TABLE 1

An outline of the methodology criteria constituting the quantitative proteomic workflows specific to each method for the quantitative analysis of the set of UGT enzymes

Methodological Criteria	SIL-Based Proteomic Method		QconCAT-Based Proteomic Method
Peptide selection method	Theoretical and in silico design—selected based on published criteria^{^a} and in silico predictions		Theoretical and experimental design—selected based on analysis of pooled HLM samples and theoretical considerations^{^a}
Standards	Stable isotope-labeled (SIL) peptide standards (AQUA QuantPro Peptides, Thermo)		Quantitative concatenation (QconCAT) standard (designed and produced in-house^{^b})
Selected peptide standards^{^c}	UGT1A1	DGAF[¹³C,¹⁵N]YTLK	TYPVPFQR[¹³C]
	UGT1A3	YLSIP[¹³C,¹⁵N]TVFFLR	YLSIPTVFFLR[¹³C]
	UGT1A4	YLSIPAVFFWR[¹³C,¹⁵N]	YIPCDLDFK[¹³C]
	UGT1A6	DIVEV[¹³C,¹⁵N]LSDR	VSVWLLR [¹³C]
	UGT1A9	GILCHYLEEGAQCPAPLSYVPR[¹³C,¹⁵N]	ESSFDAVFLDPFDNCGLIVAK[¹³C]
	UGT2B4	FSPGYAIEK[¹³C,¹⁵N]	ANVIASALAK[¹³C]
	UGT2B7	ADVWLIR[¹³C,¹⁵N]	TILDELIQR[¹³C]
	UGT2B15	FSVGYTFEK[¹³C,¹⁵N]	WIYGVSK[¹³C]
Sample preparation	In-solution denaturation with heating at 60°C for 40 min and in-solution proteolysis (trypsin: 5% w/w, 37°C, 4 h)		In-gel protein solubilization with 5% w/v SDS, in-gel proteolysis (lysyl endopeptidase: 1% w/w, 30°C, 4 h; trypsin: 1% w/w, 37°C, 18 h)
LC-MS/MS	Nano flow LC–nanoAcquity (Waters) with QTRAP 5500 hybrid MS system (SCIEX)		Nano flow LC–nanoAcquity (Waters) with TSQ Vantage triple quadrupole MS system (Thermo)
Data acquisition and analysis	MRM quantification using MultiQuant 2.0 (SCIEX)		MRM quantification using Xcalibur 2.06 SP 1 (Thermo), data analysis with Skyline 4.0.4222 (MacCoss Laboratory)

↵^a Details of theoretical selection criteria can be found in Fallon et al. (2008), Kamiie et al. (2008), and Pratt et al. (2006).
↵^b Details of QconCAT design, expression and quality control can be found in Russell et al. (2013) and Achour et al. (2015).
↵^c At least two peptide sequences were selected for each UGT enzyme in each of the two laboratories; the sequences shown here are those selected for the quantification process (Fallon et al., 2013a; Russell et al., 2013).