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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Dataset's files

Source:

			Action	DRS
	20230703_155155_20230308 MStern vs ureaHLC wlibrary uHLC Only.sne	Other
	20230703_160207_20230308 MStern vs ureaHLC wlibrary OM Only.sne	Other
	20230727_103331_20230308_uHLC.sne	Other
	20230727_103421_20230308_MStern.sne	Other
	A-HILIC1.wiff	Wiff

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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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