Lecture delivered during CSI Symposium -2016

1. Agilent 1100 LC MSD Model
In 1997 HP introduces its first benchtop
quadrupole LC/MS the 1100 Series LC/MSD
Journal of the American Society forMassSpectrometry 2001, 12 , 428–438
An Electrospray Ionization Mass Spectrometry Investigation of
1-Anilino-8-NaphthaleneSulfonate (ANS) Binding to Proteins
Learning Biological Mass Spectrometry

2. 1989 Physics W. PAUL and H. G. DEHMELT
…………for the development of the ion trap technique
1922 Chemistry F. W. ASTON
…………discovery, by means of the mass spectrograph, of
isotopes, in a large number of non-radioactive elements,
and for the enunciation of the whole-number rule.
1906 Physics J. J. THOMSON
…...........theoretical and experimental investigations on the
conduction of electricity by gases
2002 Chemistry K.TANAKA and J. B. FENN
…………development of soft desorption ionisation methods for
mass spectrometric analyses of biological
macromolecules
Mass Spectrometry

3. Prof. John Fenn
2002 NOBEL PRIZE IN CHEMISTRY
http://www.chem.ed.ac.uk/teaching/undergrad/chemistry4/lectures/moduleE/msd/PB/PBmsdlecture.pdf
Science 1989, 246,64-71
Fenn J B, ‘Electrospray wings for
molecular elephants’ (Nobel lecture),
Angew. Chem. Int. Ed., 42, 3871 (2003)
R.Sinatra etal., Science 2016,354,596

4. Serum
Red blood cells
Osmotic lysis releases
Hemoglobin molecules
Blood
Sample
Centrifuged
Distilled H2O
Human serum albumin
Human hemoglobin
Extracellular protein
Intracellular protein
Proteins can be chemically modified by reactive
molecules generated in cells under disease conditions

5. Mass (M) : 66438 Da
 chain
15126Da
 chain
15865 Da
Human Hemoglobin
m/z800 1000 12000
40
100
934.2
757.2
946.3
1009.3
1134.3
1164.4
(+14)
(+16)
(+13)
(+17)
(+15)
Human Serum Albumin
Electrospray Mass Spectra: Proteins

6. Molluscivorous (Prey --- Mollusc)
Piscivorous (Ingestion)
Marine cone snail venom may contain several hundred peptides that
target membrane receptor and channels. Conotoxins are upto 50
residues in length and contain multiple
disulfide bonds
Prof.K.S.Krishnan ( 1946-2014)

7. C.Amadis
C.Araneosus
C.Loroisii C.Monile C.Ebraeus
C.Miles
C.Frigidus
C.Lividus C.Litteratus
Vermivorous.
Molluscivorous

8. “CONOTOXINOMICS” (Olivera B. M., Toxicon 39, 7-14, 2001)
SINGLE
DISULFIDE
Conopressin Contryphan
Peptide Toxins Modifying Enzymes
Many Snails, Many Peptides and Many Enzymes

9. Target neuropathic pain
potent blocker of neuronal nAChRs ( α 9 α 10 )
Vc1.1 clinical trials discontinued after phase 2A. Efficacy concerns, lower affinity for human
receptors compared to mouse receptors
α-conotoxin Vc1.1
NMR solution sructure (PDB:2H8S)
Nicotinic acetylcholine receptors antagonists ( Alpha Conotoxins)
GCCSVPPCIANHPELCG
DCCSRPPCRWSHPELCS
ECCSHPACNVDHPEICR
GCCSVPPCIANHPELCV
Vc1.1 c.victoriae
PeIA c.pergrandis
LvIA c.lividus
AmIA c.amadis
AmIB c.amadis
AmIIA c.amadis
AmIIB c.amadis
Eline K. M. Lebbe, Steve Peigneur, Isuru Wijesekara and Jan Tytgat ,
Conotoxins Targeting Nicotinic Acetylcholine Receptors: An Overview,
Mar. Drugs 2014, 12, 2970-3004;

10. Sequencing peptides from natural mixtures
Complex mixtures Purified peptides
HPLC
Edman sequencingMass spectrometry
Difficulties with
post translational
modifications
Difficulties with
de novo
sequencing
Absence of adequate
numbers of fragment ions
to facilitate unambiguous
residue assignment
Liquid chromatography /
Mass spectrometry LC-MS
electrospray ionisation permits
ready coupling of HPLC and
Mass spectrometry
Conus venom contains a complex mixture of
heavily post translationally modified peptides

11. 0 10 20 30 40 50 60 Time [min]
0
1
2
3
4
7x10
Intens.
620.6
625.9
626.2
626.6
626.9
627.2
627.6
627.9
0.00
6
Intens.
620 621 622 623 624 625 626 627 628m/z
724.8
725.3
725.8
726.3
724.3
0.0
0.2
0.4
0.6
0.8
6x10
Intens.
724.0 724.5 725.0 725.5 726.0 726.5 727.0 m/z
Demonstration of High Sensitivity at High Resolution
Nano LC MS Profile of Crude venom of C. araneosus
5 mg Crude Venom
in 1000 ul
(M+3H)+3, M:1874.7Da (M+2H)+2
M:1446.6 Da
Diluted to 100000 times
Injection volume: 1ul
~ < 0.000000005 mg

12. REDUCTIVE ALKYLATION OF DISULFIDES
S
S
SH
SH
S
SpH 3
TCEP
pH 3
ΔM: 2*126.1= 252.2
N C C
CH2
S
O
H
H
M(RES)= 228
NEM
Alkylating agents M ΔM Mres
N-Ethylmaleimide 125.1 126.1 228
Iodoacetamide 184.96 58 160
2-Vinylpyridine 105.14 106.1 208

