Accurate quantitation of phospholamban phosphorylation by immunoblot

Abstract

We have developed a quantitative immunoblot method to measure the mole fraction of phospholamban (PLB) phosphorylated at Ser16 (X(p)) in biological samples. In cardiomyocytes, PLB phosphorylation activates the sarcoplasmic reticulum calcium ATPase (SERCA), which reduces cytoplasmic Ca(2+) to relax the heart during diastole. Unphosphorylated PLB (uPLB) inhibits SERCA at low [Ca(2+)] but phosphorylated PLB (pPLB) is less inhibitory, so myocardial physiology and pathology depend critically on X(p). Current methods of X(p) determination by immunoblot provide moderate precision but poor accuracy. We have solved this problem using purified uPLB and pPLB standards produced by solid-phase peptide synthesis. In each assay, a pair of blots is performed with identical standards and unknowns using antibodies partially selective for uPLB and pPLB, respectively. When performed on mixtures of uPLB and pPLB, the assay measures both total PLB (tPLB) and X(p) with accuracy of 96% or better. We assayed pig cardiac sarcoplasmic reticulum (SR) and found that X(p) varied widely among four animals, from 0.08 to 0.38, but there was remarkably little variation in the ratios of X(p)/tPLB and uPLB/SERCA, suggesting that PLB phosphorylation is tuned to maintain homeostasis in SERCA regulation.

Fig. 1

PLB oligomeric state does not affect immunoblot intensity. Typical immunoblots and standard curves are shown for samples pre-heated to 100° (red) and unheated (blue) for uPLB (“u” on blots, circles on graphs) or pPLB (“p” on blots, squares on graphs) are shown for Ab285, Ab8A3, Ab2D12, and AbA1. Corresponding western blots are above each graph.

Fig. 2

Validation of the method used to determine X_p and tPLB. (A) Western blots of 5, 10, and 15 ng of uPLB or pPLB standards, and 12 ng of uPLB/pPLB mixtures (duplicates) of known X_p (0.00 to 1.00, as indicated). (B) Typical standard curves from a for uPLB (squares) and pPLB (circles), from blots incubated with the indicated primary antibody. Resulting slopes (proportionality constants ε_ij in Eq. 1 and Eq. 2) are indicated. (C) Accuracy and precision of X_p values: X_p(app)/X_p ± SEM, for mixtures of known X_p. Antibody a is Ab8A3 (square), Ab2D12 (circle), AbA1 (triangle). (D) Accuracy and precision of tPLB values: tPLB(app)/tPLB + SEM, for the same mixtures as in C.

Fig. 3

Comparison of methods for measuring X_p and tPLB for the same sample (X_p = 0.5). The line at 1.00 indicates the accurate value. The antibody a (partially selective for uPLB) was Ab8A3 (squares), Ab2D12 (circles), or AbA1 (triangles). Methods 1 through 4 and their assumptions are described in the text. Only Method 4 (ours) returns accurate values for both tPLB and X_p.

Fig. 4

Application to pig cardiac SR. (A) Western blots of standards (2.5, 7.5 and 15ng), control mixtures (X_p = 0.25, 0.5 and 0.75), and 0.5µg of pig CSR from pigs 1–4 from left to right. (B) tPLB vs. X_p (C) PLB/SERCA ratios vs. X_p for uPLB, pPLB, and tPLB.