Comparison of Heavy Labeled (SIL) Peptide versus SILAC Protein Internal Standards for LC-MS/MS Quantification of Hepatic Drug Transporters

Abstract

We studied the precision of quantification of organic anion-transporting polypeptide 1B1 (OATP1B1), OATP1B3, OATP2B1, and P-glycoprotein (P-gp) in human livers by surrogate peptide based LC-MS/MS approach using two different internal standards: stable isotope labeled peptide (SIL) versus stable isotope labeled protein (SILAC). The SIL peptides were procured commercially and the SILAC proteins were generated in-house by labeling arginine and/or lysine residues in cells expressing these transporters. Liver tissue (n = 20) was homogenized and the membrane fraction was isolated. The membranes were trypsin digested and the peptides were analyzed using LC-MS/MS under optimized conditions. The precision in the quantification of proteins in three independently trypsin digested samples from each liver was calculated as the standard deviation of the log transformed protein concentration. The precision of the SIL internal standard method was either slightly (P < 0.05, paired t-test) better than that of the SILAC method (OATP1B1, OATP1B3, and P-gp) or not different (OATP2B1). Trypsin digestion, as measured by the response of the labeled peptide derived from the SILAC protein, was consistent across liver samples. These results indicate that when maximum trypsin digestion is ensured, the SIL internal standard method can be used with confidence for quantification of drug transporters.

Figure 1

Comparison of SILAC versus SIL internal standard methods. SILAC protein is added before trypsin digestion, while SIL peptide is added just before LC-MS analysis. Key: DTT, dithiothreitol; IAA, iodoacetamide; EB-II, extraction buffer II.

Figure 2

LC-MRM chromatograms of a representative trypsin digest of liver tissue membrane sample. The chromatograms show elution patterns and selectivity for P-gp, OATP2B1, OATP1B3, and OATP1B1 peptides (Table 1), respectively (left to right). Q3-1 and Q3-2 represent two different MRM transitions of a peptide. The top two channels show analyte peptides and the bottom two channels show internal standards (IS) (a). Representative calibration curves (peak area calibrator/internal standard versus amount on-column of the calibrator) and LLOQs (femtomoles, on-column) of OATP1B1, OATP1B3, OATP2B1, or P-gp (b). Limits of detection (LODs) were 3–5-fold lower than LLOQs.

Figure 3

Comparison of formation of the monitored peptides (unlabeled (∆) and labeled (filled circle)) during trypsin digestion of OATP1B1 (a), OATP1B3 (b), OATP2B1 (c), and P-gp (d). Data are presented as mean ± SD. At 24 h, additional trypsin was added (see method). Data are as a percent of trypsin digestion at 24 h (mean ± of n = 3).

Figure 4

Standard deviation (a to d) and expression (e to h) of OATP1B1, OATP1B3, OATP2B1, and P-gp in individual liver sample (mean of triplicates) as determined by SIL (open bars) or SILAC (filled bars) internal standard methods. The last bar represents the population mean value. The individual hepatic expression data using SIL method are from our previous publication [15].