13. 1000 1500 2000 2500 3000 3500 4000
CRUDE
CRUDE_TCEP_NEM
1249.4
1312.4
1753.4
1447.3
1730.4
1755.4
1813.4
1859.6
2203.6
2486.6
2512.6
2569.6
1816.5
2615.8
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Intens.[a.u.]
0
2000
4000
6000
8000
Intens.[a.u.]
2500 3000 3500 4000
3471.33471.3
3599.2
3724.2
3850.2
3975.2
REDUCTIVE ALKYLATION OF CRUDE VENOM
MAJOR
INCOMPLETE ALKYLATIONΔM = 756
3S-S
ΔM = 756
3S-S

14. Mass: Mono:1368.681 / Average:1369.518 C63H96N14O20
+ + +- -- -
RNH2
RCOOH
RNH3
+
RCOO-
pKa
pKa
Distribution of multiple of charged states (M+nH)n+
In ESI-MS spectra determined by number of basic, protonatable sites
(memory of the solution state is retained)
In MALDI spectra Na+ / K+ adducts determined by number of RCOO-

15. H2N-CHR1-CO-NH-CHR2-CO-NH-CHR3-CO-NH-CHR4-CO2H
b1 b2 b3a1 a2 a3c1 c2 c3
y3 y2 y1x3 x2 x1z3 z2 z1
The Types of Ions Generating from Backbone
Cleavage of Protonated Linear Peptides
b2 – a2 = 28 Da

16. 1813.634
1755.615
1816.653
1835.582
1845.672
1759.646
1798.523
1777.551
1795.593
1829.676
1787.592
1838.595
1821.768
1771.615
1842.699
1850.620
1768.642
1780.579
1832.688
1783.604
1790.871
1857.597
4_DHB 0:M21 MS Raw
0
1
2
3
4
5
4x10
Intens.[a.u.]
1869.325
1855.308
1811.322
1883.336
1797.308
1891.300
1877.288
1906.306
1833.299
1826.333
1819.285
1886.328
1858.313
1894.288
1909.280
1849.286
1897.345
1901.312
1800.312
1837.288
1846.314
4_ACETYLATION 0:G15 MS Raw
0
1000
2000
3000
Intens.[a.u.]
1760 1780 1800 1820 1840 1860 1880 1900
m/z
0
2
4
6
1825 1850 1875 19
ACETYLATION
INFERENCE :
1 ACETYLATION SITE
(K, Amino Terminus)
4 ESTERIFICATION SITES
(D, E, Carboxyl terminus)
AMINO & CARBOXYLIC ACID GROUP MODIFICATION
ΔM:42 ΔM:42
ESTERIFICATION
1827.4
1841.4
1855.4
1869.4
ΔM:14
ΔM:28 ΔM:42
ΔM:56

17. 679.7347
878.2567
907.2597
1171.3387
631.2625
11th july--c. Ar-crude-repeat_1_01_1814.d: +MS, 29.7-30.0min #3487-3523
0
1
2
3
4
5
5x10
Intens.
600 800 1000 1200 1400 1600 1800 m/z
878.2567
878.7579
879.2572
879.7566
880.2560
880.7555
907.2597
11th july--c. Ar-crude-repeat_1_01_1814.d: +MS, 29.7-30.0m
0
1
2
3
4
5
5x10
Intens.
880 885 890 895 900 905
M+2H
907.2597
907.7611
908.2603
908.7598
909.2590
909.7586
910.2584
11th july--c. Ar-crude-repeat_1_01_1814.d: +MS, 29.7-30.0min #3487-3523
900 905 910 915 920 925 m/z
M+2H
585.8376
586.1723
586.5056
586.8382
587.1717
587.5046
11th july--c. Ar-crude-repeat_1_01_1814.d: +MS, 29.7-30.0min #3487-3523
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens.
584 585 586 587 588 589 590 591 m/z
M+3H
603.8115
604.1443
604.4784
604.8130
605.1726
605.5077
605.8407
606.1744
606.5070
606.8410
11th july--c. Ar-crude-repeat_
0
2000
4000
6000
Intens.
603 604 605 606 607 608
M+3H
2+
585.8
3+
2+
2+ 2+ 3+ 3+
SEQUENCE IS LIKELY TO CONTAIN TWO CHARGEABLE SITES
(K,R,H, Amino Terminus)
V. LOW INTENSITY
LC/ESI-MS (Q-TOF) SPECTRA OF CRUDE CONUS ARANEOSUS
VENOM
IONS CORRESPONDING TO PEPTIDE 1813 & 1755

18. X Y H V V S A N T X W S X V
b ions
y ions
274.3
2056.6
387.1
1943.6
550.1
1779.6
687.2
1642.6
786.3
1543.6
G P
885.7
942.5
1039.5
1339.5
1426.5
1497.4
1611.4
1712.6
1825.8
2011.8
2098.8
2211.8
1387.6
1290.5
H Y
1176.5
Partial Sequence Information Obtained through Chemical Derivatization
Unsolved Problems
1. N-terminal residues
2. Distinction between Ile (I), Leu (L) and Hyp (O) at three positions(X)
X Leucine (L) 113.1 Da Isoleucine (I) 113.1 Da Hydroxyproline (O) 113.2
NH
O
CH3
CH3 NH
O
CH3
CH3
N
O
OH
Isobaric amino acids
May be resolved through
high resolution mass measurements
FT-ICR MS, Orbitrap
??

19. X Y H V S A N T X W S X V
b ions
y ions
274.2
2056.6
387.1
1943.6
550.1
1779.6
687.2
1642.6
786.3
1543.6
R P
942.5
1039.5
1339.5
1426.5
1497.4
1611.4
1712.6
1825.8
2011.8
2098.8
2211.8
1387.6
1290.5
X = I/L/O
H Y
1176.5
Improved Sequence Information through MS3 Experiment
Unsolved Problems
1. N-terminal residues ( W or S-V- or V-S-) (186.9 Da)
2. Distinction between Ile (I), Leu (L) and Hyp (O) at three positions
186.9
S??

