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

Abstract

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. The workflow was applied to an acute kidney injury (AKI) pilot study. Urine from clinical samples and a pooled sample was subjected to automated sample preparation in a KingFisher™ Flex magnetic handling station using the novel 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 analyzed by LCMS in data-independent acquisition (DIA) mode using a Dionex Ultimate 3000 UPLC coupled to a Sciex 5600 mass spectrometer. The data were searched in Spectronaut™ 17. 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 manual processing steps. Lower peptide and protein coefficient of variation was observed in the uHLC technical replicates. Following statistical analysis, candidate protein markers were filtered, at ≥8.35-fold change in abundance, ≥2 unique peptides and ≤1% false discovery rate, and revealed 121 significant, differentially abundant proteins, some of which have known associations with kidney injury. The pilot data derived using this novel workflow provide information on the urinary proteome of patients with AKI. Further exploration in a larger cohort using this novel high-throughput method is warranted.

Candidate protein biomarkers in chronic kidney disease: a proteomics study

“…In plasma, for instance, highly abundant proteins such as immunoglobulins and albumin, which can vary by over 10 orders of magnitude in concentration, can obscure the detection of low-abundant proteins, complicating the effectiveness of the MS method 26 . Similarly, the masking effect of highly abundant proteins can be observed when analyzing the urinary proteome of CKD patients, who have elevated levels of urinary albumin and other abundant proteins, making the identification of low-concentration urinary proteins challenging 27 , 28 .…”

Proteomic insights into the pathophysiology of hypertension-associated albuminuria: Pilot study in a South African cohort

“…Protein samples were digested on-bead using multimode magnetic microparticles (MagReSyn ® HILIC, ReSyn Biosciences) in a KingFisher Duo™ system (Thermo Fisher Scientific), as previously described (33, 34), with minor modifications. Briefly, magnetic hydrophilic affinity microparticles (20 μl, 200 μg) were equilibrated in 200 μl 100 mM NH 4 Ac pH 4.5, 15% MeCN.…”