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International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012
Citation preview
Measuring active cysteine
residue number in glutenin
subunits by MALDI-TOF
Wujun Ma
Western Australia Department of
Agriculture & Food
Overview
MALDI-TOF
Matrix Assisted
Laser Desorption
Ionization Time of
Flight Mass
Spectrometry
Overview
Peptide mass fingerprinting
(Protein identification)
Overview
MALDI-TOF approach for analysing
glutenins – intact proteins
Glutenin
Subunit / Allele
identification
Glutenin
extracts
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
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
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
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
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
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
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
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
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.
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.
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
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
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
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.
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….
Acknowledgements
Dr Ke Wang
Junhong Ma
Dr Shunli Wang
Dr Shahidul Islam
Dr Frank Bekes
Yueming Yan
Rudi Appels
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