International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012

1. Measuring active cysteine
residue number in glutenin
subunits by MALDI-TOF
Wujun Ma
Western Australia Department of
Agriculture & Food

2. Overview
MALDI-TOF
Matrix Assisted
Laser Desorption
Ionization Time of
Flight Mass
Spectrometry

3. MALDI-TOF introduction

4. Overview
Peptide mass fingerprinting
(Protein identification)

5. Overview
MALDI-TOF approach for analysing
glutenins – intact proteins
Glutenin
extracts
Glutenin
Subunit / Allele
identification

6. Results HMWGS -- Mr comparison
MS Deduced by DNA Difference Difference SDS-PAGE A-PAGE
HMW-GS
（Da） sequences（Da） (Da) （%） rank rank
1Dx5 87901 87189 +712 +0.81 4 1
(1) (2)
1Ax1 87575 87680 -5 -0.01 1 3
(2) (1)
1Ax2* 86899 86317 +582 +0.67 2 4
(3) (4)
1Dx2 86802 86987 -185 -0.21 3 2
(4) (3)
1Bx7 82741 82524 +217 +0.26 5 5
(5) (6)
1Bx14 6 6
1By8 74811 75157 -346 -0.46 7 7
(7) (7)
1Dy12 68590 68711 -121 -0.18 9 8
(8) (8)
1Dy10 67483 67473 +10 +0.02 8 9
(9) (9)

7. Overview
Results HMWGS -- HMW glutenin analysis
Voyager Spec #1=>B C =>N R (30.00)[B P = 82406.7, 251]
82215.70 251.3
100
90
Dy2 Bx7
80
67327.71 Ax2* Dx5
70
88075.03
60
By18
% Intensity
86331.40
50
40
73377.07
30
90571.74
20
10
0 0
60022.0 67021.6 74021.2 81020.8 88020.4 95020.0
Mass (m /z)
Voyager Spec #1=>B C =>N R (30.00)[B P = 82996.8, 518]
100
Bx7oe 82850.49 518.1
90
80
70
60
Ax2* Dx5
% Intensity
50
40 88025.70
30 Dy2 By18 86096.26
67335.29 80837.26
20
75019.50
10
0 0
60022.0 67021.6 74021.2 81020.8 88020.4 95020.0
Mass (m /z)

8. Overview
High resolution
Voyager Spec #1=>BC=>NR(20.00)[BP = 82418.1, 666]
82438.49 666.4
100
90
80 87826.97
% Intensity
70 82064.57
67395.38
Yitpi 60
50
40
30 50545.07
53610.99 75102.38
20 56790.55 92508.05
71669.47
10
0 0
50000.0 60000.6 70001.2 80001.8 90002.4 100003.0
Mass (m/z)
<<HMW_26_0001>> Voyager Spec #1=>BC=>NR(20.00)=>BC[BP = 82452.3, 204]
82437.99 204.0
100
90
80 88003.25
% Intensity
70
60 67399.76 81831.88
50
Frame 40
30
67702.78
20 51032.81 80452.46 84553.29
54789.03 75073.13 92514.64 96447.26
89224.29
10 66167.16
0 0
50000.0 60000.6 70001.2 80001.8 90002.4 100003.0
Mass (m/z)

9. Chinese spring
LMW -G S
38. 00 43. 00

10. Background
Glu-D3 gene characterisation
Gene and gene haplotypes and their distribution
among alleles
1 2 3 4 5 6
Cultivar ale
11b 12 21/22 23 31 32 41c 42 43 5 6
C Spring a + + + + + +
BT2288A e + + + + + +
Silverstar b + + + + + +
Sunco b + + + + + +
Aroona c + + + + + +
Norin61 d + + + + + +
Tasman a + + + + + +
Hartog e + + + + + +
Conclusion: forget about the AS-markers for GluD3 locus

11. Al GluD3/b
(33555+33621+33783)Da+(38261+38462+38666+38
756)Da+40986Da
V er S #1=>B =>N (2. 00)[B = 33625. 1, 11836]
oyag pec C R P
100
33621 1 .2 E+4
90 33555
80
70 40135
% Intensity
60
50
33783
41859
40 38660
33028 38462 40483
30
32665 33987
20 35988 37682 40986
31092 34553 36111
10
43459
0 50 0
30000 33000 36000 39000 42000 45000
Mass (m /z)

12. Application
Measuring the HMWGS expression level
Voyager Spec #1=>B C =>N R (2.00)[B P = 78539.4, 1006]
78531.12 1006.4
100
90
80
70
60
% Intensity
87014.30
75052.20
50
68484.97
77965.87 83005.68
87229.10
40 75278.41
83161.93 86214.91
30
20
56886.48 63862.85
71217.09
10
0 0
55015.0 62015.8 69016.6 76017.4 83018.2 90019.0
Mass (m /z)
Voyager Spec #1=>B C =>N R (2.00)[B P = 83190.7, 652]
83184.50 651.9
100
90
80
70 75229.37
87269.56
60 78698.70 87426.37
% Intensity
50 82561.65
68615.31 87730.18
64086.70
40
78179.64
63644.60
30 86469.40
59169.87 67718.64 71197.37 79620.67
20
84629.30
10
0 0
55012.0 62011.8 69011.6 76011.4 83011.2 90011.0
Mass (m /z)

13. Overview
MALDI-TOF
• Two studies have been conducted to compare
MALDI-TOF technology with other analysing
platforms:
1. Characterization and Comparative Analysis of Wheat High Molecular Weight Glutenin
Subunits by SDS-PAGE, RP-HPLC, HPCE, and MALDI-TOF-MS. Journal of Agricultural
and Food Chemistry (2010) 58 (5), 2777–2786 (IF 2.562)
2. Comparison of low molecular weight glutenin subunits identified by SDS-PAGE, 2-DE,
MALDI-TOF-MS and PCR in common wheat (2010) BMC Plant Biology 10:(124)
doi:10.1186/1471-2229-10-124.
• Results revealed that MALDI-TOF is a reliable
technology with high-throughput & resolution

14. Overview
Cysteine Residue

15. Overview
Cysteine Residue
• Glutenin matrix is formed and stabilised through
disulphide bonds;
• The cysteine residue is the molecular basis of
disulphide bonds; the number of cysteine residue in
HMWGS is positively correlated with dough quality;
• Accurately measuring the number of cysteine
residue is important to predict quality.

