Article Figures & Data
Figures
- Fig. 1.
Proteomic analysis of microsomal subproteome in the HLM samples showing the total number of identified and quantified proteins in the fraction the number of all drug-metabolizing enzymes (DME), drug-metabolizing cytochrome P450 enzymes (CYP450), and drug-metabolizing UGT enzymes (UGT) (B); Spearman correlation with linear regression of measurements of drug-metabolizing P450 and UGT enzymes using the label-free method described in this article and measurements of the same enzymes in three of the analyzed samples using the QconCAT targeted method (C); fold difference in abundance of enzymes of label-free measurements (method 1) in each sample relative to QconCAT measurements (method 2) expressed as a ratio ([ x1,i / x2,i] for enzyme i), with all pairs of measurements within approximately 2.5-fold (gray box) (D). Average fold error (AFE) is a measure of bias in the data, whereas absolute average fold error (AAFE) is a measure of scatter or spread of measurements; the closer these two measures are to 1, the lower the bias and scatter in measurement. Limited bias was seen in the two methods with a level of spread in the data (see Supplemental Table 3). (C) Abundances are expressed in units of pmol mg−1 HLM protein.
- Fig. 2.
Patterns of expression of DMEs in liver samples: cytochrome P450 enzyme abundances compared with literature values (A), UGT abundances compared with literature values (B), patterns of expression of quantified DMEs in the HLM samples (C), and heat map of the expressed P450 and UGT enzymes with samples classed using rank-order clustering (D). (A and B) Gray highlights indicate literature-derived ranges, bars indicate literature means and scatter points indicate experimentally derived values in this study. (C) BLQ is assigned for values below the limit of quantification. (D) The abundance values are normal log modified. Abundances are expressed in units of pmol mg−1 HLM protein
- Fig. 3.
The abundance of specific membrane markers of hepatocytes (asialoglycoprotein receptor 1, ASGR1), endoplasmic reticulum (calnexin), plasma membrane (ATP1A1, CD81), mitochondria (COX4), and peroxisomes (PEX14) (A), subcellular localization of all identified proteins (B), and drug-metabolizing enzymes (C) in analyzed samples, providing an indication of the presence of membrane fractions from these organelles in HLMs. (A) Abundances are expressed in units of pmol mg−1 HLM protein. (B and C) Percentages represent the proportions of proteins identified in each subcellular location to the total number of identified proteins, the sum of which adds up to more than 100% as a result of overlap in the localization of protein expression as shown in the Supplemental Material.
- Fig. 4.
Contribution of the 10 most abundant proteins to total HLM protein mass from four human livers (A) and simulated effect of variability of the top 10 proteins in HLM samples on CYP3A4 abundance in 2000 human livers (B). When the amount of CYP3A4 within tissue in simulated livers is kept constant, the abundance of CYP3A4 changes on average 1.4- to 1.7-fold, representing the effect of overall random variability in the most abundant proteins. Simulations were based on data obtained from this experimental study and a meta-analysis of available literature. (B) Abundances are expressed in units of pmol mg−1 HLM protein.
Tables
- TABLE 1
Demographic and clinical details of the individual liver donors of samples used in this study
The final column shows the suppliers of samples. HLM samples were prepared by the suppliers using differential centrifugation of hepatic tissue homogenates.
Patient Sample Age (yr) Ethnicity Gender Cause of Death Smoking Alcohol Use Medical History Medication Supplier HLM01 31 C F Motor vehicle accident Yes No None None BD Gentest HLM02 62 C F Head trauma No No Hypertension Hypertension medications BD Gentest HLM03 41 H F CVA No Occasional Hypertension,mild stroke Atenolol, dobutamine, morphine, Nuprin BD Gentest HLM04 50 C M CVA No No Healthy None Vitron C, Caucasian; CVA, cerebrovascular aneurysm; H, Hispanic; F, Female; M, male.
