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. 2015;2015(Suppl 14):003.
Epub 2015 Dec 21.

Optimizing Urine Processing Protocols for Protein and Metabolite Detection

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Free PMC article

Optimizing Urine Processing Protocols for Protein and Metabolite Detection

Nazema Y Siddiqui et al. J Proteomics Bioinform. .
Free PMC article

Abstract

Background: In urine, factors such as timing of voids, and duration at room temperature (RT) may affect the quality of recovered protein and metabolite data. Additives may aid with detection, but can add more complexity in sample collection or analysis. We aimed to identify the optimal urine processing protocol for clinically-obtained urine samples that allows for the highest protein and metabolite yields with minimal degradation.

Methods: Healthy women provided multiple urine samples during the same day. Women collected their first morning (1st AM) void and another "random void". Random voids were aliquotted with: 1) no additive; 2) boric acid (BA); 3) protease inhibitor (PI); or 4) both BA + PI. Of these aliquots, some were immediately stored at 4°C, and some were left at RT for 4 hours. Proteins and individual metabolites were quantified, normalized to creatinine concentrations, and compared across processing conditions. Sample pools corresponding to each processing condition were analyzed using mass spectrometry to assess protein degradation.

Results: Ten Caucasian women between 35-65 years of age provided paired 1st morning and random voided urine samples. Normalized protein concentrations were slightly higher in 1st AM compared to random "spot" voids. The addition of BA did not significantly change proteins, while PI significantly improved normalized protein concentrations, regardless of whether samples were immediately cooled or left at RT for 4 hours. In pooled samples, there were minimal differences in protein degradation under the various conditions we tested. In metabolite analyses, there were significant differences in individual amino acids based on the timing of the void.

Conclusions: For comparative translational research using urine, information about void timing should be collected and standardized. For urine samples processed in the same day, BA does not appear to be necessary while the addition of PI enhances protein yields, regardless of 4°C or RT storage temperature.

Keywords: Boric acid; First morning void; Metabolomics; Protease inhibitors; Proteomics; Random void; Urine; Urine protein degradation.

Figures

Figure 1
Figure 1
Median normalized protein concentrations in μg/μl for all processing groups. AM = 1st AM void; random = random clinic void; noadd = no additive; BA = boric acid, PI = protease inhibitor; [group]_0h refers to groups where samples were immediately placed at 4°C ; [group]_4h refers to groups where samples were left at room temperature for 4 hrs. The addition of PI resulted in significantly higher protein concentrations, p < 0.001 by Kruskal-Wallis. Individual pairwise comparisons show that groups with PI have significantly more normalized protein than groups without PI.
Figure 2
Figure 2
Protein degradation by processing condition. Pooled samples were digested with trypsin and individual peptides were identified via mass spectrometry. For identifications from each pool, we assessed the percent of all peptides with a semitryptic end (defined as only one end with amino acids R or K versus fully tryptic with both termini being R or K). Percentages of peptides with semi-tryptic cleavage sites were compared across processing conditions and used as a proxy for protein degradation.
Figure 3
Figure 3
Comparison of random voids with addition of PI: median normalized protein concentrations (μg/μl) from no additive ± boric acid (BA) vs. protease inhibitor (PI) ± BA.

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