGATVACD reads: 49
CSTVYVGGKETNFC reads: 5251
TVGQGLNVVSNGLSAFQC reads: 4739
CSDKTSNNGALNTVGQGLN reads: 1887
GGKETNFCSDKTSNN reads: 3771GGRETNFCSDKTSNN reads: 10
CSKKLGEKCNYHC reads: 769CSKKLGEKCDYHC reads: 111
Evidence exists in spider.q17 reads for paralogs missing from the assembly.
N to D supported in MS/MSmatched as N to n deamidation NVVVNGFSAFQC reads: 25
Karl ClauserProteomics and Biomarker Discovery 10
Alignment of CECCG 8 Cys containing groups 1,11,29
Groups 1,11,29 are highly related.Lower level variant reads not assembled with group 1 may be the missing N-term for Group 11,29
ProteinGroup
1.1 1.111.111.129.1
Karl ClauserProteomics and Biomarker Discovery 11
Alignment of CECCG 8 Cys containing groups 1,11,12
ProteinGroup12.112.1 1.1 1.1
ProteinGroup 1.1 1.1 11.1 11.1
Group 1 and 11 are highly related.Lower level variant reads not assembled with group 1 may be the missing N-term for Group 11
Karl ClauserProteomics and Biomarker Discovery 12
Alignment of CC, CXC, CXC 8 Cys containing groups 5,13,15,20,21,26
5,13,15,20,21
CC, CXC, CXC
ProteinGroup13.113.115.121.121.120.1 5.1 5.1 5.1
ProteinGroup26.126.126.115.113.113.121.1 5.1 5.120.1
Karl ClauserProteomics and Biomarker Discovery 13
Coverage of CC, CXC, CXC 8Cys containing groups 5,20
Karl ClauserProteomics and Biomarker Discovery 14
Coverage of CC,CXC,CXC 8Cys containing groups 13,15,21,26
ProteinGroup13.113.115.121.121.1
26
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Coverage of CC, CYIC 8Cys containing group 8,24
24
Karl ClauserProteomics and Biomarker Discovery 16
Alignment of 10 Cys containing protein groups 3,4,10,19
ProteinGroup 4.1 4.1 4.1 4.119.1 3.1 3.1 3.210.110.1
Groups 3,4,10 too divergent to be paralogs.Examine group 19 to determine spectral coverage of variant AAs
Group Proposed names4.1 mu1-hexatoxin-Hi1a19.1 mu1-hexatoxin-Hi1b3.1 mu1-hexatoxin-Hi2a3.2 mu1-hexatoxin-Hi2b10.1 mu1-hexatoxin-Hi3a
Karl ClauserProteomics and Biomarker Discovery 17
Coverage of 10 Cys containing groups 3,4,10,19
Karl ClauserProteomics and Biomarker Discovery 18
PSM Overlap for Group 10.1
Group 10.1 37_1680.3d.comp799_c0_seq1_F2_R4 Hi_3day_Q17_37 unknown q T C G G P D D C G E G S C C V G S F S R K C L L L S R E G A S C Q P E Y Q H T G G I/Y/S Y/A/C/P C E N/G Y I C S P I N R C Q K A 21 65 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.5243.5243.7.pkl.spo T C G G P D|D|C|G E|G|S|C|C\V\G\S\F 22 18 46 CID K120223_Hi_ri_ff4f 4f4_pccc_01.3930.3930.2.pkl.spo T C\G G P D D\C G E G S C C V G/S/F 22 18 46 HCD K120223_Hi_ri_ff4f4f4_pccc_01.3931.3931.2.pkl.spo T C/G G/P D|D C/G E/G S C C V G S F S R K 22 21 43 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4075.4075.5.pkl.spo T C/G|G P|D D\C G|E|G/S C\C V|G|S|F|S|R\K 22 21 43 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4077.4077.5.pkl.spo