20. e
CH4
e
-
(M+2H)+2(M+2H)
+
(M+H)+N-C bond cleavage
N
N
O
N
OR2
R3H
H
H
Fluoranthene
Fluoranthene
radical anion
Generation of Fluoranthene radical anion
Electron Transfer Dissociation (ETD)
c ions
z ions
Transfer of e

21. Identification of Missing N-terminus Fragment
z20
2039.9
2116.9
2199.0
2226.0
2249.0
2271.0
2286.0
2303.0
2313.0
0.0
0.2
0.4
0.6
0.8
1.0
4x10
Intens.
2050 2100 2150 2200 2250 2300 m/z
z19
z17
-S-V-
S-
c18
Ser-Val (S-V)

22. 2040.6
0
200
400
600
800
Intens.
2036 2040 2044 2048 2052 m/z m/z
2115.9
2116.9
2117.9
2118.9
0
1000
2000
3000
4000
5000
Intens.
2105 2110 2115 2120 2125 2130m
Presence of these ions negates the possibility of Hydroxyproline at the positions of X
N
O
OH
OH
N
O
OH
OH
N-C bond cleavage at Xxx- Pro/Hyp segment
c & z ions absent

23. 877.9
d+2
16(L)
998.5
w+2
17(L)
1084.9
db+2
19(I)
b+2
16
913.5
1106.5
b+2
19
High Energy CID Specta of Bt 2328 (BRUKER micrOTOF Q II)

24. S-V-S-L-Y-H-V-R-P-H-Y-S-A-N-T-L-W-S-I-V
Bt 2328: Final Sequence
No match with known protein sequences found through the BLAST analysis
1 10 20

25. 1166.775
1509.962
1537.950
109.347
1696.150
1651.083
871.666
1281.810
570.698
2410.393
1997.588
1623.115
2509.445
1879.371
479.761
158.233
250.079
1811.272
778.715
592.715
1057.715
665.689
364.913
300.960
728.663
2439.806
200.113
528.736
1006.740
1400.790
1246.758
427.815
963.690
1121.792
820.689
893.730
1350.844
2209.091
2112.754
1720.148
1307.861
1936.444
85.453
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens.[a.u.]
250 500 750 1000 1250 1500 1750 2000 2250 2500
m/z
1813 2569
TCEP
NEM
ΔM: 756, 6CYS (3S-S)
MALDI MS/MS OF MH+ 2569
-159
HS
756.6
W
186
D
115
W
186
D
115
D
115
W
186
- D - W - D - W - C* - D - H
W
186
455.8
D
115
C*
228
D
115

26. Number of Conus peptide Expected from a single species: ~100
Number of Conus peptide Obtained from a single species: ~37
(~ 100 x 1/3)
Crude venom
HPLC
Mass spectrometry
Chemical Modification
Insensitivity off-line purifications
Imperfection of chemical reactions
H Teralu, BM Olivera Physiol. Rev. 2004, 84, 41
BM Ueberheide, D Fenyo, PF Alewood, BT Chait PNAS 2009, 106, 6910

27. Isolated
mRNA
Complementary
DNA
Transcriptome
Library
Next Generation
DNA Sequencing
454 Pyrosequencing
Conus Species
Venom
Duct
Illumina 1000 seq

28. conus araneosus
MALDI analysis of HPLC peaks establishes large number of peptide
Conus araneosus – Rameswaram, Tamil Nadu
De novo sequencing is difficult
mRNA
cDNA
NGS
Assembly
Conotoxin gene
>Conotoxin
MKLTCVVIAVLLLTACQLITADDSRGTQKHALRSTTKLSTSTRCKGKGAKCSRLMYDCCTGSCRSGKCG
Masses expected to be present in NGS
Mass spectral sequencing

29. Reading frame 4
Stop codon
Recognizing conotoxin gene related sequences
CC…..C…..C…C…C
signal pro region toxin
Proteolytic cleavage site
Post translation modification sites
Translation: 6 reading frames

30. Collecting shells and genes
C.loroissiC.amadis C.monileC.araneosus C.ebraeus C.lividus
Full
genes
Partial
genes
60
7
Total
toxin
sequences
67
49
20
69
61
20
81
36
20
56
6567
16
83
Post translational modifications create further diversity
72
7
C.Miles C.LitterattusC.Frigidus
93
15
78 49
9
58
53
14
67

31. MLKMGVVLFIFLVLFPLATLQLDADQPVERYAEYKRLLNPDERRGIILHALG
KRCCDWDWCDHLCTCCGG*
signal pro
cleavage site
Mature toxin
Precursor gene sequence for peptide 1813
Mature toxin
CCDWDWCDHLCTCCGG*
Calculated Mass ( oxidized )
M-Superfamily – III ( CC-C-C-CC )
1812
Search fragment from Mass spectral sequence : DWDWCD or DCWDWD

32. 1166.775
1509.962
1537.950
109.347
1696.150
1651.083
871.666
1281.810
570.698
2410.393
1997.588
1623.115
2509.445
1879.371
479.761
158.233
250.079
1811.272
778.715
592.715
1057.715
665.689
364.913
300.960
728.663
2439.806
200.113
528.736
1006.740
1400.790
1246.758
427.815
963.690
1121.792
820.689
893.730
1350.844
2209.091
2112.754
1720.148
1307.861
1936.444
85.453
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens.[a.u.]
250 500 750 1000 1250 1500 1750 2000 2250 2500
m/z
1537.9
C*- C* - D - W - D - W - C* - D - H - L - C* - T - C* - C* - G - G
570.7
1166.8
1509.9
1696.2
1651.1
1281.8
871.7
1997.6
1879.4
2209.1
1811.3
1029.7
1057.7

33. C*- C* - D - W - D - W - C* - D - H - L - C* - T - C* - C* - G - G
EXPECTED FRAGMENTS FOR PEPTIDE 1813 (2569)
NEXT EXERCISE: ASSIGNING THE MASS SPECTRUM USING THE KNOWN SEQUENCE