- TABLE 2
Rank orders, abundance levels, and primary subcellular localization of the 10 most abundant proteins in the analyzed HLM samples and those of drug-metabolizing cytochrome P450 enzymes
Overall Rank Protein (Gene Name) Overall Abundance HLM01 HLM02 HLM03 HLM04 Mean ± S.D.a [pmol mg−1] Mean ± S.D.b [pmol mg−1] Mean ± S.D.b [pmol mg−1] Mean ± S.D.b [pmol mg−1] Mean ± S.D. b [pmol mg−1] (Rank) (Rank) (Rank) (Rank) Top 10 HLM proteins 1 Liver carboxylesterase 1c 403.14 ± 92.8 485.94 ± 24.31 (1) 396.81 ± 31.63 (1) 275.38 ± 13.45 (5) 454.42 ± 19.15 (1) 2 Cytoplasmic actin 1 (ACTB)d 316.94 ± 74.63 325.51 ± 50.06 (8) 363.32 ± 21.94 (2) 370.02 ± 24.37 (2) 208.93 ± 23.54 (10) 3 Protein disulfide isomerase (P4HB)c 304.25 ± 101.48 415.74 ± 12.23 (3) 324.86 ± 18.57 (3) 169.96 ± 4.02 (9) 306.44 ± 12.18 (2) 4 78-kDa glucose-regulated protein (HSPA5)c 284.23 ± 62.67 352.90 ± 10.52 (6) 284.86 ± 24.83 (5) 201.29 ± 2.06 (8) 297.86 ± 10.86 (3) 5 ATP synthase subunit β (ATP5B)e 264.10 ± 96.66 140.23 ± 1.59 (24) 270.66 ± 25.62 (6) 376.41 ± 19.64 (1) 269.09 ± 2.31 (5) 6 Protein disulfide isomerase A3 (PDIA3)c 262.73 ± 108.03 387.29 ± 12.61 (4) 252.14 ± 17.28 (7) 127.46 ± 2.23 (27) 286.05 ± 12.36 (4) 7 Calreticulin (CALR)c 257.40 ± 86.90 372.06 ± 7.27 (5) 223.98 ± 36.44 (10) 166.32 ± 13.55 (11) 267.26 ± 4.77 (6) 8 Haptoglobin (HP)f 253.47 ± 155.76 457.21 ± 34.92 (2) 292.00 ± 22.60 (4) 117.54 ± 10.34 (30) 147.11 ± 1.66 (21) 9 Endoplasmin (HSP90B1)c 243.64 ± 78.95 335.09 ± 9.03 (7) 231.93 ± 17.82 (9) 144.52 ± 15.96 (14) 263.01 ± 6.13 (7) 10 Cytochrome b5 (CYB5A)c 226.79 ± 18.36 251.61 ± 26.34 (11) 209.61 ± 15.40 (12) 217.01 ± 7.03 (6) 228.93 ± 36.13 (8) Drug-metabolizing P450 enzymesc 44 CYP3A4 80.87 ± 58.48 126.83 ± 4.43 (26) 134.71 ± 13.65 (26) 19.32 ± 1.63 (231) 42.64 ± 1.02 (122) 53 CYP2E1 76.28 ± 14.78 73.50 ± 6.23 (57) 74.36 ± 8.85 (65) 60.84 ± 6.38 (78) 96.43 ± 13.80 (34) 101 CYP2C9 50.37 ± 30.63 94.26 ± 13.22 (43) 29.89 ± 0.73 (192) 28.82 ± 0.40 (171) 48.49 ± 2.83 (106) 108 CYP4F 48.31 ± 19.96 28.37 ± 2.56 (165) 68.91 ± 8.19 (70) 34.32 ± 1.79 (142) 61.63 ± 2.33 (80) 109 CYP2A6 48.11 ± 43.44 108.60 ± 13.01 (36) 38.44 ± 3.53 (147) 5.11 ± 0.57 (597) 40.30 ± 1.37 (127) 180 CYP3A5 31.07 ± 9.68 37.92 ± 4.13 (117) 24.22 ± 1.54 (231) — — 186 CYP1A2 30.44 ± 7.19 24.25 ± 2.59 (184) 28.72 ± 2.81 (196) — 38.33 ± 5.88 (134) 201 CYP2C8 27.57 ± 25.42 64.37 ± 3.03 (66) 23.22 ± 1.72 (244) 6.92 ± 0.77 (516) 15.78 ± 1.25 (296) 262 CYP2B6 20.98 ± 1.22 20.98 ± 1.22 (225) — — — 425 CYP2D6 12.45 ± 4.84 10.74 ± 1.07 (372) 8.70 ± 1.72 (538) 17.91 ± 1.79 (245) — 560 CYP3A7 9.21 ± 0.62 9.21 ± 0.62 (413) — — — 815 CYP2C19 5.47 ± 0.69 5.96 ± 0.17 (542) — — 4.98 ± 0.76 (604) 1066 CYP3A43 1.06 ± 0.40 0.66 ± 0.11 (711) 1.20 ± 0.12 (814) 1.04 ± 0.11 (706) 1.46 ± 0.05 (707) 51 NADPH cytochrome P450 reductase (POR)c 77.95 ± 19.18 85.88 ± 3.27 (53) 100.12 ± 5.52 (35) 55.97 ± 8.61 (87) 69.85 ± 3.27 (62)
Data Supplement
Files in this Data Supplement:
- Supplemental Data -
Supplemental Methods
Supplemental Table 1 - An outline of the methodological steps constituting the quantitative proteomic workflows specific to each methodology used for quantitative analysis
Supplemental Figure 1 - Experimental workflow used in the label-free proteomic analysis of HLM samples
Supplemental Figure 2 - Protein concentration in HLM samples (A), and the amount of protein digested compared to the amount of peptide recovered for LC-IMS-MS/MS analysis (B)
Results
Supplemental Figure 3 - Linear regression analysis of protein abundances measured using myoglobin as a standard...
Supplemental Table 2 - Appraisal of the protein standards used for the label-free quantitative analysis
Supplemental Table 3 - The precision (%CV) of label-free measurements, their relative error (%RE) compared to QconCAT measurements and differences between the two sets of measurements
Supplemental Figure 4 - Venn diagrams showing the overlap between all quantified human liver microsomal proteins...
Supplemental Figure 5 - Venn diagrams of the subcellular localization of all proteins...
Supplemental Figure 6 - Results of ion mobility spectrometry (IMS) in sample HLM01 illustrating effective separation of peptide ions in the mobility domain...
References
- Supplemental Data -
