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Measuring active cysteineresidue number in glutenin subunits by MALDI-TOF            Wujun Ma  Western Australia Departmen...
OverviewMALDI-TOF            Matrix Assisted            Laser Desorption            Ionization Time of            Flight M...
MALDI-TOF introduction
OverviewPeptide mass fingerprinting  (Protein identification)
Overview    MALDI-TOF approach for analysing       glutenins – intact proteinsGluteninextracts                            ...
Results HMWGS -- Mr comparison          MS     Deduced by DNA   Difference   Difference   SDS-PAGE   A-PAGEHMW-GS         ...
OverviewResults HMWGS -- HMW glutenin analysis                                                 Voyager Spec #1=>B C =>N R ...
Overview                                                       High resolution                                            ...
Chinese springLMW -G S 38. 00               43. 00
Background        Glu-D3 gene characterisationGene and gene haplotypes and their distribution               among alleles ...
Al GluD3/b                       (33555+33621+33783)Da+(38261+38462+38666+38                                     756)Da+40...
ApplicationMeasuring the HMWGS expression level                                                                           ...
Overview                               MALDI-TOF• Two studies have been conducted to compare  MALDI-TOF technology with ot...
OverviewCysteine Residue
Overview              Cysteine Residue• Glutenin matrix is formed and stabilised through  disulphide bonds;• The cysteine ...
Outline of the cysteine residue number         determination procedure• An alkylation reagent, 4-vinylpyridine (4-vp) has ...
Detecting the cysteine number in HMWGS                                                 Voyager Spec #1=>BC=>NR(7.00)[BP = ...
Extend the application to other proteins                                   eg, lupin seed storage proteins                ...
Developed a fast procedure to measure the number of  Detecting the cysteine number in HMWGS        cysteine residues in HM...
Look forward• It has been noticed that some HMW non-prolamin proteins  possess similar characteristics of glutenin protein...
Procedure    •Treat the total protein extracts    with 4-vp;    •Develop tools to monitor the    position shifts of the 2-...
Acknowledgements   Dr Ke Wang   Junhong Ma   Dr Shunli Wang   Dr Shahidul Islam   Dr Frank Bekes   Yueming Yan   Rudi Appels
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Measuring active cysteine residue number in glutenin subunits by MALDI-TOF

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

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Measuring active cysteine residue number in glutenin subunits by MALDI-TOF

  1. 1. Measuring active cysteineresidue number in glutenin subunits by MALDI-TOF Wujun Ma Western Australia Department of Agriculture & Food
  2. 2. OverviewMALDI-TOF Matrix Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry
  3. 3. MALDI-TOF introduction
  4. 4. OverviewPeptide mass fingerprinting (Protein identification)
  5. 5. Overview MALDI-TOF approach for analysing glutenins – intact proteinsGluteninextracts Glutenin Subunit / Allele identification
  6. 6. Results HMWGS -- Mr comparison MS Deduced by DNA Difference Difference SDS-PAGE A-PAGEHMW-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. 7. OverviewResults 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. 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 67395.38Yitpi 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 50Frame 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. 9. Chinese springLMW -G S 38. 00 43. 00
  10. 10. Background Glu-D3 gene characterisationGene and gene haplotypes and their distribution among alleles 1 2 3 4 5 6Cultivar ale 11b 12 21/22 23 31 32 41c 42 43 5 6C 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. 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. 12. ApplicationMeasuring 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. 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. 14. OverviewCysteine Residue
  15. 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. 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. 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. 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. 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. 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. 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. 22. Acknowledgements Dr Ke Wang Junhong Ma Dr Shunli Wang Dr Shahidul Islam Dr Frank Bekes Yueming Yan Rudi Appels

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