Comparison of Depletion Strategies for the Enrichment of Low-Abundance Proteins in Urine

Abstract

Proteome analysis of complex biological samples for biomarker identification remains challenging, among others due to the extended range of protein concentrations. High-abundance proteins like albumin or IgG of plasma and urine, may interfere with the detection of potential disease biomarkers. Currently, several options are available for the depletion of abundant proteins in plasma. However, the applicability of these methods in urine has not been thoroughly investigated. In this study, we compared different, commercially available immunodepletion and ion-exchange based approaches on urine samples from both healthy subjects and CKD patients, for their reproducibility and efficiency in protein depletion. A starting urine volume of 500 μL was used to simulate conditions of a multi-institutional biomarker discovery study. All depletion approaches showed satisfactory reproducibility (n=5) in protein identification as well as protein abundance. Comparison of the depletion efficiency between the unfractionated and fractionated samples and the different depletion strategies, showed efficient depletion in all cases, with the exception of the ion-exchange kit. The depletion efficiency was found slightly higher in normal than in CKD samples and normal samples yielded more protein identifications than CKD samples when using both initial as well as corresponding depleted fractions. Along these lines, decrease in the amount of albumin and other targets as applicable, following depletion, was observed. Nevertheless, these depletion strategies did not yield a higher number of identifications in neither the urine from normal nor CKD patients. Collectively, when analyzing urine in the context of CKD biomarker identification, no added value of depletion strategies can be observed and analysis of unfractionated starting urine appears to be preferable.

Fig 1. Protein amounts at different steps of the analysis as estimated by Bradford measurements.

ND: not determined due to measurements being below the limit of detection (i.e. concentration < 0.2 μg/μL).

Fig 2. Representative SDS-PAGE results for fractionated and non-fractionated samples (normal and CKD).

The figure represents initial urine, flow-through and elution for each of the depletion kits applied. The fractions representing depleted sample and albumin as a common protein depleted by all the kits are marked. I—Initial urine (non-fractionated sample); F—Flow-through fraction; E—Elution. The same protein amounts were loaded onto the gels for initial sample (lane 2 in all cases). Any observed differences in staining intensities are attributed to differences in the silver staining procedure.

Fig 3. Average number of peptides identified per method.

Fig 4. Coefficient of variation for 50 most abundant proteins and whole dataset for A) Normal, B) CKD urine.

Normal samples appear having higher variability compared to the CKD samples, nevertheless this difference is not significant. Additionally and as expected, the variability increases when low-abundance proteins are included in the CV calculations.

Fig 5. Relative abundance of 20 most abundant proteins derived from undepleted urine and comparison of their abundance with corresponding depleted fractions for urine from healthy controls and CKD patients.

Efficient depletion of target proteins is observable for all methods, with the exception of albumin for ProteoSpin in CKD sample. * Denotes significant changes compared to initial urine. ABMP: protein AMBP, ALBU: albumin, IGHG1: Ig gamma-1 chain region, UROM: uromodulin, KNG1: kininogen 1, APOD: apolipoprotein D, OSTP: osteopontin, PTGDS: prostaglandin-H2 D-isomerase, P3IP1: phosphoinositide-3-kinase-interacting protein 1, RNAS1: ribonuclease pancreatic, THRB: prothrombin, AMY2B: alpha-amylase 2B, CD59: CD59 glycoprotein, ZA2G: zinc-alpha-2-glycoprotein, MASP2: mannan-binding lectin serine protease 2, IGHA1: Ig alpha-1 chain C region, CD44: CD44 antigen, EGF: pro-epidermal growth factor, RNAS2: non-secretory ribonuclease, VASN: vasorin, A1AT: alpha-1-antitrypsin, TRFE: serotransferrin, HPT: haptoglobin, HEMO: hemopexin, A1AG1: alpha-1-acid glycoprotein 1, RET4: retinol-binding protein 4, VTDB: vitamin D-binding protein, FETUA: alpha-2-HS-glycoprotein, IGLL5: immunoglobulin lambda-like polypeptide 5, APOA1: apolipoprotein A-I, A1AG2: alpha-1-acid glycoprotein 2, APOH: beta-2-glycoprotein 1, CERU: ceruloplasmin.