T C/G|G P/D\D\C\G|E\G S C\C V/G|S|F|S|R\K 22 21 43 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4064.4117.4.pkl.spo T/C|G/G|P D/D|C/G E/G S C C V/G S F S R K 22 21 43 CID K120223_Hi_ri_ff4f4f4_pccc_01.3098.3098.3.pkl.spo T C G/G P D D C G/E/G/S C C V/G/S/F/S R K 22 21 43 ETD K120223_Hi_ri_ff4f4f4_pccc_01.3100.3100.3.pkl.spo T C/G/G P D|D C G E G/S C C V G/S/F/S R\K 22 21 43 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4061.4061.3.pkl.spo T C\G G/P D D/C/G E/G/S/C/C/V/G/S/F/S R K 2 2 21 43 HCD K120223_Hi_ri_ff4f4f4_pccc_01.3099.3099.3.pkl.spo T/C/G/G/P D|D|C/G E|G S/C C V/G S F S R K C L L L 22 25 39 CID K120223_Hi_ri_ff4f4f4_pccc_01.5269.5269.3.pkl.spo T C G/G P D D C G E G S C C V G/S/F/S/R K C L L\L 22 25 39 ETD K120223_Hi_ri_ff4f4f4_pccc_01.52 71.5271.3.pkl.spo T C G G P D D C G E G S C C V G S F S R K C L L L S R E G A S C Q P E Y Q H T G G I Y S\Y/A|C/P C/E/N G Y I C S P I N R C Q K A 22 64 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.5227.5227.8.pkl.spo T C G G P D D C G E G S C C V G S F S R K C L/L L S R E G A S C Q P E Y Q H T G G I\Y\S Y A\C|P C E N/G Y I C S P I N R C Q K A 22 64 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.5292.5292.7.pkl.spo T C G G P D D\C G E G S C C V G S F S R K C L L L S R E G A S C Q P E Y Q H/T/G G I/Y S Y/A/C/P C E N/G Y I/C S/P I N R C Q K A 22 64 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.5191.5191.6.pkl.spo T C G\G P D\D C G E G S C C V G S F S R K\C L L L S R E G A S C Q P E Y Q H T G G/I Y S Y A C P C E/n G Y I C S P I n R C/Q K A 22 64 0 ETD K120223_Hi_ri_ff4f4f4_pccc_ 01.5264.5264.8.pkl.spo C/V/G/S/F/S|R\K 35 8 43 ETD K121115_Hi_dsrak_ff4f4f4C HEsa_cccc_01.3004.3121.2.pkl.spo C/V/G/S/F/S R K 35 8 43 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.3003.3120.2.pkl.spo V/G|S|F S R\K 36 7 43 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2885.2969.2.pkl.spo V/G/S/F S/R\K 36 7 43 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2887.2971.2.pkl.spo V/G|S|F S R/K 36 7 43 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2886.2970.2.pkl.spo S R K C L L L S R E G A S C Q\P E Y Q H T G G I\Y\S\Y\A|C/P C/E/N G Y I C S P I N R C Q K A 40 46 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.4775.4775.7.pkl.spo S R\K\C\L\L/L\S\R\E\G\A\S\C|Q P E\Y Q\H T|G|G\I Y S Y A C P C E/N G/Y I C/S P I N/R C/Q/K A 40 46 0 ETD K120223_Hi_ri_ff4f4f4_pccc_01.4777.4777.7.pkl.spo S R K C L L L S R E G A S C Q P E Y Q\H\T\G\G\I\Y\S\Y A C/P C E N G Y I/C/S P I N R C Q K A 40 46 0 HCD K120223_Hi_ri_ff4f4f4_pccc_01.4776.4776.7.pkl.spo S R K C L L L S R E G A S C Q P E Y\Q\H/T|G G\I|Y/S/Y/A/C/P C E N/G Y/I/C S P I N R C Q K A 40 46 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.4798.4798.5.pkl.spo S R K C\L L L\S\R\E G A