Age and gender differences in the urinary proteome of healthy subjects using the advantages of quantitative proteomics.

Background: Advances in quantitative proteomics have enabled accurate and comprehensive quantification of proteins. In this study, urine from healthy volunteers was analyzed using this method to confirm the depth of protein identification and quantitative stability, and to examine changes in abundance with age and sex, as well as the amounts of proteins known to be urinary biomarkers of kidney damage.

Methods: Urine samples were collected from 89 healthy male and female volunteers in their 20s to 70s. Proteins were precipitated with methanol/chloroform, digested with trypsin, and peptides were identified and quantified by mass spectrometry. The quantitative relative value of a protein in the total protein was multiplied by 1,000,000 to obtain a normalized relative value (ppm). The stability of the quantitative proteomics was examined by measuring quality control samples prepared from healthy volunteer urine mix (HVmix) or diabetic urine mix (DMmix) 5 times in every 20 sample measurements.

Results: The number of proteins identified was stable at approximately 3000 in five measurements of HVmix and DMmix, as quality control samples. The average coefficient of variation (CV) of the quantitative values was 12.6% for HVmix and 14.8% for DMmix. Approximately 90% of the proteins did not change significantly with age, while approximately 10% of the proteins increased or decreased with age. The quantitative values of some proteins, such as KLK3 in men and A2ML1 in women, differed significantly between the sexes. In addition, several proteins that have already been reported as biomarkers of kidney damage were also quantified.

Conclusion(s): This study demonstrated that advanced proteomics enabled comprehensive quantification of urinary proteins, some proteins varied in abundance with age and gender, and provided insight into protein structure.