measuring active cysteine residue number in glutenin subunits by maldi-tof
DESCRIPTION
International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012TRANSCRIPT
![Page 1: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/1.jpg)
Measuring active cysteine
residue number in glutenin
subunits by MALDI-TOF
Wujun Ma
Western Australia Department of
Agriculture & Food
![Page 2: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/2.jpg)
Overview
MALDI-TOF
Matrix Assisted
Laser Desorption
Ionization Time of
Flight Mass
Spectrometry
![Page 4: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/4.jpg)
Overview
Peptide mass fingerprinting
(Protein identification)
![Page 5: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/5.jpg)
Overview
MALDI-TOF approach for analysing
glutenins – intact proteins
Glutenin
Subunit / Allele
identification
Glutenin
extracts
![Page 6: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/6.jpg)
Results HMWGS -- Mr comparison
HMW-GS
MS
(Da)
Deduced by DNA
sequences(Da)
Difference
(Da)
Difference
(%)
SDS-PAGE
rank
A-PAGE
rank
1Dx5
87901
(1)
87189
(2)
+712
+0.81
4
1
1Ax1
87575
(2)
87680
(1)
-5
-0.01
1
3
1Ax2*
86899
(3)
86317
(4)
+582
+0.67
2
4
1Dx2
86802
(4)
86987
(3)
-185
-0.21
3
2
1Bx7
82741
(5)
82524
(6)
+217
+0.26
5
5
1Bx14
6
6
1By8
74811
(7)
75157
(7)
-346
-0.46
7
7
1Dy12
68590
(8)
68711
(8)
-121
-0.18
9
8
1Dy10
67483
(9)
67473
(9)
+10
+0.02
8
9
![Page 7: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/7.jpg)
Overview
Results HMWGS -- HMW glutenin analysis
60022.0 67021.6 74021.2 81020.8 88020.4 95020.0
Mass (m /z)
0
251.3
0
10
20
30
40
50
60
70
80
90
100%
Inte
nsity
Voyager Spec #1=>BC=>NR(30.00)[BP = 82406.7, 251]
82215.70
67327.71
88075.03
86331.40
73377.07
90571.74
60022.0 67021.6 74021.2 81020.8 88020.4 95020.0
Mass (m /z)
0
518.1
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
Voyager Spec #1=>BC=>NR(30.00)[BP = 82996.8, 518]
82850.49
88025.70
86096.26
80837.2667335.29
75019.50
Ax2* Dx5
Bx7
By18
Dy2
Dy2 By18
Bx7oe
Ax2* Dx5
![Page 8: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/8.jpg)
Overview
High resolution
Yitpi
Frame
50000.0 60000.6 70001.2 80001.8 90002.4 100003.0
Mass (m/z)
0
204.0
0 10 20 30 40 50 60 70 80 90
100
% I
nte
ns
ity
<<HMW_26_0001>> Voyager Spec #1=>BC=>NR(20.00)=>BC[BP = 82452.3, 204] 82437.99
88003.25
67399.76 81831.88
67702.78
80452.46 51032.81 84553.29 54789.03 92514.64 75073.13 96447.26 89224.29 66167.16
50000.0 60000.6 70001.2 80001.8 90002.4 100003.0
Mass (m/z)
0
666.4
0 10 20 30 40 50 60 70 80 90
100
% I
nte
nsit
y
Voyager Spec #1=>BC=>NR(20.00)[BP = 82418.1, 666]
82438.49
87826.97 82064.57
67395.38
50545.07 53610.99 75102.38 92508.05 56790.55 71669.47
![Page 9: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/9.jpg)
Chinese spring
0.00
0.00
0.01
0.01
0.02
0.02
0.03
0.03
0.04
13.00 18.00 23.00 28.00 33.00 38.00 43.00 48.00
HMW-GS LMW-GS
![Page 10: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/10.jpg)
Background
Glu-D3 gene characterisation
Gene and gene haplotypes and their distribution
among alleles
Cultivar ale
1 2 3 4 5 6
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
![Page 11: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/11.jpg)
Al GluD3/b
(33555+33621+33783)Da+(38261+38462+38666+38
756)Da+40986Da
30000 33000 36000 39000 42000 45000
Mass (m /z)
0
1.2E+4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
Voyager Spec #1=>BC=>NR(2.00)[BP = 33625.1, 11836]
50
33621
33555
40135
33783
4185938660
33028 404833846232665 33987 4098635988 37682
34553 361113109243459
![Page 12: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/12.jpg)
Application
Measuring the HMWGS expression level
55015.0 62015.8 69016.6 76017.4 83018.2 90019.0
Mass (m /z)
0
1006.4
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
Voyager Spec #1=>BC=>NR(2.00)[BP = 78539.4, 1006]
78531.12
87014.3075052.20
68484.97
77965.87 83005.6887229.10
75278.41 86214.9183161.93
63862.8556886.4871217.09
55012.0 62011.8 69011.6 76011.4 83011.2 90011.0
Mass (m /z)
0
651.9
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
Voyager Spec #1=>BC=>NR(2.00)[BP = 83190.7, 652]
83184.50
75229.37
87269.56
78698.70 87426.37
82561.6587730.1868615.3164086.70
78179.64
63644.6086469.40
59169.87 71197.3767718.64 79620.67
84629.30
![Page 13: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/13.jpg)
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
![Page 15: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/15.jpg)
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.
![Page 16: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/16.jpg)
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.
