FUNCTIONAL PEPTIDOMICS OF AMPHIBIAN VENOMS The dermal granular - PowerPoint PPT Presentation

FUNCTIONAL PEPTIDOMICS OF AMPHIBIAN VENOMS

The dermal granular (venom) gland The dermal granular (venom) gland � � The venom producing cells of the Stimulation/injury causes opening of the granular gland produce secretions, granular gland and emptying of the which are stored in the cytoplasmic syncitium (containing cellular continutiy of the syncytium constituents) onto the dermal surface of the frog; this includes poly A+ mRNA

Focused interests Focused interests - antimicrobials � - antimicrobials � - anticancer anticancer � - � - novel novel neurohormones neurohormones � - � - vasoactives vasoactives � - � - cell growth regulators cell growth regulators � - � - anthelmintics anthelmintics � - � - insecticides insecticides � - � - molecular targets in pathology molecular targets in pathology � - �

Pharmaceutical Biotechnology Pharmaceutical Biotechnology Research Group, UUC Research Group, UUC TM “Classic” Ion Trap � 1 x LCQ 1 x LCQ TM “Classic” Ion Trap � � 1 x Voyager DE MALDI 1 x Voyager DE MALDI - - TOF TOF � � 1 x QToF 1 x QToF Ultima Ultima �

LCQ Ion Trap LCQ Ion Trap

Fractionation of venom Fractionation of venom RT: 0.00 - 70.02 SM: 7B NL: 56.81 44.46 45.55 100 5.43E8 TIC MS 59.47 Feb25_Pac e 80 c 46.29 n 63.14 hymed_dac a d nic_5mg- n 48.49 60 u 500ul b 55.50 A e v i 66.08 40 40.63 t 34.95 a l e 26.53 R 54.48 27.15 36.48 20 33.82 17.42 6.50 19.41 5.54 8.08 15.65 0 NL: 26.46 42.79 46.07 50.10 56.39 100 1.00 33.87 59.25 Analog 1 Analog 80 17.36 Feb25_Pac 27.07 34.90 hymed_dac 63.07 60 nic_5mg- 36.40 14.22 500ul 27.85 40 19.36 54.58 65.93 5.77 20 6.59 31.36 19.96 11.23 4.04 0 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Time (min)

LC- -UV UV- -MS Analysis of MS Analysis of Litoria aurea Litoria aurea skin secretion skin secretion LC RT: 0.01 - 240.00 124.99 100 LC-MS chromatogram 90 80 Relative Abundance 70 60 112.42 50 129.37 40 30 20 97.41 10 37.15 73.77 132.54 3.44 88.21 2 24.77 151.80 176.14 208.29 223.50 43.24 63.87 198.30 0 3.47 24.55 100 90 80 LC-UV chromatogram 70 60 (214nm) 50 28.86 40 124.86 30 37.04 122.40 20 126.17 40.69 4.25 85.58 98.08 10 13.42 43.13 73.68 132.46 58.78 165.45 174.80 187.03 210.99 220.73 2 0 20 40 60 80 100 120 140 160 180 200 220 Time (min) 200ul injection. 0-80% ACN in 0.05% TFA water in 240 minutes.

LC- -UV UV- -MS Analysis of MS Analysis of Litoria aurea Litoria aurea skin secretion skin secretion LC Expanded area RT: 10.73 - 44.08 37.15 100 90 LC-MS chromatogram 37.28 80 34.89 Relative Abundance 70 60 50 37.45 40.78 32.41 24.77 33.14 40 36.23 25.16 31.19 38.39 30 25.47 27.16 41.04 21.96 38.59 17.56 30.40 19.79 20 22.69 16.45 13.49 11.05 10 0 24.55 100 90 LC-UV chromatogram 80 70 60 (214nm) 50 28.86 40 30 37.04 27.59 20 34.81 32.85 40.69 10 36.17 10.77 13.42 38.27 4 19.65 31.36 16.72 15.12 21.75 24.18 0 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Time (min) 200ul injection. 0-80% ACN in 0.05% TFA water in 240 minutes.

