TABLE 1

An outline of the methods constituting the QTAP workflow specific to each laboratory for the absolute quantification of Na/K-ATPase, P-gp, and BCRP

CriteriaThe University of ManchesterBertin Pharma
Peptide selectionIn silico: CONSeQuence programaIn silico: Tohoku University algorithmb
Standard generationQuantitative concatenation (QconCAT)Absolute quantification (AQUA)
Selected peptidesNa/K-ATPase: IVEIPFNSTN[K13C]P-gp: AGAVAEEVLAAI[R13C]BCRP: VIQELGLD[K13C]Na/K-ATPase: AAVPDA[V13C,15N]GKP-gp: FYDPL[A13C,15N]GKBCRP: SSL[L13C,15N]DVLAAR
DigestionDeoxycholate denaturation, Lys-C plus trypsin-based digestionMS2Plex-based process including trypsin-based digestion
LC-MS/MSNanoflow LC: nanoAcquity (Waters) with TSQ Vantage (Thermo Fisher), MRMNormal flow LC: Flexar LC (PerkinElmer) with API5500 (AB SCIEX) MRM

  • ↵a Details of CONSeQuence given in Eyers et al. (2011).

  • ↵b Details of peptide selection criteria given in (Kamiie et al. (2008).