S C Q P E Y Q H T G\G I Y S Y A C P C E N G Y I C S P I\N R C Q K A 40 46 0 ETD K120223_Hi_ri_ff4f4f4_pccc_01.4800.4800.5.pkl.spo S R K C\L\L L S\R E G A S C Q P E Y Q H T G\G I Y S Y A C P C E N G Y I C/S P I n/R C Q K A 40 46 0 ETD K120223_Hi_ri_ff4f4f4_pccc_01.4774.4774.5.pkl.spo S R K C L L L S R E G A S C Q P E Y\Q\H\T\G/G\I\Y\S Y A C P C E n G Y/I/C S P I N R C\Q K A 40 46 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.4772.4772.5.pkl.spo C/L|L/L S R|E|G|A\S C|Q P E Y|Q H T G G I Y\S|Y A\C P C 43 28 15 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6424.6424.4.pkl.spo C L\L\L S R\E G A S C|Q P|E\Y|Q H/T G\G I Y\S\Y A C P C\E n G Y I C S P I n|R C 43 40 3 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6704.6704.7.pkl.spo C L|L/L S\R\E\G A S C|Q P\E|Y\Q H T G|G I Y S|Y A\C P C E n G Y I\C S P I\n/R C/Q\K A 43 43 0 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6494.6494.7.pkl.spo C L L L S R E G A S C Q\P E Y Q H T G G I\Y\S\Y\A\C\P C E n G Y I C S P I n R C Q K A 43 43 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.5246.5246.5.pkl.spo C L\L L S\R\E\G A S C Q P\E Y Q H T G G I Y S Y A C P C E n G Y I C S P I n/R C/Q K A 43 43 0 ETD K120223_Hi_ri_ff4f4f4_pccc_01.5248.5248.5.pkl.spo S R E G A S C Q P E Y Q H T G G I\Y\S\Y|A|C|P C E N G Y I C S P I N R C Q K A 47 39 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.4752.4752.5.pkl.spo S Y/A|C|P/C/E/N/G Y/I/C S P I N R C Q K A 65 21 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.4044.4099.3.pkl.spo S Y\A\C P C E N G Y I C S P I/N R C Q K A 65 21 0 HCD K120223_Hi_ri_ff4f4f4_pccc_01.4045.4100.3.pkl.spo A C|P/C/E/N/G Y I/C S P I N R C Q K A 67 19 0 CID K120223_Hi_ri_ff4f4f4_pccc_01.3631.3680.3.pkl.spo A C P C E|N/G|Y/I C|S P I N/R C/Q K A 67 19 0 ETD K120223_Hi_ri_ff4f4f4_pccc_01.3633.3682.3.pkl.spo A C|P C E N G Y/I/C/S/P I/N R C Q K A 67 19 0 HCD K120223_Hi_ri_ff4f4f4_pccc_01.3632.3632.3.pkl.spo E N/G/Y I/C/S/P I N R C Q K A 71 15 0 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4825.4825.4.pkl.spo E/N\G|Y\I/C|S P I|N|R\C|Q|K A 71 15 0 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4827.4827.4.pkl.spo E N/G Y I/C/S/P I N R C Q K A 71 15 0 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4826.4826.4.pkl.spo E/N/G/Y/I/C/S/P I N R C Q K A 71 15 0 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4819.4849.3.pkl.spo E/N G/Y/I C|S P I|N|R C|Q\K\A 71 15 0 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4821.4851.3.pkl.spo E/N/G\Y/I/C/S P I N R C Q K A 71 15 0 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4820.4850.3.pkl.spo S/P|I/N R C Q K\A 77 9 0 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2469.2469.3.pkl.spo S P/I|N|R\C|Q|K A 77 9 0 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2471.2471.3.pkl.spo S/P|I/N R C Q K\A 77 9 0 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2470.2470.3.pkl.spo S P|I|N R\C Q|K A 77 9 0 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2460.2460.2.pkl.spo
