Nazema Y Siddiqui1, Laura G DuBois2, Lisa St John-Williams2, Thompson J Will2, Carole Grenier3, Emily Burke4, Matthew O Fraser5, Cindy L Amundsen1, Susan K Murphy3. 1. Division of Urogynecology & Reconstructive Pelvic Surgery; Department of Obstetrics & Gynecology, Duke University, Durham, NC, USA. 2. Center for Genomic & Computational Biology; Duke University, Durham, NC, USA. 3. Division of Gynecologic Oncology; Department of Obstetrics & Gynecology, Duke University, Durham, NC, USA. 4. Department of Biostatistics and Bioinformatics, Duke University, Durham, NC, USA. 5. Division of Urology; Department of Surgery, Duke University, Durham, NC, USA.
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.
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.
Entities:
Keywords:
Boric acid; First morning void; Metabolomics; Protease inhibitors; Proteomics; Random void; Urine; Urine protein degradation
Authors: Stefan Schaub; John Wilkins; Tracey Weiler; Kevin Sangster; David Rush; Peter Nickerson Journal: Kidney Int Date: 2004-01 Impact factor: 10.612
Authors: Stéphane Decramer; Anne Gonzalez de Peredo; Benjamin Breuil; Harald Mischak; Bernard Monsarrat; Jean-Loup Bascands; Joost P Schanstra Journal: Mol Cell Proteomics Date: 2008-07-30 Impact factor: 5.911
Authors: Dan Theodorescu; Stefan Wittke; Mark M Ross; Michael Walden; Mark Conaway; Ingo Just; Harald Mischak; Henry F Frierson Journal: Lancet Oncol Date: 2006-03 Impact factor: 41.316
Authors: Abdul-Hamid Emwas; Claudio Luchinat; Paola Turano; Leonardo Tenori; Raja Roy; Reza M Salek; Danielle Ryan; Jasmeen S Merzaban; Rima Kaddurah-Daouk; Ana Carolina Zeri; G A Nagana Gowda; Daniel Raftery; Yulan Wang; Lorraine Brennan; David S Wishart Journal: Metabolomics Date: 2014-11-21 Impact factor: 4.290
Authors: Sophie Hepburn; David A Cairns; David Jackson; Rachel A Craven; Beverley Riley; Michelle Hutchinson; Steven Wood; Matthew Welberry Smith; Douglas Thompson; Rosamonde E Banks Journal: Proteomics Clin Appl Date: 2015-02-26 Impact factor: 3.494
Authors: Carlos Gevers-Montoro; Mar Romero-Santiago; Lisa Losapio; Francisco Miguel Conesa-Buendía; Dave Newell; Luis Álvarez-Galovich; Mathieu Piché; Arantxa Ortega-De Mues Journal: Front Integr Neurosci Date: 2022-04-12