34. 1166.775
1509.962
1537.950
109.347
1696.150
1651.083
871.666
1281.810
570.698
2410.393
1997.588
1623.115
2509.445
1879.371
479.761
158.233
250.079
1811.272
778.715
592.715
1057.715
665.689
364.913
300.960
728.663
2439.806
200.113
528.736
1006.740
1400.790
1246.758
427.815
963.690
1121.792
820.689
893.730
1350.844
2209.091
2112.754
1720.148
1307.861
1936.444
85.453
0.00
0.25
0.50
0.75
1.00
1.25
4x10Intens.[a.u.]
250 500 750 1000 1250 1500 1750 2000 2250 2500
m /z
822.074
3373.871
3489.230
199.895
342.536
1293.081
1964.464
266.799
1380.145
109.168
1236.069
3257.728
1050.071
455.407
2201.131
1115.173
777.088
1608.307
567.228
886.952
1836.379
780.079
2267.769
2862.376
3077.012
2093.499
2404.972
3455.434
2727.332
3303.489
3188.405
2637.873
0
100
200
300
400
500
Intens.[a.u.]
500 1000 1500 2000 2500 3000 3500
m/z
526.324
1024.868
693.065
1484.911
316.702
599.187
454.369
379.591
878.923
3965.201
1584.808
1382.730
209.930
1240.769
1155.782
127.898
4128.429
1795.083
2112.759
1908.142
2846.520
2225.557
2714.066
3228.290
2388.204
0
50
100
150
Intens.[a.u.]
500 1000 1500 2000 2500 3000 3500 4000
500 1000 1500
500 1000 1500 2500
25002000
2000 3000 3500
PEPTIDE 1813(2569)
PEPTIDE 2775(3531)
CCDWDW CD H LCTCCGG
500 1000 1500 25002000 3000 3500 4000
*
*
*
ACRKKWE FCIVPIIGFIYCCPGLICGPFVCV
DCLPIGSSCHSSEQCCSGWC PPQRVC
*
*
PEPTIDE 3471(4227)

35. +16
Pro
P
Hyp
O
+33
1551.563
1553.573
1584.536
1555.574
1567.570
1568.568
1588.539
1585.535
1573.562
1591.535
1575.548
0.0
0.5
1.0
1.5
2.0
4x10
Intens.[a.u.]
1550 1555 1560 1565 1570 1575 1580 1585 1590
m/z
> C.Am 1584.5
MMSKLGVLLIICLLLFPLTAVPLDGDQPADRPAERMQV
DIPTEHHPMFGAVRGCCGAWACMAGCRPCCG*
GCCGAWACMAGCRPCC - NH2
MH+=1584.5
MH+=1551.6
+Na
+K
GCCPALACAMGCRPCC-NH2
MALDI crude venom
C. Amadis: Two genes, three peptides

36. 570.807
513.776
396.788
709.880
681.860
1794.345
325.809
281.851
610.819
794.903
1011.927
354.737
1213.995
865.955
726.898
1737.143
424.834
1142.930
370.832
2307 0:L15 LIFT 2307.000
250
500
750
1000
1250
1500
Intens.[a.u.]
400 600 800 1000 1200 1400 1600 1800
m/z
b3
y3
b4
b5
b6
b7
b13y4
954.9
y5
y6
y7
y8
1442.1
y9
y13
y4-17
b2
preferential cleavage of XXX-PRO bonds
C.Amadis 1551 assignment of MALDI spectrum
??

37. 17 18 19 20 21 22 23
0.0
0.2
0.4
0.6
0.8
7x10Intens.
23.0 23.5 24.0 24.5 25.0 25.5 26.0 26.5 Time
0.0
0.2
0.4
0.6
0.8
1.0
1.2
70Intens.
27 28 29 30 31 32 33
0.5
1.0
1.5
7
34 35 36 37 38 39 40 41 42Time
05
1.0
1.5
7x10
Intens.
43 44 45 46 47 48 49 Time [min]
0
2
4
6
6x10
Intens.
Time
0.5
1.0
1.5
2.0
2.5
3.0
6x10Intens.
Conus amadis
LC-MS Q-TOF
Mass detected HPLC
Total Ion chromatogram
I II
III IV
V
VI
I - VI
> 40 peaks
ACN / H20
C18

38. 17 18 19 20 21 22 23Time [
0.0
0.2
0.4
0.6
0.8
7x10
Intens.
C AMADiS CRUDE_1_01_2482.d: BPC +All MS
Expanded view of HPLC segment I C.Amadis-Q-TOF
3+
2972.2
2+
1982.9
2+
1982.9

39. 17 18 19 20 21 22 23Time [
0.0
0.2
0.4
0.6
0.8
7x10
Intens.
C.AMADIS CRUDE_1_01_2482.d: BPC +All MS
Expanded view of HPLC segment I C.Amadis-Q-TOF
3484.083+
3598.143+,2+,4+
975.442+
3666.063+,4+,2+
1317.52+
2280.843+,4+
4082.276+,5+
3156.323+,2+,4+
1698.663+,2+,4+
1244.463+,2+,4+
1656.723+,2+,4+
1636.683+,2+,4+
1680.662+,3+,4+
1682.943+,2+
1670.73+,2+
3233.683+,4+,5+
2561.043+,2+,4+,5+

40. 3156.29
3912.65
5+
4+
3+
2+
632.27 (5+)
790.08 (4+)
TVDCGGVPCEFGCCRIIDGKEKCREIDCD
1 10 20 29C.Amadis 3156.29
NGS derived sequence
Peptide mass 3156.29
detected in crude venom
3156.29 charge state distribution
Isotopic separation
0.20 Da
Isotopic separation
0.25 Da
TCEP - NEMReduction
Alkylataion
ΔM = 756.36
3 S-S

41. T V D CGGVPCEFGCCRIIDGKEKCREIDCD
C.Amadis-Q-TOF-Crude venom (oxidised peptides) M+ = 3157 (oxidised)
1019.43 (3+) =2842.2 (1+)
986.40 (3+) =2957.2 (1+)
948.06 (3+) =3056.3 (1+)
Confirming NGS derived sequences by MS fragmentation of intact disulfides
1019.43
986.40
948.06
MS/MS of 790.33(4+)
19.2 Min
NGS derived sequence (29 residues)
y28
y27
y26