Karl ClauserProteomics and Biomarker Discovery 19
Coverage of 6Cys containing groups 6, 9
Karl ClauserProteomics and Biomarker Discovery 20
Coverage of 6Cys groups 17, 22, 25, 27, 28
27
22
25
28
17
Karl ClauserProteomics and Biomarker Discovery 21
Group 18 - 6Cys - only intact MS/MS z5-10
(R)M F C K P L D\Q|Q\C\N K|D L H\C C|K P L/K C/R|R/S/N/N/G/R/K Y C/K P(-) (R)M F\C\K P L D\Q|Q\C N K\D L H\C\C|K P/L/K C/R/R/S/N/N/G R/K Y C/K P(-) (R)M F\C\K P L D\Q|Q\C N K\D L H\C C K P L K C|R/R/S/N/N/G/R/K/Y C/K P(-) (R)M F C\K P L D\Q\Q\C N K\D/L H\C\C|K P L K\C/R/R S/N/N/G/R/K/Y C K P(-) (R)M/F C/K P L\D\Q|Q\C N K D L H C C K P L K\C\R\R/S/N/N/G/R/K Y C K P(-)
z9z8z7z6z5
(R)M/F/C\K/P L D\Q Q C N K D|L H/C C K P L K C R R S N N G R K\Y C K\P(-) z6z5(R)M/F/C|K/P L D\Q Q/C N/K/D|L H C C K P L K C R R S N N G R K\Y C K\P(-)
(R)M F C|K/P/L/D/Q/Q/C/N/K/D/L H/C C K P L K C R R S N N G R K\Y C K P(-) z5
ETD
CID
HCD
CysIodoacetamide
56spectra
2peptides
Karl ClauserProteomics and Biomarker Discovery 22
Group 18 - 6Cys - only intact MS/MS z5-10
CysIodoacetamide
56spectra
2peptides
MFCKPLDQQCN reads: 6
CRRSNNGRKYCKP reads: 12
CNKDLHCCKPLKC reads: 1
CCKPLKCRR reads: 11CRKPLKKRL reads: 66
Karl ClauserProteomics and Biomarker Discovery 23
Group 18 - 6Cys - only intact MS/MS z8-9 ETD(R)M F C K P L D\Q|Q\C\N K|D L H\C C|K P L/K C/R|R/S/N/N/G/R/K Y C/K P(-)
(R)M F\C\K P L D\Q|Q\C N K\D L H\C\C|K P/L/K C/R/R/S/N/N/G R/K Y C/K P(-)
Karl ClauserProteomics and Biomarker Discovery 24
Group 18 - 6Cys - only intact MS/MS z6-7, ETD(R)M F\C\K P L D\Q|Q\C N K\D L H\C C K P L K C|R/R/S/N/N/G/R/K/Y C/K P(-)
(R)M F C\K P L D\Q\Q\C N K\D/L H\C\C|K P L K\C/R/R S/N/N/G/R/K/Y C K P(-)
Karl ClauserProteomics and Biomarker Discovery 25
Group 18 - 6Cys - only intact MS/MS z5(R)M/F C/K P L\D\Q|Q\C N K D L H C C K P L K\C\R\R/S/N/N/G/R/K Y C K P(-)
(R)M F C|K/P/L/D/Q/Q/C/N/K/D/L H/C C K P L K C R R S N N G R K\Y C K P(-)
ETD
HCD
Karl ClauserProteomics and Biomarker Discovery 26
BLAST2GO – Functional Annotation
• Export FASTA of Valid Hits from SM• Run BLAST step• Run GO mapping step• Run annotation step
• Run InterProScan, SIGNALP• Run GO-Slim• Export results to SM categories file
Karl ClauserProteomics and Biomarker Discovery 27
Venom Toxin Nomenclature
King GF, Gentz MC, Escoubas P, Nicholson GM A rational nomenclature for naming peptide toxins from spiders and other venomous animals.Toxicon 52 (2008) 264–276.
Karl ClauserProteomics and Biomarker Discovery 28
Next Steps
• Improve paralog inclusion in RNA-Seq Transcript Assembly.• Call full length gene, predict signal & propeptide.
