Unveiling the rat urinary proteome with three complementary proteomics approaches

Abstract

Urine is a suitable biological fluid to look for markers of physiological and pathological processes, including renal and nonrenal diseases. In addition, it is an optimal body sample for diagnosis, because it is easily obtained without invasive procedures and can be sampled in large quantities at almost any time. Rats are frequently used as a model to study human diseases, and rat urine has been analyzed to search for disease biomarkers. The normal human urinary proteome has been studied extensively, but the normal rat urinary proteome has not been studied in such depth. In light of this, we were prompted to analyze the normal rat urinary proteome using three complementary proteomics platforms: SDS-PAGE separation, followed by LC-ESI-MS/MS; 2DE, followed by MALDI-TOF-TOF and 2D-liquid chromatography-chromatofocusing, followed by LC-ESI-Q-TOF. A total of 366 unique proteins were identified, of which only 5.2% of unique proteins were identified jointly by the three proteomics platforms used. This suggests that simultaneous proteomics techniques provide complementary and nonredundant information. Our analysis affords the most extensive rat urinary protein database currently available and this may be useful in the study of renal physiology and in the search for biomarkers related to renal and nonrenal diseases.

Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets

“…Recently, we have also identified more than 2000 proteins in the skin mucus of GSB using 1-DE/MS approaches [ 99 ], when the digested protein fragments were matched against our protein database with a high coverage of GSB protein-codifying sequences (more than 15,000 unique sequences in Swissprot). These numbers are in the same order of magnitude or even higher than those reported for other mucosal tissues and body fluids in humans and other animal models [ 100 – 102 ], and in skin mucus in GSB [ 103 , 104 ]. The recent use of the iTRAQ technique to characterise the proteins in bile and intestinal mucus of Nile tilapia [ 105 ] was also able to discriminate more than 2700 peptide fragments, but only 319 (corresponding to 179 different proteins) were properly identified.…”

Skin Mucus of Gilthead Sea Bream (Sparus aurata L.). Protein Mapping and Regulation in Chronically Stressed Fish

“…However, by using our gilthead sea bream protein database we identified 2,466 spectra with a much higher protein score (≥20). This number was significantly reduced to 2,060 when a protein score filter of 30 was applied (Table S1 ), but even in this case, the number of identified proteins was relatively high compared to the proteins that compose other mucosal tissues and body fluids in humans (de Souza et al, 2006 ; Lee et al, 2009 ; Marimuthu et al, 2011 ) and other animal models (Sánchez-Juanes et al, 2013 ; Bennike et al, 2014 ; Winiarczyk et al, 2015 ). Certainly, this was favored by the use of a homologous protein database derived from a reference transcriptome with a high coverage of protein-codifying sequences (more than 15,000 unique sequences in Swissprot database), which first increased the consistency of annotation in parallel with the number of protein isoforms or subunits of a given enzyme or protein complex represented in the analyzed samples (e.g., enzyme subunits of the mitochondrial respiratory chain; protein subunits of the eukaryotic translation initiation factor; ribosomal proteins; proteasome subunits, etc.).…”

NMR-Based Metabonomic Analysis of Physiological Responses to Starvation and Refeeding in the Rat

“…The pattern observed in the urinary levels of creatinine may also be informative about the renal functional responses to starvation because alterations in glomerular filtration rate and polyuria are induced in starved animals. , Therefore, the increased levels of creatinine at t 1 may have derived not only from the skeletal muscle protein turnover but also from changes in renal filtration induced by starvation, as this osmolyte is neither secreted nor reabsorbed by the renal tubule in the female rat . Accordingly, NAC levels were higher in the t 1 than in the t 2 or t 3 metabotype, which may have resulted from protein or peptide mobilization during starvation because the presence of urinary proteins is a response to the stress produced by food deprivation . Regarding the excess of urinary creatine after refeeding in t 2 , it may be derived from either intestinal absorption of dietary creatine or de novo creatine biosynthesis via kidney–liver–skeletal muscle axis …”

“…42 Accordingly, NAC levels were higher in the t 1 than in the t 2 or t 3 metabotype, which may have resulted from protein or peptide mobilization during starvation because the presence of urinary proteins is a response to the stress produced by food deprivation. 43 Regarding the excess of urinary creatine after refeeding in t 2 , it may be derived from either intestinal absorption of dietary creatine or de novo creatine biosynthesis via kidney−liver− skeletal muscle axis. 40 In addition, the amino acid taurine is involved in skeletal muscle homeostasis, and several physiological functions have been described for it as conjugating for bile acids, osmoregulator, modulator of calcium homeostasis and signaling, endogenous antioxidant, and anti-inflammatory compound in various tissues.…”