Study On Post - Translational Modification Of Urinary Protein And Urine Protein Preserved On Nitrocellulose Membrane By Heating Elution

Biomarker is the measurable change associated with a physiological or pathophysiological process. Its nature is change. Contrast to the blood, which has mechanisms to maintain a homeostatic internal environment, urine is more likely to reflect changes in the body. So it is more sensitively to detect changes in the abundance of many proteins in urine than in plasma. Urine as the valuable biological sample, its preservation along with the corresponding medical records will make it easier to conduct large-scale biomarker research and population-wide validations, which in turn will lead to generation of high-quality biomarkers. It’s possible to store and archive urine samples economically on a large scale for long-term, as the simple method to preserve urinary proteins onto a membrane established recently.The preservation of urinary proteins on a membrane plays a vital role in biomarker research, and the efficient elution of proteins preserved on nitrocellulose membrane (NC membrane) determines the application of this method. But the elution of the proteins from the membrane was not optimized, and it became a bottle neck restricting its application. Firstly, it took at least 10min intense vortexing to make the NC membrane completely dissolve. Secondly, the NC membrane solution was too viscous to be separated thoroughly from proteins and would form membrane again, which impaired the elution efficiency.To solve these problems, we put forward the heating elution procedure. By raising the temperature to reduce the intense vortexing time, and keeping gentle rotating while precipitation to prevent nitrocellulose reformation. We used SDS-PAGE and LC-MS/MS to analyze the urinary proteins prepared by heating elution procedure, intense vortexing elution procedure and acetone precipitation method. There was no degradation of proteins prepared by heating elution procedure. Compared with proteins prepared by heating elution method and acetone precipitation method, the overlapping rates of the proteins was almost same (92.6% and 96.8%, respectively) and the ratios of CV values (<20%) of the proteins were both high (85.2% and 94.4%, respectively). The heating elution procedure achieved good technical reproducibility, and was much simpler and more efficient than the previous one. It can facilitate the popularization and application of the preservation of urinary proteins on membrane.Post-translational modifications (PTMs) can determine proteins’properties and functions, then modulate many biological functions. Hence searching biomarkers on protein PTMs level gains more information than merely on protein level. However only glycosylation of urinary proteins was studied and applied in biomarker studies. Compared to 400 forms of PTMs detected in proteins, what we have known about PTMs of urine proteins is just the "tip of the iceberg".6 types of lysine PTMs, including succinylation, crotonylation, propionylation, butyrylation, trimethylation, acetylation which commercial antibodies were available, were reported to play a key role in the regulation of diverse cellular functions. We explored the status of these PTMs in urine from healthy controls and kidney diseases patients. Western blot analysis revealed that these 6 types of PTMs existed in almost all urine samples.And the patterns of PTMs, which were different from each other, were much simpler than those of total proteins. Enrichment or visualization of PTMs provides ways to sampling part of urinary proteome. It could show the differences among diseases and healthy controls better, which might not be shown easily in comparison of total urinary proteins. The patterns of PTMs among healthy and kidney diseases were summarized.