• 10 Cys toxins lack propeptide?• CCC 8 cys toxins propeptide so long that the assembly
doesn’t extend upstream enough to cover signal peptide?• Group 23 looks like a fusion toxin, misassembled?
• 12 Cys, 2 pairs of adjacent CC’s• perhaps 2 concatenated 6 Cys toxins• Present in 2 libraries
• Run SM homology searches• Improve SM monoisotopic m/z assignment for z>4.• Obtain ETD MS/MS of intact toxins after MeAziridine Cys
mod.• Assemble spectra de novo.• Name novel toxins.
Karl ClauserProteomics and Biomarker Discovery 29
Adding Charge to Cys for better ETD
Fig. 1. Overview of the de novo sequencing strategy. (I) UV trace of HPLCseparation of crude venom extract from C. textile. (II) MALDI TOF MS offraction i after no treatment, reduction, and alkylation. (III) On-line LC ESIMS/MS using CAD and ETD on reduced and alkylated aliquots of fraction i. The final step shows the conversion of Cys residues to dimethylated Lys analogs followed by ETD fragmentation; MS/MS is shown for the (M5H)5 ion of the 1,889.715 Da species in II. c ions are indicated by and z ions by . Shell image Copyright 2005, Richard Ling.
Ueberheide BM, Fenyo¨ D, Alewood PF, and Chait BT. PNAS 2009 106 , 6910–6915 .
2-Methylaziridine
Karl ClauserProteomics and Biomarker Discovery 30
Lys-C Cleaves at MeAziridine Cys mod
Group 4.1 u1-hexatoxin-hi1a 37_1680.3d.comp452_c0_seq2_F2_R3 Hi_3day_Q17_37 C G|D|Q|V C|G\A|G|T\C 37 11 59 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.0830.0857.3.pkl.spo C G|D|Q|V C|G|A|G|T\C 37 11 59 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2261.2381.3.pkl.spo C G/D/Q/V C G\A G\T\C 37 11 59 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.0810.0947.2.pkl.spo C G/D/Q|V C\G\A\G\T\C 37 11 59 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2177.2361.2.pkl.spo C G/D/Q/V C G\A\G\T\C 37 11 59 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.2391.2427.2.pkl.spo C G|D|Q V\C G|A\G|T\C\C|A/E|Y P|E I\H\C\K 37 21 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4477.4477.6.pkl.spo C G D Q V C G A G T C C/A E/Y/P E I H C K 37 21 49 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4476.4476.6.pkl.spo C|G|D|Q V\C|G|A G\T|C/C A E|Y P|E|I|H\C\K 37 21 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4381.4431.5.pkl.spo C|G|D|Q V\C\G|A G\T\C|C|A\E|Y P|E|I|H\C\K 37 21 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4491.4491.5.pkl.spo C/G|D|Q\V\C|G|A G\T|C|C\A E|Y P|E|I|H\C\K 37 21 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4531.4531.5.pkl.spo C/G|D|Q V C|G/A\G\T|C\C\A|E|Y P|E|I|H\C\K 37 21 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4411.4461.4.pkl.spo C G D/Q V/C G A G T C C/A E/Y|P E\I\H\C\K 37 21 49 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4522.4522.3.pkl.spo C/G/D/Q V C\G\A G|T C C A E|Y P|E|I/H\C\K 37 21 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4387.4441.3.pkl.spo C/G/D/Q V C|G A G/T C C A E|Y P|E/I/H\C\K 37 21 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4471.4481.3.pkl.spo G A/G T C C A E/Y/P E|I H C K 43 15 49 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4225.4299.4.pkl.spo G/A/G/T\C|C|A|E|Y P|E|I\H|C\K 43 15 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4227.4301.4.pkl.spo G A\G T C C A E/Y/P E I H C K 43 15 49 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4226.4300.4.pkl.spo G