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.
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
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