Application of Offgel and Dimethylation Labeling As Quantitative Proteomic Analysis Procedure to Investigate Protein Changes in Fruit During Ripening and in Response to Postharvest Treatment

Proteomics is a systematic approach to study changes in proteins. It provides an essential linkage between the transcriptome and metabolome. Despite recent developments, fruit proteomics is still facing major challenges that limit in-depth study. In addition to limited protein sequences available for most fruits, high throughput, reliable and quantitative protein analysis procedure for fruit is also lacking. As a sample preparation step, OFFGEL isoelectric focussing (IEF) offers the ability to fractionate peptides or proteins prior to LC/MS analysis, which has become one of the more popular sample preparation procedures in proteomic research. In order to develop and improve the quantitative fruit proteomic research platform, we conducted a detailed investigation to exam the advantages of OFFGEL in fruit proteomic research using apple, banana and strawberry fruit. We provided technical details concerning sample preparation, trypsin digestion, OFFGEL-IEF, reversed phase nano-LC separation. We demonstrated that proteomic protocol employing OFFGEL–IEF as a crucial sample preparation step can significantly improve the proteome coverage and quantitative proteomic workflows. Our results demonstrated that the use of OFFGEL is an effective method for peptide fractionation and increased significantly the number of proteins identified by at least 10 times as compared with unfractionated samples, with more than 900, 800, and 928 proteins in apple, banana and strawberries respectively. With improved and developed protocol, we further combined the OFFGEL-IEF with quantitative proteomic procedure such as isotope dimethylation labeling to investigate the protein changes in banana fruit after ethylene and high temperature treatment. We identified and quantified more than 220 proteins that changed significantly (at least two fold for up- and down-regulation) in these treatments. The link between the changes of these proteins with their biological functions is also discussed.