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 CriteriaSIL-Based Proteomic MethodQconCAT-Based Proteomic Method
Peptide selection methodTheoretical and in silico design—selected based on published criteriaa and in silico predictionsTheoretical and experimental design—selected based on analysis of pooled HLM samples and theoretical considerationsa
StandardsStable isotope-labeled (SIL) peptide standards (AQUA QuantPro Peptides, Thermo)Quantitative concatenation (QconCAT) standard (designed and produced in-houseb)
Selected peptide standardscUGT1A1DGAF[13C,15N]YTLKTYPVPFQR[13C]
Sample preparationIn-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/MSNano 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 analysisMRM 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).