42. a
Charge state distribution of 3912.25 ( Reduced / Alkylated) C.Amadis 3156.25
5+
5+
4+
3+
4+
3+

43. 286.0927
298.1274
316.10071+
326.11711+
343.1077
362.10311+
385.1724
401.11551+
414.18261+
426.14471+
442.17761+
458.13531+
477.13111+
499.20201+
529.20861+
541.17361+
559.18421+
574.2354583.2218
602.23091+
612.24971+
630.25881+
640.24231+
658.25311+
672.3003680.78222+
700.31574+
729.32811+
757.32354+
773.34072+
790.33844+
806.83872+
830.85232+
853.04353+
875.35501+
902.06613+
933.41363+
945.07843+
964.4123
1011.44101021.09883+
1053.45113+
1103.41591+
1158.5132
1231.50451+
1287.5672
1352.6023
1. +MS2(783.9329), 35.7eV, 15.4min #1812
0
1
2
3
4x10
Intens.
400 600 800 1000 1200 m/z
1360.5
b3 y2
y3
y22
b6
y18
y19
y20
y21
y22
y7
y8
y9
b6-H20
b6-18
y22
739.31+
b7
b7
+ + + + +
y11
y11
MS/MS 783.935+ ( 3912.25)
Observed fragments confirms
sequence of C.Am 3156.25

44. HS CC SH
H
+ S CC S
H
ΔM = -2Da
Number of
Disulfide Bonded
Cysteines
Number of
Disulfide Isomers
4 3
6
8
15
105
n (n-1).(n-3).(n-5)…..(n-(n-1))
34
S S
SS
S S
SS
S S
SS
Disulfide Isomers: Same sequence different connectivity
.. ..
Oxidation
Reduction
46620662575398912000

45. G-V-C1-C2-G-V-S-F-C3-Y-O-C4
Ar1248 : Two Disulfide Bonds
1 6 12
..C1C2 …….C3..C4 ..C1C2…….C3..C4 ..C1C2……...C3...C4
..C1C2 + C3….C4
..C1C2
….C3..C4
..C1C2
….C3..C4
..C1
….C3
+
C2
..C4
..C1
..C4
+ C2
….C3
One Cleavage
Two Cleavages
3 Possible Disulfide Connectivities

46. Determination of Disulfide Connectivity in Polypeptides
Separation of Cystines
X1C1 X2..X3C2X4….X5C3 X6…X7 C4 X8
+
Peptide Bond Cleavage
Chemical Cleavage
Reagents:
Acid Hydrolysis (H2SO4, Oxalic)
CNBr (Met – Xxx Bond)
Enzymatic Cleavage
Reagents:
Trypsin (R/K – X bond)
Glu-C (E/D – X bond)
Asp-N (X – D bond)
Poor Selectivity
Limited Selectivity Range
X1C1 X2
X5C3 X6
X3C2 X4
X7C4 X8

47. Determination of Disulfide Connectivity in Polypeptides
Protocols of Partial Reduction & Alkylation
C1 .. C2…….C3 … C4
1st Reduction Step
C1 .. C2…….C3 … C4
SH SH
Chemical labeling (X)
C1 .. C2…….C3 … C4
X X
Next Reduction & labeling (Y)
X X
C1 .. C2…….C3 … C4
Y Y
Peptide Sequencing (Edman / Mass Spectrometry)
Chemical
Tris (2-carboxyethyl)phosphine
(TCEP)
Burns, JA; Butler, JC;
Moran, J; Whitesides, GM
J. Org. Chem. 1991, 56, 2648
Gray, WR
Protein Sci. 1993, 2, 1732
Limitations:
1. Difficulties in
achieving selective
Reduction
2. Thiol disulfide
interchange

48. H+
C S
S C
H
-H2S2(66 Da)
HS
Dehydroalanine
Residue Mass: 69Da
Cysteinpersulfide
Residue Mass: 135Da
Path A
H+
H
+
Cysteine
Residue Mass: 103Da
Cysteinthioaldehyde
Residue Mass: 101Da
Path B
Fragmentation at Disulfide Bridges Under CID Conditions
Disulfide Bridges
(Cystine)
-H2S(34 Da)

49. Direct Fragmentation of a Two Disulfide Bonded Conus peptide

50. Moitrayee
Bhattacharyya
Working Protocol
of DisConnect

51. Determination of Disulfide Connectivity through DisConnect

52. J. Biol. Chem. 2007, 282, 30699
Anal. Chem. 2011, 84, 262
CCNCSSKWCRDHSRCC*
CCNCSSKWCRDHSRCC*
KIIIA- A Conotoxin: Controversy over Disulfide Connectivity
C1-C4/C2-C5/C3-C6
C1-C5/C2-C4/C3-C6

53. Detection of conotoxin disulfide isomers in C.amadis venom
>C.Am1820
MMYKLGVLLIICLLLFPLTAVPQDGDQPADRPAERMQDDISFEHDRFFDPVKR CCKYGWTCWLGCSPCCG
CCKYGWTCWLGCSPCCG
CCKYGWTCWLGCSPCC*
CCKYGWTCWLGCSOCC*
proteolysis
C- terminal amidation
proline hydroxylation
1862.65
1804.64
M
1820.64
F11
F13
F12
F10
F14
2+ 2+
CCKYGWTCWLGCSOCC* 2576.64
Reduction / Alkylation
TCEP/NEM
Delta M =756
C = Cys - NEM
Residue mass = 228
Linearized for MS-MS sequencing
911.30
911.81
912.31
912.81
913.31
913.81
914.30
911 912 913 914m/z
911.30
911.81
912.31
912.81
913.30
913.81
914.31
911 912 913 914 m/z
HPLC 280/216 nm detection
m/z 911.3 (2+) is detected in
two distinct HPLC peaks.
Two disulfide isomers ???
HPLC/MS
of crude
venom