16. Outline of the cysteine residue number
determination procedure
• An alkylation reagent, 4-vinylpyridine (4-vp) has the
ability to combine with cysteine residue. For every
cysteine residue in a protein, this chemical reaction
increases the molecular mass value of 105.14 Da.
• The mass difference before and after the 4-vp
treatment can be reliably determined by MALDI-TOF.
• The measured mass difference can be used to
determine the number of active cysteine residue.

17. Detecting the cysteine number in HMWGS
Voyager Spec #1=>BC=>NR(7.00)[BP = 79182.4, 554] Voyager Spec #1=>BC=>NR(2.00)[BP = 88608.5, 240]
79145.20 68223.63 88633.08
88633.08
100 79145.20 553.6 100
68223.63
84196.69 239.9
68325.47
68325.47 84196.69
90 90
80 80
88834.41
88834.41 88109.80
70 70
87972.33
% In ten sity
% In ten sity
60 60 84715.20
75931.99
50 50
76059.15
76059.15
75931.99
83386.00
75799.45
40 86899.93
86899.93 40 87625.67
30 30
85434.48
69385.06
20 65915.01 20
72035.62 61936.09 70845.02
66344.86 78629.90 91400.14
10 10
0 0 0 0
59999.0 66999.4 73999.8 81000.2 88000.6 95001.0 60013.0 67012.4 74011.8 81011.2 88010.6 95010.0
Mass (m/z) Mass (m/z)
Bumper (2*, 17+18, 5+10) Add 4vp Shan229 (N, 20+20, 5+10) Add 4vp
Voyager Spec #1=>BC=>NR(2.00)[BP = 88116.4, 372]
67615.89 Spec #1=>BC=>NR(5.00)[BP = 67597.7, 892]
Voyager
88108.26
88108.26
67615.89 892.2 100 371.9
100 78695.98
78695.98
90 90
67488.13
67488.13
80
88316.74
80 84007.12
84007.12
87668.12
88316.74
70 70 84210.06
% In ten sity
60 60
75324.47 87426.09
% Intensity
75324.47 78158.26
50 50
67241.74
86491.78
86491.78
75163.24
75163.24
40 40
66747.36
30 30
66597.97 75510.21 85038.22
79982.66 86012.76 68582.55
20 71210.50 73895.88 20
77889.57
84397.70
63890.17 70855.23
10 10 91180.16
91418.61
0 0 0 0
59999.0 66999.4 73999.8 81000.2 88000.6 95001.0 60013.0 67012.4 74011.8 81011.2 88010.6 95010.0
Mass (m/z) Mass (m/z)
Bumper (2*, 17+18, 5+10) No 4vp Shan229 (N, 20+20, 5+10) No 4vp

18. Extend the application to other proteins
eg, lupin seed storage proteins
21395.03 25909.51
100 100
90 90
80 80 C
70 70
20896.18
60 A 60
% intensity
% intensity
50 50
40 40
19176.39 25805.34
30 30
20 22167.76 20 26011.82
10 26114.11
10
0 0 0 0
17479.0 18745.4 20011.8 21278.2 22544.6 23811.0 25419 25601 25783 25965 26147 26329
Mass (m/z) Mass (m/z)
21403.43 26022.98
100
100
90
90
80
80
70 B D
% intensity
70
% intensity
60 20919.75
60
50
50
40
40
30 25912.66
19286.88 30
20 22189.21 26123.54
20
10 26218.35
10
0 0
17479.0 18745.4 20011.8 21278.2 22544.6 23811.0 0 0
25419 25601 25783 25965 26147 26329
Mass (m/z)
Mass (m/z)

19. Developed a fast procedure to measure the number of
Detecting the cysteine number in HMWGS
cysteine residues in HMW glutenins
•Typically only requires 1 pmol
proteins;
•Very accurate and sensitive;
•High throughput

20. Look forward
• It has been noticed that some HMW non-prolamin proteins
possess similar characteristics of glutenin proteins and can be
integrated into the glutenin matrix.
• We conducted 3 proteomics studies in the past three years and
have concluded that a high number of non-prolamins are related
to quality.
• Recently, based on a proteomics studies, we found a few
avenin-like proteins that usually contain18 to 19 cysteine
residues expressed significant differential expressions subject to
various abiotic stresses.
• Isolating the sub-proteome of the cysteine residue containing
proteins will lead to discovery of novel factors in relation to
quality.
• We are currently developing procedures for measuring and
screening cysteine containing proteins in seed proteome.

21. Procedure
•Treat the total protein extracts
with 4-vp;
•Develop tools to monitor the
position shifts of the 2-D protein
spots;
•Determine cysteine numbers of
protein spots based on the
position variation caused by 4-vp
treatment;
•Or, label the 4-vp chemical….

22. Acknowledgements
Dr Ke Wang
Junhong Ma
Dr Shunli Wang
Dr Shahidul Islam
Dr Frank Bekes
Yueming Yan
Rudi Appels