Abstract
Biomarker discovery approaches in urine have been hindered by concerns for reproducibility and inadequate standardization of proteomics protocols. In this study, we describe an optimized quantitative proteomics strategy for urine biomarker discovery, which is applicable to fresh or long frozen samples. We used urine from healthy controls to standardize iTRAQ (isobaric tags for relative and absolute quantitation) for variation induced by protease inhibitors, starting protein and iTRAQ label quantities, protein extraction methods, and depletion of albumin and immunoglobulin G (IgG). We observed the following: (a) Absence of protease inhibitors did not affect the number or identity of the high confidence proteins. (b) Use of less than 20 μg of protein per sample led to a significant drop in the number of identified proteins. (c) Use of as little as a quarter unit of an iTRAQ label did not affect the number or identity of the identified proteins. (d) Protein extraction by methanol precipitation led to the highest protein yields and the most reproducible spectra. (e) Depletion of albumin and IgG did not increase the number of identified proteins or deepen the proteome coverage. Applying this optimized protocol to four pairs of long frozen urine samples from diabetic Pima Indians with or without nephropathy, we observed patterns suggesting segregation of cases and controls by iTRAQ spectra. We also identified several previously reported candidate biomarkers that showed trends toward differential expression, albeit not reaching statistical significance in this small sample set.
Highlights
Biomarker discovery approaches in urine have been hindered by concerns for reproducibility and inadequate standardization of proteomics protocols
Urine proteomics is complicated by low urine protein concentration, variations in pH, and high concentrations of salts and urea or other urine components that interfere with sample processing
Before utilizing isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics for urine biomarker discovery, we evaluated the impact of variation in several processing steps on iTRAQ protein identification and quantitation
Summary
Biomarker discovery approaches in urine have been hindered by concerns for reproducibility and inadequate standardization of proteomics protocols. (e) Depletion of albumin and IgG did not increase the number of identified proteins or deepen the proteome coverage Applying this optimized protocol to four pairs of long frozen urine samples from diabetic Pima Indians with or without nephropathy, we observed patterns suggesting segregation of cases and controls by iTRAQ spectra. With ongoing advances in mass spectrometry (MS) and proteomics technology, proteomics analysis is progressively occupying a central position in biomarker discovery platforms. Biofluids such as urine and blood are the preferred media for proteomics analysis because of their ease of collection and extensive history of use in clinical laboratory prac-. No systematic analyses of urine sample collection and processing have been reported for iTRAQ
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