A comparative analysis of methods used for quantification of transporter-protein absolute abundances across research groups
| Group | Peptide Selection Method | Standard Generation (P-gp Standard) | Tissue/Cell Fractionation (DC/KIT/FASP) | Detergents/ Chaotropic/ Reducing Agent | Digestion Strategy | Standard Pre/ Postdigestion | LC-MS/MS System | Source |
|---|---|---|---|---|---|---|---|---|
| Tohoku University, Boehringer Ingelheima | In silico: In-house and MS/MS verification | AQUA Isotope label (FYDPLAGK) | DC: PM fraction; whole brain capillaries | Guanidinium hydrochloride | In-solution trypsin (16 h, 37°C) E:S, 1:100 | Postdigestion | Multiplex HPLC: normal flow and nanoflow MS: API5000 /QTRAP5500 (AB SCIEX) or 4000 Q trap (Applied Biosystems) | Ohtsuki et al. (2012) Sakamoto et al. (2011)a Uchida et al., 2014) |
| Pfizer | Prospector: UCSF and MS/MS verification | AQUA Isotope label (NTTGALTTR) | Kit extraction: native membrane (Calbiochem) | DTT; DOC | In-solution trypsin (16 h, 37°C) E:S, 1:20–50 | Predigestion or postdigestion | Single and multiplex HPLC: normal flow MS: API4000 (Applied Biosystems)f | Li et al. (2009) Zhang et al. (2011) Balogh et al. (2013) Qiu et al. (2013) |
| University of Paris Descartes | Linked to Tohoku University | AQUA Isotope label (NTTGALTTR) | Whole brain capillaries | Guanidinium hydrochloride | In-solution trypsin (16 h, 37°C) E:S, 1:100 | Postdigestion | HPLC: normal flow and nanoflow MS: 4000 QTRAP/API 5000 (Applied Biosystems) | Shawahna et al. (2011) |
| AstraZeneca | MS fragmentation: MASCOT search and MS/MS verification | AQUA Isotope label (AGAVAEEVLAAIR) | DC: TM fraction | PPS | In-solution trypsin (6 h, 37°C) E:S, 1:20 | Predigestion | Single UHPLC: normal flow MS: 6460 (Agilent Technologies) | Miliotis et al. (2011) |
| TNO | See Tohoku University entry | AQUA Isotope label | DC: PM fraction | DTT | In-solution trypsin (o/n, +2 h, 37°C) E:S, 1:100 | Postdigestion | Multiplex UHPLC: normal flow MS: Xevo-TQ-S (Waters) | van de Steeg et al. (2013) |
| The University of Washington | Prospector: UCSF and Skyline and MS/MS verification Links to Pfizer’s proteomic laboratory | AQUA Isotope label (NTTGALTTR) | Kit extraction: native membrane (Calbiochem) | DTT | In-solution trypsin (14–24 h, 37°C) E:S, 1:25 | Predigestion (Deo et al., 2012) or postdigestion | Multiplex UHPLC: normal flow MS: See Pfizerf or 6460A (Agilent Technologies) | Deo et al. (2012) f (Prasad et al. (2014) |
| The University of Uppsala | Links to Pfizer and Max Planck Institute proteomic laboratoriesd | AQUA Isotope label P-gp quantification not reported to date | Kit extraction: native membrane (Calbiochem) | DOC | In-solution trypsin (16 h, 37°C) E:S, 1:20 | Postdigestion | See Pfizer entry | Karlgren et al. (2012) Vildhede et al. (2014) |
| Max Plank Institute | Total protein nontargeted | DDA Label-free | DC: TM fraction | SDS/Urea | In-solution FASP Lys-C (o/n) room temp trypsin: (2 h, 37°C) | n/a | Total protein array HPLC: nanoflow Orbitrap LTQ (Thermo Scientific) | Karlgren et al. (2012) Wisniewski et al. (2014) |
| The University of Greifswaldb | Linked to Tohoku University | P-gp quantification not reported to date | DC: PM fraction | Not stated | In-solution trypsin (16 h, 37°C) E:S, 1:100 | Postdigestion | Multiplex HPLC: normal flow and nanoflow MS: QTRAP5500 (AB SCIEX) or 4000 Q trap (Applied Biosystems) | Niessen et al. (2010) |
| The University of Greifswaldb | Expasy program and MS/MS verification | AQUA Isotope label (AGAVAEEVLAAIR) | Kit extraction: native membrane (Calbiochem) | DTT | In-solution trypsin (16 h, 37°C) E:S, 1:40 | Postdigestion | Multiplex HPLC: normal flow MS: API4000 (Applied Biosystems) | Groer et al. (2013) |
| Bertin Pharma | Linked to Tohoku University | AQUA Isotope label [FYDPLAGK, see Sakamoto et al. (2011)] | DC: PM fraction | Unknowne | In-solutione trypsin | Postdigestion | Multiplex HPLC: normal flow MS:API5000 (AB SCIEX) | Kunze et al. (2014) |
| The University of Manchester | SRM Atlas CONSeQuence program: UoM and MS/MS verification | QconCAT Isotope label (AGAVAEEVLAAIR) | DC: PM fraction | DOC-DTT | In-solution Lys-C (4 h, 30°C) Trypsin: (18 h, 37°C) E:S, 1:20 | Predigestion | Multiplex HPLC: nanoflow MS: TSQ Vantage (Thermo Scientific) | Russell et al. (2013) |
| University of Kansas/ Stanford University, and Eli Lilly | In silico: in-house and MS/MS verification | AQUA Isotope label (NTTGALTTR) | Kit extraction: native membrane (Millipore) | DOC-DTT | In-solution trypsin (18 h, 37°C) E:S, 1:20 | Postdigestion | Multiplex UPLC: normal flow MS: Xevo TQ-S (Waters) | Peng et al. (2015) |
| Bristol Myers Squibb | AQUA Isotope label | Kit extraction: native membrane (Calbiochem) | DOC-DTT | In-solution trypsin (16 h, 37°C) E:S, 1:20 | Postdigestion | Multiplex unknown UPLC: normal flow MS: API6500 (AB SCIEX) | Shen et al. (2015) | |
| The University of Dundeec | n/a | S-tag: Antibody label-free | DC:TM fraction | n/a | n/a | n/a | n/a | Tucker et al. (2012) |
DC, differential centrifugation; DDA, data-dependent acquisition; E:S, enzyme-to-substrate ratio for trypsin; DOC-DTT, sodium deoxycholate-dithiothreitol; DOC, sodium deoxycholate; DTT, dithiothreitol; FASP, filter-aided sample preparation; n/a, not applicable; o/n, overnight; PM, plasma membrane; PPS, a silent surfactant; TM, total membrane; UCSF, University of California, San Francisco.
↵a Boehringer Ingelheim, Japan collaborated with Tohoku University.
↵b Two distinct groups at The University of Greifswald.
↵c Professor Coughtrie’s group is now based at the University of British Columbia.
↵d The targeted proteomic strategy at the University of Uppsala used the Pfizer laboratory and techniques described in Balogh et al. (2013).
↵e An “off-the-shelf” MS2Plex kit is used.
↵f Deo et al. (2012) performed LC-MS/MS analysis at Pfizer Ltd.
n/a, not applicable.