A/G T C C A E|Y|P E\I H\C\K 43 15 49 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4259.4259.3.pkl.spo G/A/G/T C\C|A|E|Y P|E|I\H\C\K 43 15 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4261.4304.3.pkl.spo G A/G T C/C/A E|Y|P E\I\H\C\K 43 15 49 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4292.4292.2.pkl.spo G A G/T C C/A/E/Y P\E\I H C\K 43 15 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4294.4294.2.pkl.spo G A/G T C/C/A/E|Y|P E I|H|C\K 43 15 49 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4293.4293.2.pkl.spo C/A|E|Y/P/E|I\H|C\K 48 10 49 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4134.4237.3.pkl.spo R V\G\Q|L|Y|D|I C V D S E A T K 58 16 33 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6342.6489.4.pkl.spo R/V|G|Q/L|Y/D|I\C/V|D|S|E|A|T\K 58 16 33 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6344.6491.4.pkl.spo R V\G\Q\L\Y/D/I/C/V D/S E/A/T K 58 16 33 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6343.6490.4.pkl.spo R V G Q\L\Y\D|I|C/V/D|S E\A T\K 58 16 33 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6329.6526.3.pkl.spo R/V/G|Q|L|Y/D|I\C|V|D|S|E|A\T\K 58 16 33 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6331.6528.3.pkl.spo R V G Q\L\Y\D\I|C/V/D/S E A/T/K 58 16 33 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6330.6527.3.pkl.spo R V G Q L|Y|D|I\C|V|D|S E\A\T\K 58 16 33 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6336.6486.2.pkl.spo R/V/G/Q L\Y\D I C\V\D\S\E\A\T\K 58 16 33 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6338.6488.2.pkl.spo R V G Q L\Y\D|I\C V|D|S E|A|T|K 58 16 33 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6337.6487.2.pkl.spo L Y/D|I|C/V D|S E A T K 62 12 33 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6372.6416.2.pkl.spo L/Y/D/I C/V|D|S\E\A\T\K 62 12 33 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6374.6418.2.pkl.spo L Y|D|I|C/V D/S E/A/T/K 62 12 33 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6373.6417.2.pkl.spo Y/D|I|C/V D|S/E/A/T K 63 11 33 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6346.6496.2.pkl.spo Y/D/I C/V D|S|E\A\T\K 63 11 33 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6348.6498.2.pkl.spo Y D|I|C/V D/S/E/A/T/K 63 11 33 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6347.6497.2.pkl.spo D/I|C/V/D|S E/A\T K 64 10 33 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6359.6469.2.pkl.spo D/I C/V/D/S|E\A\T\K 64 10 33 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6361.6471.2.pkl.spo D I|C/V/D/S E/A/T/K 64 10 33 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6360.6470.2.pkl.spo D/S/G|N/H/L|F\F\C P C|D|E|G|m|Y\C 74 17 16 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6534.6591.4.pkl.spo D/S/G/N/H\L|F|F C P\C|D|E\G|m\Y\C 74 17 16 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6514.6514.3.pkl.spo D/S/G/N/H|L|F/F\C P\C|D|E/G|m\Y\C 74 17 16 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6548.6588.3.pkl.spo D/S/G/N H|L|F\F\C P C D E G M\Y\C|D|m\N/S W S\C 74 24 9 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.7231.7231.4.pkl.spo D S G N H\L F|F\C P C|D|E|G\m Y\C|D/m N|S W\S\C 74 24 9 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6944.6944.5.pkl.spo D/S G N H L F F C/P C D E G m\Y C D m\N\S\W\S\C 74 24 9 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6936.6936.4.pkl.spo