Optimization Of Centrifugation Conditions For The Extraction Of Urine Protein By Acetone Precipitation And Its Application In Individual Differences In Urinary Proteome

Background Urine is an easily available and non-invasive body fluid.Proteomics studies based on mass spectrometry have shown that urine contains thousands of proteins,and is a potential source of biomarkers for various diseases.However,in addition to proteins,there are also many compounds in urine that interfere with the mass spectrometry analysis of urinary proteins,so the extraction of urine proteins is an essential step in the analysis of urine proteomics.Therefore,it is particularly important to explore the effect of centrifugal speed and temperature on urine proteome after acetone precipitation.In addition,the individual differences of the normal urine proteome are related to the stability of the urine proteome,this is related to whether it can be used in the study of disease biomarkers,which is of great significance for the clinical study of urine proteomicsMethods In this study,human urine samples after acetone precipitation were treated with different centrifugal speeds(4 000 ×g-and 12 000 ×g)and centrifugal temperatures(4? and 20?),and urine proteins were extracted for mass spectrometry analysis.The optimum conditions for urine protein centrifugation after acetone precipitation were determined by comparing the number of proteins,polypeptides and spectra identified by the four methods,as well as the isoelectric point,molecular weight distribution,miss-cutting rate and post-translational modification.The above optimal centrifugation conditions were used for urine proteome analysis of 36 normal subjects(male:female=1:1)and the individual differences,technical differences and minimum sample size of the normal urine proteome were studied by using the label-free quantitative methodResults The experimental results showed that under the condition of low temperature and high rotation speed(4?,12 000 xg),the number of protein polypeptides was the largest,and the identification effect was the best.In the urine proteomic analysis of normal people,the individual difference was 0.635.The minimum sample size is 16.Conclusions In this study,urine protein extraction efficiency and mass spectrometry results were compared at different centrifugal speeds and temperatures,and the optimal method of urine protein centrifugation was determined.Moreover,the minimum sample size required for the analysis of urine proteome and the differences between individuals were evaluated using the label-free quantitative method under optimal centrifugation.This study provides an optimal centrifugation scheme for the extraction of urinary protein in the future and lays a foundation for the clinical application of urinary proteome.