54. 202.06
272.09
342.10
457.11
587.181+
675.22
748.26
806.31
959.31
1072.36
1488.54
1620.63
1874.69
5. +MS2(1290.4737), 49.8eV, 27.9min #3263
0
500
1000
1500
2000
2500
Intens.
200 400 600 800 1000 1200 1400 1600 1800 m/z
FRACTION 13
1289.47
1289.97
1290.47
1290.97
1291.47
1291.97
1292.471290 1291 1292 m/z
859.98
860.31
860.64
860.98
861.31
861.65
861.98
860 861 862 m/z
CCKYGWTCWLGCSOCC* 2576.64
C C K Y G W T C W L G C S O C C
Y3
Y4
B4
Y9
Y9
Y10
B2
B5

55. 201.05
246.08
314.11
342.10
429.12
474.14
570.16
587.191+
636.30
674.22
748.27
805.28
872.37
942.26
959.30
992.371+
1072.38
1153.44
1201.42
1290.472+
1320.47
1506.55
1588.56
1619.64
1774.66
1906.71
1991.73
2088.76
2123.84
16.
+MS2(1289.4677), 49.8eV, 24.0min #2811
0
1000
2000
3000
4000
5000
6000
Intens.
200 400 600 800 1000 1200 1400 1600 1800 2000 m/z
859.98
860.31
860.64
860.98
861.31
861.64
861.97
862.31
862.64860.0860.5861.0861.5862.0 m/z
1289.46
1289.97
1290.47
1290.97
1291.47
1291.97
1292.47
1289 1290 1291 1292 m/z
C C K Y G W T C W L G C S O C C
FRACTION 11
CCKYGWTCWLGCSOCC* 2576.64
Y3
Y4
B4
Y6
Y9
B8B2 B5
B1
B9
B10
Y11 B12
B13

56. Establishing the identity of the MS/MS fragmentation patterns
201.05
246.08
314.11
342.10
429.12
474.14
570.16
587.191+
636.30
674.22
748.27
805.28
872.37
942.26
959.30
992.371+
1072.38
1153.44
1201.42
1290.472+
1320.47
1506.55
1588.56
1619.64
1774.66
1906.71
1991.73
2088.76
2123.84
16.
+MS2(1289.4677), 49.8eV, 24.0min #2811
0
1000
2000
3000
4000
5000
6000
Intens.
200 400 600 800 1000 1200 1400 1600 1800 2000 m/z
FRACTION 11
FRACTION 13
202.06
272.09
342.10
476.14
587.181+
675.22
748.26
806.31
959.31
1072.36
1488.54
1620.63
1874.69
5.
+MS2(1290.4737), 49.8eV, 27.9min #3263
0
500
1000
1500
2000
2500
Intens.
200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

57. 24 25 26 27 28 29 Time [min]
0
1
2
3
60
ns.
911.3140
911.8154
912.3151
912.8149
913.3147
913.8146
0.0
0.5
1.0
1.5
5x10
Intens.
912 914 916m/z 911.3140
911.8155
912.3152
912.8150
913.3146
913.8145
0.0
0.5
1.0
1.5
2.0
5x10
Intens.
912 914 m/z
911.3134
911.8150
912.3146
912.8145
913.3144
913.8139
914.9022
915.4037
915.9055
0.0
0.5
1.0
1.5
2.0
5x10
Intens.
910 912 914 m/z
911.3151
911.8166
912.3161
912.8158
913.3153
913.8148
0
2
4
6
5x10
Intens.
911 912 913 914 m/z
903.0517
903.3057
903.5521
903.8023
904.0524
904.3035
904.5526
904.8031
905.0523
905.3121905.3810
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Intens.
903.0 903.5 904.0 904.5 905.0 m/z
903.3168
903.8174
904.3175
904.8176
905.3182
905.8187
0
2
4
6
4x10
Intens.
903 904 905 m/z
903.8170
904.3169
904.8166
905.3168
905.8173
906.3168
903.3158
0
1
2
3
4x10
Intens.
903 904 905 906 m/z
903.8187
904.3182
904.8184
905.3180
905.8181
906.3182
903.3172
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Intens.
903 904 905 906 907 m/z
911.32 Pro
903.32 Hyp
20 25 30 35 40 45 Time [min]
0.0
0.5
1.0
1.5
7x10Intens. Identifying biosynthetic precursors
(extracted ion chromatograms)
2+

58. Mj TIM tetramer
Crystal Structure of Methanococcus Triosephosphate Isomerase
Oxidizable residues
6 free Cysteine
3 Methionine

59. Component Relative
abundance
(A )23245.36 100
(B)23276.61 26
ESI-MS of Native MjTIM
∆M = 32 (2 O)
Probable Cys or Met Oxidation
750.831+(A)
775.830+(A)
802.629+(A)
831.228+(A)
861.927+(A)
895.026+(A)
930.825+(A)
969.524+(A)
1011.723+(A)
1057.622+(A)
1107.921+(A)
1163.220+(A)
1224.519+(A)
1296.318+(F)
1368.417+(A)
1453.716+(A)
1550.715+(A)
1661.414+(A)
0
250
500
750
1000
1250
1500
Intens.
800 900 1000 1100 1200 1300 1400 1500 1600 m/z

60. 969.5
970.8926
0.5
1.0
1.5
2.0
2.5
3.0
4
x10
24+
24+
1011.6
1013.1
0.5
1.0
1.5
2.0
2.5
3.0
4x10
1010 1014 1018 1022 1026 m/z
23+
23+
m/z
0.0 966 968 970 972 974 976 978 980 982 984 986z
1057.6
1059.1
0.0
0.5
1.0
1.5
2.0
2.5
3.0
4x10
1052 1054 1056 1058 1060 1062 1064 1066 1068 1070 1072
22+
21+
1107.9
1109.5
0.0
0.5
1.0
1.5
2.0
2.5
3.0
4x10Intens.
1106 1108 1110 1112 1114 1116 1118 1120 1122 1124 m/
21+
21+
Individual Charge States Showing Satellite Peaks Due to Oxidation Products

