Optimizing protein recovery for urinary proteomics, a tool to monitor renal transplantation

Abstract

Despite attractiveness of urine for biomarker discovery for systemic and renal diseases, the confounding effect of the high abundance plasma proteins in urine, and a lack of optimization of urine protein recovery methods are bottlenecks for urine proteomics. Three methods were performed and compared for percentage protein yield, yield consistency, ease and cost of analysis: (i) organic solvent precipitation, (ii) dialysis/lyophilization, and (iii) centrifugal filtration. Urine samples were subjected to an immunoaffinity column to deplete high abundance proteins. Difference gel electrophoresis was performed to assess use of depletion strategy for detection of low abundance proteins. Urine from healthy volunteers (n = 10) and kidney transplant recipients with proteinuria (n = 11) were used. Centrifugal filtration performed best for analysis ease and yield consistency. Highest percentage yield was obtained from dialysis/lyophilization but was laborious and residual salt interfered with subsequent gel electrophoresis. Organic solvent precipitation was inexpensive, but suffered from varying yield consistency. Increased spot intensity for some low abundance and previously undetected proteins were noted after depletion of high abundance proteins. In conclusion, we compare the pros and cons of different protein recovery methods and reveal an increase in the dynamic range of protein detection after depletional strategy that could be critical for biomarker discovery, particularly with reference to processing human study samples from clinical trials.

Optimizing a Proteomics Platform for Urine Biomarker Discovery

“…total amount of protein extracted) (Table I). However, unlike twodimensional gels (7,11), with iTRAQ, the largest number of proteins were identified in the samples precipitated with methanol ( Fig. 3a and supplemental Fig.…”

“…Impact of Abundant Protein Depletion-Depletion of abundant urine proteins such as albumin increases the number of identified protein spots on a two-dimensional gel (1,7,16). However, the effect of such depletion on the urine proteome, as perceived by iTRAQ, has not been addressed.…”

“…One such method, isobaric tags for relative and absolute quantitation (iTRAQ) 1 (2), combines relative and absolute peptide quantification with multiplexing ability to enable an increased throughput as well as simultaneous comparison of up to eight samples within one experimental run. Variations induced by urine sample processing have been systematically evaluated for proteomics analyses using two-dimensional gel electrophoresis (3)(4)(5)(6), differential gel electrophoresis (7), and liquid chromatography-coupled mass spectrometry (LC-MS) (5,8,9). However, no systematic analyses of urine sample collection and processing have been reported for iTRAQ.…”

Optimization for Peptide Sample Preparation for Urine Peptidomics

“…In this context, urine could prove to be an important proximal fluid in providing biomarkers that could be tested noninvasively [2]. The choice of urine is particularly useful in the context of kidney and urinary system related diseases such as acute and chronic kidney diseases, diseases with either the urinary bladder or prostrate, and kidney transplantation [2][3][4][5][6][7][8][9]. Circulating peptides in urine are to be considered potentially useful as biomarkers as they are both abundant and easily accessible.…”

Mass spectrometric detection of candidate protein biomarkers of cancer cachexia in human urine

“…These latter results are consistent with our own findings. Improvements in methodology, such as the use of storage agents (41); alternative strategies of protein precipitation (42); a return to 2D PAGE (43); or bioinformatics (43) may all increase the yield of potential biomarkers.…”