Dataset Information

Urine-HILIC – An automated sample preparation for bottom-up urinary proteome profiling in clinical proteomics

ABSTRACT: Urine provides a diverse source of information related to health status and is ideal for clinical proteomics because of its ease of collection. To date, there is no standard operating procedure for reproducible and robust urine sample processing for mass spectrometry-based clinical proteomics. To address this need, a novel workflow was developed based on an on-bead protein capture, clean up, and digestion without the requirement from pre-processing steps such as precipitation. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample were subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using a novel urine-HILIC (uHLC) approach based on MagReSyn® HILIC microspheres. For benchmarking, the pooled sample was also prepared using a published protocol based on an on-membrane (OM) protein capture and digestion workflow. Peptides were analysed by LCMS in DIA mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. Data was searched in Spectronaut™ 17. Following statistical analysis, candidate protein markers were filtered at ≥ 2-fold change in abundance, ≥ 2 uniques peptides and ≤ 1% false discovery rate. Both workflows showed similar peptide and protein identifications in the pooled sample. The uHLC workflow was easier to set up and complete, having less hands-on time than the OM method. With fewer manuel processiing steps, a lower peptide and protein CV was observed in the uHLC technical replicates. Analysis of clinical samples revealed many significant, differentially abundant kidney injury-associated urinary proteins. The pilot data derived using this novel workflow provides information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.

INSTRUMENT(S): TripleTOF 5600

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Urine

SUBMITTER: Previn Naicker

LAB HEAD: Previn Naicker

PROVIDER: PXD043925 | Pride | 2024-01-26

REPOSITORIES: Pride

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Publications

Urine-HILIC: Automated Sample Preparation for Bottom-Up Urinary Proteome Profiling in Clinical Proteomics.

Govender Ireshyn Selvan IS Mokoena Rethabile R Stoychev Stoyan S Naicker Previn P

Proteomes 20230928 4

Urine provides a diverse source of information related to a patient's health status and is ideal for clinical proteomics due to its ease of collection. To date, most methods for the preparation of urine samples lack the throughput required to analyze large clinical cohorts. To this end, we developed a novel workflow, urine-HILIC (uHLC), based on an on-bead protein capture, clean-up, and digestion without the need for bottleneck processing steps such as protein precipitation or centrifugation. Th ...[more]

PMID: 37873871

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Project description:Objectives: Secretion of extracellular vesicles (EV) and associated micro-RNAs (miR) is altered during cellular stress and may serve as biomarkers of organ injury. We hypothesized that measuring changes in urinary levels of EV and miR will predict the onset of acute kidney injury in cardiac surgery patients. Design: Predictive accuracy biomarker study performed in the cohort of the REVAKI-2 trial Setting: Single center ICU between September 2015 and September 2018 Interventions: Intravenous sildenafil citrate 12.5 mg kg-1 over 150 min or dextrose 5% at the commencement of surgery. Measurements and main results: Urine samples were collected before and 24 hours after the procedure from 93 cardiac surgery patients. Urine EV concentrations and size distribution were assessed using NanoSight. EV derivation and levels were measured using flow cytometry. Samples from 10 selected patients were sequenced to detect differentially expressed miR. Verification was performed with advanced TaqMan assays in samples from all patients. Urine EV concentrations significantly increased in patients with AKI after surgery, with the percentage of EV positive for aquaporin-2 and β1-integrin also increasing. Pre surgery podocalyxin-positive EV were significantly lower, and β1-integrin EV werehigher in patients with AKI. The levels of the former correlated with the severity of theinjury. miR-125a-5p was expressed at higher levels in urine from patients with AKI stage 2/3. Levels of miR-10a-5p decreased after surgery in AKI patients; its levels correlated with the severity of the injury. Preoperative levels of podocalyxin EVs and miR-125a-5p had moderate AKI predictive value and, in a logistic model together with ICU lactate levels, offered good (AUC = 80.9%) AKI prediction. Conclusions: Lower levels of podocalyxin-positive EV at baseline predict the severity of post-surgery AKI. Urine EV concentrations and miR expression offer excellent predictive accuracy when combined with commonly measured biomarkers.

Dataset Information

Urine-HILIC – An automated sample preparation for bottom-up urinary proteome profiling in clinical proteomics

Publications

Urine-HILIC: Automated Sample Preparation for Bottom-Up Urinary Proteome Profiling in Clinical Proteomics.

Similar Datasets

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