Serotonin- -based Compounds based Compounds Serotonin Serotonin Serotonin R = NH 2 H O R N N’ Methyl Methyl serotonin N’ H R=NHCH 3 Bufotenine Bufotenine N’N’N’ Trimethyl serotonin Trimethyl serotonin N’N’N’ R = N(CH 3 ) 2 R = N + (CH 3 ) 3

MS/MS of isobaric compounds MS/MS of isobaric compounds m/z 160.1 CID product 160.1 CID product m/z Trimethyl serotonin Trimethyl serotonin ion ion m/z 219.1 219.1 m/z Energy Energy m/z m/z 59.1 CID fragment 59.1 CID fragment - N + (CH 3 ) 3 5- -MeO MeO DMT DMT 5 m/z 174.1 CID product 174.1 CID product m/z m/z 219.1 219.1 m/z ion ion Energy Energy m/z 45 CID fragment 45 CID fragment m/z -N(CH N(CH 3 ) 2 - 3 ) 2

MS S#: 1-36 RT: 0.02-0.70 AV: 36 NL: 1.20E7 219 F: + c Full ms [ 50.00 - 500.00] 219.2 100 CH 3 95 analysis of + 90 H O N CH 3 85 80 CH 3 75 N’N’N’ 70 65 N Relative Abundance 60 55 trimethyl H 50 45 40 N’N’N’ trimethyl 35 serotonin 160 30 25 serotonin 160.2 20 220.2 15 10 391.1 5 413.3 161.2 284.4 352.2 369.3 421.3 453.3 149.2 221.2 249.2 313.2 481.3 495.3 74.0 95.2 115.1 132.2 190.2 215.2 and 0 50 100 150 200 250 300 350 400 450 500 m/z 219 S#: 4-15 RT: 0.06-0.25 AV: 12 NL: 7.34E7 F: + c Full ms [ 50.00 - 500.00] 219.1 100 CH 3 95 90 N 5-methoxy O H 3 C 85 CH 3 80 75 70 dimethyl N 65 H + Relative Abundance 60 H 55 50 tryptamine 45 174 5-methoxy dimethyl 40 35 174.3 30 tryptamine 25 20 220.1 15 DISTINGUISHED! DISTINGUISHED! 10 175.3 5 390.2 217.3 221.1 354.8 130.3 159.3 378.1 71.1 85.0 104.1 118.2 176.3 249.1 277.0 292.9 304.0 326.5 421.2 0 100 150 200 250 300 350 400 m/z

CH 3 CH 3 H O NH H O N H + H + CH 3 N N H H MS N' methyl 5-HT MS bufotenine 191 205 -(CH 3 ) 2 NH CH 3 + + -CH 3 NH 2 H O N CH 3 H O NH 3 -45 -31 CH 3 N N H H -(CH 3 ) 3 N -NH 3 MS 5-HTQ MS 5-HT -17 219 177 -59 H O N H + H MS 2 160 -28 -CO N H + H MS 3 132 -NH 3 -17 ? MS 4 115

MS analysis of peptide mixtures Bradykinin and threonine-6 bradykinin from Bombina orientalis Mar02_eleanor_bo_fr97b#50-79 RT: 1.30-1.59 AV: 19 NL: 2.25E6 F: + c Full ms [ 150.00-1100.00] 531.0 100 90 80 70 e c n a 60 d n u b A 50 e v i t a l 40 e R +2 charge states 30 +1 charge states 538.7 20 1074.3 549.8 10 556.6 630.1 562.5 973.5 904.3 864.7 675.5 697.4 752.5 811.3 837.6 918.4 1005.5 0 550 600 650 700 750 800 850 900 950 1000 1050 m/z

MS/MS Sequencing of Bradykinin Mar02_eleanor_bo_fr98#69-92 RT: 1.31-1.82 AV: 14 NL: 5.42E3 F: + Z ms [ 526.00-536.00] 530.8 100 ZoomScan high 90 resolution mass This isolates the ion of 80 interest, even in a 70 analysis e c n complex mixture. 531.2 a 60 d n u b Fragmentation can then A 50 e v i t a take place. l 40 e R 30 531.7 20 10 534.7 532.3 535.2 527.3 530.6 532.7 526.8 529.4 528.4 533.2 533.6 526.1 527.8 535.7 0 526 527 528 529 530 531 532 533 534 535 536 m/z

