SRM assay design for tryptic peptides and isotope-labeled peptides
The P450 enzymes targeted for quantitation as well as the protein accession numbers, peptide sequences, and transitions used for quantitation of each P450 in the targeted mass spectrometric assay are outlined. The protein list was generated from a global proteomics screen of liver microsomes and tissue protein lysates isolated from 9- to 10-week-old male and female Balb/c mice. All peptides selected are specific to the protein, with the exception of CYP4A10 peptide, which is also predicted to be formed from CYP4A32. The multiple reaction monitoring (MRM) assay refers to the single peptide and transition pair, which form the complete SRM assay method. The heavy labeled (13C and 15N) synthetic peptides included in the assay for quantitation are represented in boldface with an asterisk next to the heavy labeled residue.
| Enzyme | Accession No. | Peptide Sequence | MRM (Parent/Transition) |
|---|---|---|---|
| 1A2 | NP_034123.1 | T254FNDNFVLFLQK265 | 743.390/747.476 (y6) |
| TFNDNFVLFLQK* | 747.398/755.491 (y6) | ||
| 2A12 | NP_598418.1 | E88ALVDHAEEFSGR100 | 730.344/1047.449 (y9) |
| 2B9 | NP_034130.1 | D263YIDTYLLR271 | 586.303/780.425 (y6) |
| 2B10 | NP_034129.1 | G99TVAVVEPTFK109 | 574.322/819.461 (y7) |
| GTVAVVEPTFK* | 578.329/827.475 (y7) | ||
| 2C29 | NP_031841.3 | N49ISQSFTNFSK59 | 636.815/830.404 (y7) |
| 2C37 | NP_034131.2 | Y308AILLLLK315 | 473.820/712.533 (y6) |
| 2C39 | NP_034133.2 | F126TLTTLR132 | 426.253/603.382 (y5) |
| 2C50 | NP_598905.2 | G384TNVITSLSSVLR396 | 673.886/862.499 (y8) |
| 2C54 | NP_996260.1 | E85ALVDHGDVFAGR97 | 693.344/973.449 (y9) |
| 2C67 | NP_001019890.1 | V125FTINTLR132 | 482.285/503.294 (y4) |
| 2C70 | NP_663474.2 | E85ALIDQGDEFSDK97 | 733.836/1040.417 (y9) |
| 2D9 | NP_034136.2 | D408ESVWEKPLR417 | 629.825/828.473 (y6) |
| 2D10 | NP_034135.2 | F369GDIAPLNLPR379 | 606.840/709.436 (y6) |
| 2D22 | NP_062797.3 | T263TWDPTQPPR272 | 599.796/695.383 (y6) |
| TTWDPTQPPR* | 604.800/705.392 (y6) | ||
| 2D26 | NP_083838.1 | G121VILAPYGPEWR132 | 679.367/454.302 (b5) |
| 2E1 | NP_067257.1 | G113IIFNNGPTWK123 | 623.833/963.468 (y8) |
| 2F2 | NP_031843.2 | S48QDLLTSLTK57 | 553.309/662.408 (y6) |
| 2J5 | NP_034137.1 | L50PFVGNFFQIDTK62 | 763.406/1012.510 (y8) |
| 2U1 | NP_082092.2 | E464TFIPFGIGK473 | 554.805/618.361 (y6) |
| 3A11 | NP_031844.1 | L332QDEIDEALPNK343 | 692.851/358.208 (y3) |
| LQDEIDEALPNK* | 696.858/366.223 (y3) | ||
| 3A13 | NP_031845.1 | D244VISFFTTSVER255 | 700.856/839.426 (y7) |
| 3A25 | NP_062766.2 | F447ALISIK453 | 396.255/573.397 (y5) |
| 4A10 | NP_034141.3 | T215YLQAIGDLNNLFHSR230 | 621.323/887.448 (y7) |
| 4A12A | NP_803125.2 | S214YIQAVEDLNDLVFSR229 | 934.973/1207.595 (y10) |
| 4B1 | NP_031849.1 | G125LLVLEGPK133 | 463.289/642.382 (y6) |
| 4F13 | NP_570952.1 | S453PLAFIPFSAGTR465 | 682.372/735.378 (y7) |
| 4V2 | NP_598730.1 | V391FPSVPLFAR400 | 566.829/886.515 (y8) |
| VFPSVPLFAR* | 571.833/896.523 (y8) |
* designating the last letter only, which is intended to indicate the only heavy labeled residue of the entire boldface peptide sequence.