Article Figures & Data
Figures
- Fig. 1.
Comparison of liver proteome measurements using three label-free methods. (A) Percentage of the measured total protein content relative to the nominal content (dashed line) analyzed by mass spectrometry. The amount was determined using the TPA, HiN [based on either myoglobin (MYG) or BSA as standards] and iBAQ (based on BSA). (B) Head-to-head comparison of average concentrations (of 2232 proteins) in 27 samples quantified by iBAQ (BSA) and TPA compared with HiN (BSA). (C) Correlation between mean concentrations of 57 key pharmacokinetic targets measured by TPA or iBAQ and mean abundances measured by HiN. The data show that TPA overestimates protein amounts compared with the HiN method, whereas HiN and iBAQ methods produce comparable results in most cases. The data also indicate that it is possible to estimate absolute abundances by iBAQ or TPA using conversion factors. Abundance is expressed in units of pmol mg−1 total protein.
- Fig. 2.
Correlation between protein concentrations of key pharmacokinetic targets measured by iBAQ (blue) or TPA (orange) relative to HiN method in 27 liver samples. The data show examples of drug-metabolizing enzymes and transporters. BSA was used as a standard for HiN and iBAQ, and abundance was measured in units of pmol mg−1 total protein.
- Fig. 3.
Correlation of protein concentrations of (A) cytochrome P450 and (B) UGT enzymes measured by HiN (red), iBAQ (blue), and TPA (orange) against functional activity in 27 liver samples. Activity was measured with metabolite formation assays against the substrates: phenacetin (CYP1A2), mephenytoin (CYP2B6), diclofenac (CYP2C9), mephenytoin (CYP2C19), bufuralol (CYP2D6), testosterone (CYP3A), β-estradiol (UGT1A1), chenodeoxycholic acid (UGT1A3), 5-hydroxytryptophol (UGT1A6), propofol (UGT1A9), zidovudine (UGT2B7) and S-oxazepam (UGT2B15). Abundance was measured in units of pmol mg−1 total protein; catalytic activity was measured in units of nmol metabolite min−1 mg−1 total protein.
- Fig. 4.
Comparison of abundances of (A) cytochrome P450 and (B) UGT enzymes measured using label-free methods (HiN, iBAQ, and TPA) against targeted data. Ratios of label-free measurements relative to targeted data for (C) cytochrome P450 and (D) UGT enzymes. In (A) and (B), the whiskers represent the minimum-to-maximum range, the boxes represent the 25th and 75th percentiles, the lines represent the medians, and + signs represent the means. Comparisons based on a t test against targeted data are shown in black and against HiN measurements are shown in red. * P < 0.05, ** P < 0.01, and *** P < 0.001. In (C) and (D), the dashed lines denote the twofold range, and the percentages are the proportion of label-free measurements within twofold of targeted data. Abundance was measured in units of pmol mg−1 total protein.
- Fig. 5.
Comparison of between-sample variability across 27 liver samples in the abundance of enzymes and transporters measured by HiN, iBAQ, and TPA, expressed as (A) fold difference (maximum-to-minimum ratio) and (B) percent CV (CV = SD × 100/mean). BSA was used as a standard for HiN and iBAQ.
Tables
- TABLE 1
The total amount of protein in analyte estimated by the label-free quantification methods, averaged (± SD) over 27 samples.
Method Estimated Total Protein Content Nominal protein contenta 16,667 pmol mg−1 HiN based on BSA 8916 ± 1775 pmol mg−1 HiN based on myoglobin 34,580 ± 5078 pmol mg−1 iBAQ based on BSA 19,265 ± 2937 pmol mg−1 Total Protein Approach (TPA) 24,513 ± 4620 pmol mg−1 aAmount estimated assuming an average analyte protein molecular mass of 60 kDa; units are pmol mg−1 total protein.
Data Supplement
- Supplemental Data -
Supplemental Table 1. Demographic and clinical details of the donors of the 27 liver samples.
Supplemental Table 2. Agreement in identification between technical replicates.
Supplemental Table 3. Similarity matrix between biological samples based on percent identical peptide (PIP).
Supplemental Table 4. Abundance (in pmol mg-1) of cytochrome P450 enzymes, UGT enzymes, ABC transporters, and solute carriers using HiN with BSA as a standard.
Supplemental Table 5. Abundance (in pmol mg-1) of cytochrome P450 enzymes, UGT enzymes, ABC transporters, and solute carriers using iBAQ with BSA as a standard.
Supplemental Table 6. Abundance (in pmol mg-1) of cytochrome P450 enzymes, UGT enzymes, ABC transporters, and solute carriers using the TPA method.
Supplemental Table 7. Effect of age, body mass index (BMI) and genotype on the abundance of enzymes and transporters measured using HiN, iBAQ and TPA label-free methods.
Supplemental Figure 1. Relative abundance of (A) CYPs, (B) UGTs and (C) ABC transporters in human liver determined using either HiN, iBAQ or TPA.
- Supplemental Data -
