Strategy for Identification of Phosphorylation Levels of Low Abundance Proteins in Vivo for Which Antibodies Are not Available

Protein function is mainly modulated by dynamic reversible or irreversible post-translational modifications. Among them, the identification of protein phosphorylation sites and changes in phosphorylation levels in vivo are of considerable interest for a better understanding of the protein function. Thus, effective strategies for the quantitative determination of phosphorylation degrees for low abundant proteins, for which antibodies are not available, are required in order to evaluate the functional regulation of proteins attributed to phosphorylation. In this study, we used the heart β1-adrenergic receptor (Adrb1) as a model protein and developed FLAG-Adrb1 knock-in mice, in which the FLAG tag was inserted at the N-terminus of Adrb1. The phosphorylation sites and levels of Adrb1 in the heart were elucidated by immuno-affinity purification followed by quantitative mass spectrometry analysis using ion intensity ratio of the phosphorylated peptide versus corresponding unphosphorylated peptide. The phosphorylation levels at Ser274 and Ser462 of Adrb1 were approximately 0.25 and 0.0023. This effective strategy should be useful for not only analyzing site-specific phosphorylation levels of target proteins, but also quantifying the expression levels of proteins of interest when appropriate antibodies are not available

Prediction of diuretic response to tolvaptan by a simple, readily available spot urine Na/K ratio.

BACKGROUND:Tolvaptan is vasopressin type 2 receptor antagonist that inhibits water reabsorption. It is used in combination with standard diuretics to treat ascites unresponsive to standard diuretic therapy or hyponatremia because of liver cirrhosis. This study evaluated the effectiveness and safety of tolvaptan in clinical practice and aimed to determine the factors related to its effectiveness. METHODS:Tolvaptan was administered to 88 consecutive cirrhotic patients with ascites unresponsive to standard diuretic therapy. An effective treatment response was a ≥2% reduction in body weight on day 7. The association of patient pretreatment characteristics with therapeutic effects was analyzed. RESULTS:Mean weight reduction on day 7 of tolvaptan therapy was -2.9% ± 3.2%, and treatment was effective in 52% of patients. Multivariate analysis revealed that spot urine Na/K ratio ≥2.5 at baseline was the only factor independently related to therapeutic effect, with an odds ratio of 7.85 (95% confidence interval 2.64-23.40, p = 0.0002). Weight reduction percentage on day 7 was -4.0% ± 2.8% in patients with spot urine Na/K ≥2.5, which was significantly greater than the 0.7% ± 2.7% loss in those with urine Na/K < 2.5 (p < 0.05). A spot urine Na/K ratio ≥2.5 had a sensitivity of 85% and specificity of 60% for predicting effective treatment. No adverse events of treatment led to treatment discontinuation. CONCLUSIONS:Baseline spot urine Na/K was predictive of an effective response to tolvaptan therapy. It is simple to perform and readily available and might serve as an indicator of optimal timing of tolvaptan administration in patients with inadequate response to conventional Na diuretic therapy

ROC curve analysis of urine Na/K ratio and prediction of total urine Na excretion per day.

<p>ROC analysis revealed that urine Na/K ratio was closely associated with urinary Na excretion ≥ 78 mmol/day, with the area under curve of 0.94 (95% CI: 0.85–1.0).</p

Clinical backgrounds of patients.

<p>Clinical backgrounds of patients.</p

Multivariate regression analysis assessing the effectiveness of tolvaptan.

<p>Multivariate regression analysis assessing the effectiveness of tolvaptan.</p

Urine Na/K ratio and background factors.

<p>Urine Na/K ratio and background factors.</p

ROC curve analysis of spot urine Na/K ratio and prediction of weight loss.

<p>The ROC curve shows baseline spot urine Na/K ratio to predict weight reduction ≥2% on day 7 of tolvaptan therapy. Urine Na/K ratio ≥2.5 was the best cutoff value to predict effectiveness.</p