61. 775.730+
802.529+
831.128+
861.827+
894.926+
930.725+
969.424+981.9
994.78+
1011.523+1024.5
1057.522+1071.0
1085.83+
1107.821+
1122.0
1163.120+
1178.1
1195.87+
1224.319+
1240.0
1292.218+
1308.918+
1368.2
1385.717+
1405.8
1453.616+
1472.316+
1550.515+
1570.415+
0
1000
2000
3000
4000
5000
Intens.
800 900 1000 1100 1200 1300 1400 1500 m/z
ESI-MS of Heated MjTIM
Component Relative
abundance
(A )23243.30
(Native 23245.2)
100
(B)23275.21 40
40% Oxidation
But 100% loss in
activity
Cysteine oxidation to sulfenic/ sulfinic acid or methionine oxidation to sulfo
or sulfone is not responsible for loss of enzymatic activity

62. 775.830+(A)
802.529+(A)
831.228+(A)
861.927+(A)
895.026+(A)
930.825+(A)
969.524+(A)
1011.623+(A)
1057.622+(A)
1107.921+(A)
1163.220+(A)
1185.1135
1224.419+(A)
1292.418+(A)
1368.417+(A)
0
1000
2000
3000
4000
Intens.
800 900 1000 1100 1200 1300 1400 m/z
ESI-MS of MjTIM Heated with DTT
Component Relative
abundance
(A )23245.30 100
(B)23276.21 19

63. Utility of accurate mass measurements on proteins
Could the loss of MjTIM activity be a result of intramolecular
disulfide formation between two proximal thiol groups??
Cys-SH + HS- Cys Cys-S-S-Cys ∆M= -2 Da
Measured Mass using ESI-Q-TOF ( BRUKER, micrOTOF Q II)
MjTIM native = 23245.3 (Active)
MjTIM Heated for 3 hours at 348K at pH 6.7 = 23243.3 (Inactive)
MjTIM Heated for 3 hours at 348K at pH 8.8 =23243.3 (Inactive)
MjTIM Heated for 3 hours at 348K, pH 6.7
with reducing agent (DTT) =23245.2 (Active)
Enzymatically inactive sample appear to have a mass reduced by 2 Da

64. 895.052
26+ (A)
930.813
25+ (A)
0
500
1000
1500
2000
2500
Intens.
890 900 910 920 930 940 m/z
894.967 26+ (A)
910.5711
930.734 25+ (A)
0.00
0.25
0.50
0.75
1.00
1.25
4x10
Intens.
890 900 910 920 930 940 m/z
895.057
26+ (A)
930.817 25+ (A)
0
200
400
600
800
1000
1200
Intens.
890 900 910 920 930 940 m/z
Native MjTIM
Heated MjTIM
MjTIM Heated with DTT
Charge States
Mr= 23245.3
Mr= 23245.3
Mr= 23243.2 Mr= 23243.3
Mr= 23245.3
Mr= 23245.3

65. A Cys-Gly-Cys Motif in MjTIM
Inspection of crystal structure reveals that Cys81 and Cys83 are
appropriately positioned for oxidative S-S bond formation
Aº

66. 704.83
23+
736.87
22+
771.89
21+
810.46
20+
853.07
19+ 900.39
18+
953.30
17+
976.7
1012.81
16+
1080.29
15+
1157.34
14+
1246.31
13+
1350.07
12+
+MS, 27.3-28.3min
0
2
4
6
8
6x10
Intens.
700 800 900 1000 1100 1200 1300 m/z
Native Mj TIm
Residue 1-151
Mass 16189 Da
LC-ESI MS of Tryptic Digest of Native MjTIM
10 20 30 40 50 60 Time [min]
0
2
4
6
8x10
Intens.
TRP-MJ-051009.D: TIC +All MS
Residue 1-151
Residue 152-219

67. 10 20 30 40 50 60 70 Time [min]
0.0
0.2
0.4
0.6
0.8
1.0
9x10
Intens.
MJ3OX8.8TRP6-15.09.09.D: TIC +All MS
704.77
23+ 736.76
22+
771.81
21+
810.35
20+
852.95
19+
900.28
18+
953.17
17+
1012.68
16+
1080.12
15+
1157.23
14+
+MS, 31.6-32.0min #(1573-1600)
0.0
0.5
1.0
1.5
7x10
Intens.
700 800 900 1000 1100 1200 1300 m/z
Heated MjTIM
LC-ESI MS of Tryptic Digest of Heated MjTIM
Residue 1-151
Mass 16187 Da
(∆M= -2 Da)
Residue 1-151
Residue 152-219

68. 644.011+
708.3110+
786.939+
860.2
1179.316+
1415.05+
1011.477+
885.18+
+MS, 35.1-35.4min
0
1
2
3
4
7x10
Intens.
400 600 800 1000 1200 1400 1600 m/z
644.011+
708.3210+
786.919+
1179.316+
1415.015+
1011.477+
885.118+
+MS, 30.8-31.0min
0.0
0.5
1.0
1.5
2.0
2.5
7x10
Intens.
600 700 800 900 1000 1100 1200 1300 1400 1500 m/z
Native MjTIM
Heated MjTIm
Residue 152-219
Mass 7073 Da
Residue 152-219
Mass 7073 Da

69. Mj TIM Sequence
MVIVINYKTYNESIGNRGLEIAKIAEKVSEESGITIGVAPQFVDLRMIVENVNIPVYAQHIDNINPGS
HTGHILAEAIKDCGCKGTLINHSEKRMLLADIEAVINKCKNLGLETIVCTNNINTSKAVAALSPDCIAV
EPPELIGTGIPVSKANPEVVEGTVRAVKEINKDVKVLCGAGISKGEDVKAALDLGAEGVLLASGVVK
AKNVEEAIRELIKFI
MjTIM – Expected tryptic peptides

70. 524.3
545.3
546.3
523.3
2.
0.0
0.2
0.4
0.6
0.8
1.0
6x10
Intens.
510 520 530 540 550 560 m/z
LC-ESI-MS of Tryptic Digest of Heated MjTIM
D-C-G-C-K D-C-G-C-K
S S SHSH
MH+ (Da) 523 525
Oxidised Reduced
+Na+

