Microparticle-assisted protein capture method facilitates proteomic and glycoproteomic analysis of urine samples

Serum tests have become a partial alternative to renal biopsy for diagnosing primary membranous nephropathy (pMN). However, urine tests, due to their non-invasive nature and ability to more accurately reflect glomerular diseases, hold great promise for the detection of pMN. However, the low protein concentration and the time-consuming sample preparation procedure of urine samples challenges the proteomic and glycoproteomic analysis to find urine-derived signatures associated with pMN. In this study, we presented a microparticle-assisted protein capture (MAPC) method to efficiently prepare urine samples. It was found that proteins and N-linked intact glycopeptides can be sensitively identified from urine samples of pMN patients and healthy controls by using this method. For comparison, proteins and N-linked intact glycopeptides from serum were also analyzed. Interestingly, discrepancy was found in the changing trends for proteins/intact glycopeptides between serum and urine. Moreover, urine derived proteins, N-linked intact glycopeptides, and glycans exhibited more pronounced changes between pMN and healthy control compared to serum sample. Notably, urine IgG4 not only up-regulated in pMN at global peptide level, its corresponding site-specific glycans were specifically identified in pMN urine with significantly up-regulations, suggested the potential of using glycosylated urine IgG4 for the non-invasive diagnosis of pMN.