MS/MS Sequencing of Bradykinin Mar02_eleanor_bo_fr97b#70-122 RT: 1.63-2.95 AV: 48 NL: 9.83E4 F: + c Full ms2 531.00@40.00 [ 145.00-1100.00] 904.3 MS/MS fragmentation profile 100 90 can be used to call the peptide 80 sequence 70 e c n a 60 452.8 d n u b 886.3 642.2 A 50 e v i t a l 40 e R 807.3 30 858.3 710.2 20 624.2 402.1 643.2 1001.3 175.1 906.3 555.2 370.3 501.3 333.2 254.2 10 711.2 607.2 1002.4 653.3 288.2 748.2 217.2 907.3 1004.3 0 200 300 400 500 600 700 800 900 1000 1100 m/z

SEQUEST TM TM SEQUEST STEP 1. STEP 2. SEQ 1 Mass Search of Indexed SEQ 2 Protein and DNA Databases based on the peptide SEQ 3 parent molecular weight MS and MS/MS Spectrum SEQ 4 Predicted MS/MS Spectra STEP 3. STEP 4. Compares and correlates Rank hits, predicted and sequence peptides, experimental MS/MS spectra create summary and Protein I.D. MS/MS peptide sequencing software

(a) y 8 RPPGFSPFR S#: 75-108 RT: 1.63-2.56 AV: 34 NL: 9.80E4 F: + c Full ms2 531.00 [ 145.00 - 1100.00] b 6 904.3 100 b 7 95 b 8 b 4 90 85 y 7 80 y 5 75 b 5 y 3 70 65 b 3 60 452.8 Relative Abundance b 1 55 y 6 642.2 886.3 50 905.3 45 40 807.3 35 y 4 30 b 2 y 2 y 1 25 858.3 710.2 624.2 20 402.1 643.2 404.3 15 808.3 1001.4 175.1 555.2 453.7 906.3 370.3 333.2 10 254.2 711.2 841.3 1002.4 419.2 614.3 237.2 506.4 653.3 492.4 5 288.2 217.2 748.2 1003.4 907.3 983.4 0 200 300 400 500 600 700 800 900 1000 m/z

(b) RPPGFTPFR y 8 S#: 25-33 RT: 0.41-0.65 AV: 9 NL: 6.24E4 F: + c Full ms2 538.00 [ 150.00 - 1100.00] y 5 918.3 100 95 b 6 90 b 8 85 y 7 y 3 80 75 y 4 70 y 6 656.2 65 459.9 60 y 2 Relative Abundance 900.3 55 b 7 919.3 50 b 9 45 b 4 b 5 40 821.2 b 1 35 b 2 b 3 402.1 30 515.9 25 y 1 724.2 657.2 20 612.3 872.3 508.3 411.4 822.3 15 638.2 507.5 1015.4 157.1 377.4 316.2 725.2 419.2 855.3 254.1 555.2 10 920.3 333.2 5 237.2 667.3 274.2 777.5 998.2 971.3 1017.4 0 200 300 400 500 600 700 800 900 1000 m/z

Transcriptomic strategy strategy Transcriptomic Extraction of peptide mixtures peptide mixtures from from frog frog sample sample followed by solvent followed by solvent Extraction of extraction, centrifugation, lyophilization lyophilization, etc , etc extraction, centrifugation, Fractionation of the mixtures the mixtures by HPLC by HPLC and v and verification erification of peptide mass of peptide mass Fractionation of by MALDI by MALDI- -TOF mass spectrometry TOF mass spectrometry Verification V erification of existing sequence by MS/MS (LCQ of existing sequence by MS/MS (LCQ) and d ) and determination etermination of of novel peptide sequence by Edman Edman degradation degradation novel peptide sequence by Screening of collected fraction Extraction of polyA polyA RNA/total RNA RNA/total RNA Screening of collected fraction Extraction of for biological activity for cDNA cDNA library library for biological activity for