71. 360.1
377.2
395.2
408.2
477.0
488.0
505.2
2.
0.0
0.2
0.4
0.6
0.8
1.0
5x10
Intens.
360 380 400 420 440 460 480 500 520 540 m/z
394.3
395.2
396.1
397.2
+MS2(523.3)
0
1
2
3
4
5
6
4x10
390 392 394 396 396 402m/z
MS/MS of 523.2
Expected structure of parent ion:
S S
D-C-G-C-K
m/z 395.2 and 394.3
Product Ions

72. 340 360 380 400 420 440 460 480 500 m/z
+MS3 505
0
20
40
60
80
100
Intens.
395.2
377.2
MS3 of m/z 505 (Parent ion)
276.0
282.1
292.9
+MS3(395.2)
0.00
0.25
0.50
0.75
1.00
1.25
1.503x10
Intens.
280 300 320 340 360 380 m/z290 310 330 350 370 390
377.2
MS3 of m/z 395/394 (?) (Parent ions)
S S
D-C-G-C-K
- H2O
Product ions
Product ions
S S
D-C-G-C-K -H2O (18)
-H2S2 (66)
-C02 (44)
m/z 523
m/z 523

73. S-S
H2N
H
N
O S
N
H
H
N
O
O H
HO2C
H2N
H2N
S
O
O
O
H
N
H
N
H
HO2C
H2N
O S
S
H2N
H2N
O
O
O H
+
+
S H
S
H2N
H
N
O
O
OH
NH3+
D-C-G-C-K
S S
DC
S S
m/z 523
m/z 523
m/z 394
m/z 282.1
+
S-S
D
C
G
K
C
K-C-G-C-D
S S
C-G-C-D
S S
m/z 377
m/z 505
m/z 505
S----S
D
C
G
K
CH H
HO2C
G-∆A-A-K-∆A m/z 395
-NH3
-CO
m/z 293
Gas phase cyclization
J Am Chem Soc.
2006, 128:10364
Harrison AG, Young AB,
Bleiholder C,
Suhai S, Paizs B.

74. 74
 Hyperthermophilic archaeon
 Inhihabits hydrothermal vents
A) (48- 940C)
B) Pressure 200 atm
Methanocaldococcus jannaschii Glutaminase
(MjGATase)
ATPPase
GATase is the smaller subunit of GMP synthetase
 Hydrolyses glutamine and generates ammonia (ammonia donor)
 Ammonia is tunnelled to ATPPase subunit to form GMP
Woods Hole Oceaonographic
institute
UC Berkeley EM lab.
GMP synthetase
JNCASR

75. Intens.
618.7432
34+
637.4460
33+
657.3436
32+
678.5027
31+
701.0914
725.2316
29+
751.0974
28+
778.8811
27+
808.7987
26+
841.1088
25+
876.1205
24+
914.1684
23+
955.6820
22+
1001.1340
21+
1051.1209
20+
0.0
0.5
1.0
1.5
2.0
4
x10
650 700 750 800 850 900 950 1000 1050 m/z
778.8811
27+
780.0821
27+
0.0
0.5
1.0
1.5
2.0
2.5
4
x10
Intens.
777 778 779 780 781 782 783m/z
30+
66.2kDa
45kDa
35kDa
18.4kDa
14.4kDa
21kDa
0.00.2
0.4
0.6
0.8
1.0
7
x10
Wt MjGATase
Expected Mass : 21020
Observed Mass : 21003
Mass of MjGATase lower by 17 Da.
75
188 amino acids
Loss of ammonia (NH3) a possibility?

76. 76
MjGATase
PhGATase
Sequence alignment of Pyrococcus horikoshii GATase with MjGATase shows
high degree of similarity
GLU 113
ILE 114
PHE115
GLU 111
ASP 110
2.8
3.27
SUCCINIMIDE
(ASP112 D*)
Mutational analysis to establish the sequence effects on
succinimide formation
I I D EDELFK
VDKENDLFK
PhGATase
MjGATase
PhGATase (1WL8)
crystal structure
with succinimide
Mass spec studies
MjGATse with
succinimide
109
ASP 112
H20
SUCCINIMIDE
ASN 109
N H3
SUCCINIMIDE
S/D

77. 77
Expected mass :21021Da
Observed mass :[A] 21003 Da
:[B] 21021 Da
657.0099
32+
678.1840
31+
700.7530
30+
724.8762
29+
750.7357
28+
778.4935
27+
808.4099
26+
840.7023
25+
875.6974
24+
913.7301
23+
955.2260
22+
1000.6621
21+
0.0
0.5
1.0
1.5
4
x10
Intens.
650 700 750 800 850 900 950 1000m/z
N109S
Expected mass :20993Da
Observed mass : 20993 Da
808.4099
26+
0.0
0.5
1.0
1.5
4
x10
Intens.
800 805 810 815 m/z
657.3341
32+
678.5049
31+
701.0899
30+
725.2292
29+
751.0914
28+ 808.7910
26+
841.1012
25+
876.1063
24+
914.1551
23+
955.6606
22+
1001.1227
21+
1051.1314
0
2
4
6
4x10
Intens.
650 700 750 800 850 900 950 1000 1050m/z
N109D
Substitution of N109 to S : Abolishment of succinimide
Replacement of N109 to D: like PhGATae forms succininimide
Expected mass :21021Da
Observed mas [A] : 21003 Da
[B]: 21021 Da
Mutational analysis on N109 confirms its conversion to succinimide
955.66
22+
956.49
22+
1
2
3
4
5
4x10
Intens.
956 m/z
[A]
[B]

78. Minus Succinimide
Increased floppiness
Structural collapsed
 Plus Succinimide
 Confers rigidity
Structural integrity
Increased molecular motion
High temperature
78
Succinimide : A molecular restrainer at high temperature
PhGATase (1WL8)
crystal structure
with succinimide

80. Analytical Biochemistry, 2016, 500, 45–50
Nanospray ESIMS : Triosephosphate isomerase
dimer interface mutants
Effect of Collision Energies