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
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.
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