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Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis.

ABSTRACT: Mass spectrometry-based urinary proteomics is one of the most attractive strategies to discover proteins for diagnosis, prognosis, monitoring, or prediction of therapeutic responses of urological diseases involving the kidney, prostate, and bladder; however, interfering compounds found in urine necessitate sample preparation strategies that are currently not suitable for urinary proteomics in the clinical setting. Herein, we describe the C4-tip method, comprising a simple, automated strategy utilizing a reverse-phase resin tip-based format and "on-tip" digestion to examine the urine proteome. We first determined the optimal conditions for protein isolation and protease digestion on the C4-tip using the standard protein bovine fetuin. Next, we applied the C4-tip method to urinary proteomics, identifying a total of 813 protein groups using LC-MS/MS, with identified proteins from the C4-tip method displaying a similar distribution of gene ontology (GO) cellular component assignments compared to identified proteins from an ultrafiltration preparation method. Finally, we assessed the reproducibility of the C4-tip method, revealing a high Spearman correlation R-value for shared proteins identified across all tips. Together, we have shown the C4-tip method to be a simple, robust method for high-throughput analysis of the urinary proteome by mass spectrometry in the clinical setting.

SUBMITTER: Clark DJ

PROVIDER: S-EPMC6512789 | biostudies-literature | 2019 May

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:The presence of urinary fistula after ileal conduit urinary diversion is a challenging complication, and this study investigated the role of the intra-conduit negative pressure system (NPS) in the presence of urinary fistula following ileal conduit (IC) urinary diversion as a conservative treatment. METHODS:Using the intra-conduit NPS, a minor drainage tube was placed within a silicon tube to suck urine from the conduit with consistent negative pressure. Patients with urinary fistula following IC from August 2012 to July 2017 were recorded, and the clinical characteristics and outcome were retrospectively analyzed. RESULTS:The intra-conduit NPS was used as a primarily conservative treatment for 13 patients who suffered from urinary fistula and presented with a large amount of abdominal/pelvic drainage without other significant morbidities. The median age was 60?years old (42-74?years), and 7patients were male. The median duration between the IC operation and the presence of urinary fistula was 15?days (2-28?days), and elevated creatinine levels were detected in the abdominal/pelvic drainage with a median level of 2114??mol/L (636-388??mol/L). A significant decrease in abdominal/pelvic drainage was identified in 12 patients. The median time that the NPS was used was 9?days (7-11?days). The other patient did not show any improvements after 2?days of observation and then underwent open surgery. With ureteral stenting, 2 abdominal drainage tubes and the intra-conduit NPS were placed during operation, no urine leakage was observed in the abdominal/pelvic field, and the patient was cured in 9?days. With a median follow-up of 22?months, no fistula recurrence or hydronephrosis was detected. CONCLUSION:The intra-conduit negative pressure system is a feasible and promising way to cure urinary fistula following ileal conduit urinary diversion. Because this procedure is a mini-invasive and simple approach, it might represent an alternative in selected patients.

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Simple Tip-Based Sample Processing Method for Urinary Proteomic Analysis.

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