D/S/G/N H\L|F\F\C P C\D\E\G\m Y C|D|m|N|S W\S\C 74 24 9 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6938.6938.4.pkl.spo D/S G/N H|L\F F\C P C\D\E\G\m Y\C\D|m\n/S W S C 74 24 9 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6968.6968.4.pkl.spo D/S G N H|L F/F\C P\C|D|E\G\m|Y\C|D|m|N|S W|S\C/Q\K 74 26 7 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6581.6581.6.pkl.spo D S\G N H\L\F F C P C D E G m Y C D m N S/W/S C Q K 74 26 7 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6580.6580.6.pkl.spo D S G N H\L F\F\C P C\D|E\G\m Y C|D|m|N\S\W/S\C|Q\K 74 26 7 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6541.6541.5.pkl.spo D S/G/N H|L F|F\C P C\D\E\G\m Y\C|D|m|N\S\W\S\C|Q\K 74 26 7 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6578.6578.5.pkl.spo D S\G N H L\F F\C P C D E G m/Y C D m N\S|W/S C Q K 74 26 7 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6540.6540.5.pkl.spo D S|G N H L F F C/P C D E G m Y C D m N\S|W/S C Q K 74 26 7 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6577.6577.5.pkl.spo D/S/G/N H\L|F F C P C D E G m Y C|D m\N|S\W|S\C\Q\K 74 26 7 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6538.6538.4.pkl.spo D/S G N H|L F\F\C P\C|D|E|G\m\Y\C\D\m\n|S W|S\C/Q\K 74 26 7 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6574.6574.6.pkl.spo D S\G N H L\F F C P C D E G m Y C D m n S/W/S C Q K 74 26 7 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6573.6573.6.pkl.spo D/S G N H|L F\F\C P C\D|E|G\m Y\C|D\m\n S|W/S C Q K 74 26 7 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6604.6604.5.pkl.spo D S\G n H L F/F C P C D E G m Y C D m N S/W/S C Q K 74 26 7 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6603.6603.5.pkl.spo D/S G N/H|L F|F\C P C D|E\G\m\Y\C|D m/N\S W\S C|Q|K K 74 27 6 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6228.6228.5.pkl.spo D S\G N H L F\F\C P C D E G m Y C D m N S\W/S C Q K K 74 27 6 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6227.6227.5.pkl.spo D S\G n H L F F C P C D E G m Y C D m n S W/S C Q K/K 74 27 6 HCD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.6233.6233.6.pkl.spo D m|N|S\W|S|C 91 7 9 CID K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4175.4175.2.pkl.spo D/m/N/S W\S\C 91 7 9 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.4177.4177.2.pkl.spo D/m|N|S\W\S\C|Q|K\K 91 10 6 ETD K121115_Hi_dsrak_ff4f4f4CHEsa_cccc_01.3327.3327.3.pkl.spo
Cys cleavages observed only in most abundant proteinsKinetics: K >> C
Karl ClauserProteomics and Biomarker Discovery 31
MeAziridine Cys mod Yields Great ETD Spectra(K)S/Y\W|K/G|H/G\V\C\S A\S|L|F|E|R|L|K\G\C(-)
(K)C/I|G|Q|W|Y/N|G|Q|A S C|Q|S/T\F|m|G|L\F\K(S)
Karl ClauserProteomics and Biomarker Discovery 32
MeAziridine Cys mod Yields Great ETD Spectra(K)C/N|Y|H\C|E\C\C|G/A T V\A\C|S/T|V\Y/V|G G\K(E)
(K)C Y\C/D|Y|G|L|F|G|N\C|N\C|Y\K(R)
Karl ClauserProteomics and Biomarker Discovery 33
MeAziridine Cys mod Yields Great ETD Spectra
(Q)T C/G|G P|D D\C G|E|G/S C\C V|G|S|F|S|R\K(C)
(N)W C|A|K/N\E|D\C\C\C P M\K(C)
Karl ClauserProteomics and Biomarker Discovery
CentipedesSpidersScorpions
S. Morsitans
A. xerolimniorum
L. weigensis
H. jugulans
C. westwoodi
E. rubripes
U. manicatus
L. variatus
L. buchari
C. squama
I. vescus
Thereuopoda sp.
S. foelschei
C. Tropix
H. infensa
7 scorpions4 centipedes1 spider