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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)
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
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.
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)