![Page 17: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/17.jpg)
Detecting the cysteine number in HMWGS
59999.0 66999.4 73999.8 81000.2 88000.6 95001.0
Mass (m/z)
0
553.6
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Voyager Spec #1=>BC=>NR(7.00)[BP = 79182.4, 554]
79145.20
68325.47
88834.41
76059.15
86899.93
65915.01 72035.62
60013.0 67012.4 74011.8 81011.2 88010.6 95010.0
Mass (m/z)
0
239.9
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Voyager Spec #1=>BC=>NR(2.00)[BP = 88608.5, 240]
88633.0868223.63
84196.69
88109.80
87972.33
84715.20
75931.99
83386.0075799.4587625.67
85434.4869385.06
61936.09 70845.0266344.86 78629.90 91400.14
Bumper (2*, 17+18, 5+10) Add 4vp Shan229 (N, 20+20, 5+10) Add 4vp
59999.0 66999.4 73999.8 81000.2 88000.6 95001.0
Mass (m/z)
0
892.2
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Voyager Spec #1=>BC=>NR(5.00)[BP = 67597.7, 892]
67615.8978695.98
88316.74
75324.47 78158.2667241.74
86491.78
66597.97
86012.7679982.6673895.8871210.50
84397.70
91418.61
60013.0 67012.4 74011.8 81011.2 88010.6 95010.0
Mass (m/z)
0
371.9
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
Voyager Spec #1=>BC=>NR(2.00)[BP = 88116.4, 372]
88108.26
67488.13
87668.1284007.12
84210.06
87426.09
75163.24
66747.36
75510.21 85038.22
68582.5577889.57
70855.2363890.17
91180.16
Bumper (2*, 17+18, 5+10) No 4vp Shan229 (N, 20+20, 5+10) No 4vp
86899.93
86491.78
88834.41
79145.20
78695.98
76059.15
75324.47
67488.13
67615.89
68325.47
75163.24
84007.12
88108.26
68223.63
75931.99
84196.6988633.08
88316.74
59999.0 66999.4 73999.8 81000.2 88000.6 95001.0
Mass (m/z)
0
553.6
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Voyager Spec #1=>BC=>NR(7.00)[BP = 79182.4, 554]
79145.20
68325.47
88834.41
76059.15
86899.93
65915.01 72035.62
60013.0 67012.4 74011.8 81011.2 88010.6 95010.0
Mass (m/z)
0
239.9
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Voyager Spec #1=>BC=>NR(2.00)[BP = 88608.5, 240]
88633.0868223.63
84196.69
88109.80
87972.33
84715.20
75931.99
83386.0075799.4587625.67
85434.4869385.06
61936.09 70845.0266344.86 78629.90 91400.14
Bumper (2*, 17+18, 5+10) Add 4vp Shan229 (N, 20+20, 5+10) Add 4vp
59999.0 66999.4 73999.8 81000.2 88000.6 95001.0
Mass (m/z)
0
892.2
0
10
20
30
40
50
60
70
80
90
100
% In
ten
sity
Voyager Spec #1=>BC=>NR(5.00)[BP = 67597.7, 892]
67615.8978695.98
88316.74
75324.47 78158.2667241.74
86491.78
66597.97
86012.7679982.6673895.8871210.50
84397.70
91418.61
60013.0 67012.4 74011.8 81011.2 88010.6 95010.0
Mass (m/z)
0
371.9
0
10
20
30
40
50
60
70
80
90
100
% Inte
nsity
Voyager Spec #1=>BC=>NR(2.00)[BP = 88116.4, 372]
88108.26
67488.13
87668.1284007.12
84210.06
87426.09
75163.24
66747.36
75510.21 85038.22
68582.5577889.57
70855.2363890.17
91180.16
Bumper (2*, 17+18, 5+10) No 4vp Shan229 (N, 20+20, 5+10) No 4vp
86899.93
86491.78
88834.41
79145.20
78695.98
76059.15
75324.47
67488.13
67615.89
68325.47
75163.24
84007.12
88108.26
68223.63
75931.99
84196.6988633.08
88316.74
![Page 18: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/18.jpg)
Extend the application to other proteins eg, lupin seed storage proteins
17479.0 18745.4 20011.8 21278.2 22544.6 23811.0
Mass (m/z)
00
10
20
30
40
50
60
70
80
90
10021403.43
20919.75
19286.88
22189.21
17479.0 18745.4 20011.8 21278.2 22544.6 23811.0
Mass (m/z)
00
10
20
30
40
50
60
70
80
90
10021395.03
20896.18
19176.39
22167.76
A
B
% in
tens
ity%
inte
nsity
25419 25601 25783 25965 26147 26329
Mass (m/z)
00
10
20
30
40
50
60
70
80
90
10026022.98
25912.66
26123.54
26218.35
25419 25601 25783 25965 26147 26329
Mass (m/z)
00
10
20
30
40
50
60
70
80
90
10025909.51
25805.34
26011.82
26114.11
C
D
% in
tens
ity%
inte
nsity
![Page 19: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/19.jpg)
Developed a fast procedure to measure the number of
cysteine residues in HMW glutenins
•Typically only requires 1 pmol proteins; •Very accurate and sensitive; •High throughput
Detecting the cysteine number in HMWGS
![Page 20: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/20.jpg)
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.
![Page 21: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/21.jpg)
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….
![Page 22: Measuring active cysteine residue number in glutenin subunits by MALDI-TOF](https://reader034.vdocuments.site/reader034/viewer/2022042715/559662061a28aba9258b4621/html5/thumbnails/22.jpg)
Acknowledgements
Dr Ke Wang
Junhong Ma
Dr Shunli Wang
Dr Shahidul Islam
Dr Frank Bekes
Yueming Yan
Rudi Appels