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

MALDI-TOF introduction

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

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