Assessment of peptides and fragments used to quantify each of the eight UGT enzymes

ProteinPeptide SequenceaIn Silico Score
(0 to 1)bTheoretical AssessmentcIncorporation Correction Factor (%)dMRM Transitions Monitored 
[(m/z)z]peptide → [(m/z)z]fragmente
UGT1A1D70GAFYTLK77f0.354++4.0457.73+2/524.31+1 (y4)457.73+2/671.38+1 (y5)457.73+2/742.41+1 (y6)
T78YPVPFQR850.363++2.0504.272+/547.30+1 (y4)504.272+/646.37+1 (y5)504.272+/743.42+1 (y6)
UGT1A3Y164LSIPTVFFLR174f0.514+++1.0678.392+/681.41+1 (y5)678.392+/782.46+1 (y6)678.392+/879.51+1 (y7)
UGT1A4Y175IPCDLDFK183f,g0.432+++5.0585.782+/637.32+1 (y5)585.782+/797.35+1 (y6)585.782+/894.40+1 (y7)
G184TQCPNPSSYIPK196g0.522++5.0724.852+/791.43+1 (y7)724.852+/905.47+1 (y8)724.852+/1002.53+1 (y9)
UGT1A6S103FLTAPQTEYR113f0.554+++2.0656.832+/793.38+1 (y6)656.832+/864.42+1 (y7)656.832+/965.47+1 (y8)
V250SVWLLR256h,i0.354+1.5436.772+/587.37+1 (y4)436.772+/686.43+1 (y5)
UGT1A9E139SSFDAVFLDPFDNCGLIVAK159f,g0.553++4.01172.562+/1233.63+1 (y11)1172.562+/1348.66+1 (y12)1172.562+/1461.74+1 (y13)
UGT2B4F174SPGYAIEK1820.391+++5.0506.262+/623.34+1 (y5)506.262+/680.36+1 (y6)506.262+/777.41+1 (y7)
A321NVIASALAK330f0.412+++4.0479.292+/560.34+1 (y6)479.292+/673.42+1 (y7)479.292+/772.49+1 (y8)
UGT2B7T41ILDELIQR49f0.426++2.0550.822+/658.39+1 (y5)550.822+/773.42+1 (y6)550.822+/886.50+1 (y7)
A253DVWLIR259i0.300+1.5436.752+/587.37+1 (y4)436.752+/686.43+1 (y5)436.752+/801.46+1 (y6)
UGT2B15W97IYGVSK103h0.325+3.0426.732+/553.30+1 (y5)426.732+/666.38+1 (y6)
S432VINDPVYK440f0.406++5.0517.782+/506.30+1 (y4)517.782+/735.37+1 (y6)517.782+/848.45+1 (y7)
MetCATjGVNDNEEGFFSARk0.561++++2.5721.322+/813.39+1 (y7)721.322+/942.43+1 (y8)721.322+/1056.47+1 (y9)
  • a Peptide sequences as defined by the human UniProtKB database ( Subscript number labels on the C- and N-terminal amino acids of peptide sequences denote their positions in the UGT protein sequences on the basis of their database entries. The terminal lysine (K) and arginine (R) residues were labeled using [13C6] stable isotopes in the QconCAT standard.

  • b In silico assessment was carried out using CONSeQuence algorithm on the basis of charge, hydrophobicity and secondary structure (Eyers et al., 2011).

  • c Theoretical assessment on the basis of criteria outlined in Supplemental Information; arbitrarily, +, ++, +++, and ++++ scores were assigned to peptides under assessment (highest score, +++++) by two independent analysts.

  • d The proportion of light to heavy peptide owing to inefficient incorporation of the 13C label needed to correct quantification ratios; this can be variable from batch to batch.

  • e Up to three transitions for each peptide were designed in silico using Skyline (superscript indicating charge states, z); selected fragments were then appraised on the basis of unique sequences, m/z, quality of elution profiles, and the CV of the returned quantitative ratios. In this table, only the light (native) peptide transitions are listed, where the y-ions (subscript indicates the length of the sequence) were used.

  • f Peptide selected for quantification of each UGT enzyme on the basis of the selection criteria outlined in Supplemental Information and in silico appraisal.

  • g Cysteine residues were alkylated (by carbamidomethylation), necessitating an increment of +57.0215 Da in monoisotopic mass of peptides and certain fragments.

  • h Two transitions were designed and monitored for peptides VSVWLLR (UGT1A6) and WIYGVSK (UGT2B15), which returned low scores on the basis of theoretical, in silico, and fragment assessments. These peptides were excluded from analysis.

  • i The isobaric sequences VSVWLLR (UGT1A6) and ADVWLIR (UGT2B7) were overlapped on the chromatogram owing to close retention times (with the same m/z of parents and fragments). These peptides were excluded from analysis.

  • j MetCAT: QconCAT used as a standard for the quantification of human liver P450 and UGT enzymes (Russell et al., 2013; Achour et al., 2015).

  • k Sequence of QconCAT-based internal standard used for quantification of the QconCAT.