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Table 1 In silico peptide selection criteria

From: A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice

Necessary conditions 1. The peptide is theoretically obtained by a protease, i.e., trypsin digestion of the target protein. An arginine or lysine residue occurs prior to the site of cleavage and at the C-terminus of the peptide if trypsin is used.
2. The amino acid sequence of the peptide is unique for a target protein in the peptide library that is theoretically obtained by protease digestion of all the proteins that are registered in protein databases.
3. A length of 6 to 16 amino acids (8 to 10 amino acids is preferable) for detection by QqQ MS.
4. NO methionine or cysteine residues are included.
5. NO posttranslational modification and NO single nucleotide polymorphisms are included for the quantification of the total level of the target protein.
6. NO continuous sequence of arginine or lysine residues (RR, KK, RK, KR) occurs in the digestion region for efficient digestion by trypsin.
7. The peptide does NOT include a proline residue at the C-terminal side of an arginine or lysine residue (RP or KP) in the digestion region for efficient digestion by trypsin.
8. The peptide does NOT include a transmembrane region for efficient digestion by a protease (such as trypsin).
Sufficient conditions 9. The peptide does NOT include histidine residues, which reduce peptide sensitivity in the mass spectrometer.
10. The peptide includes a glycine or proline residue to increase peptide sensitivity in the mass spectrometer.
11. The LC retention time should be predicted based on the hydrophobicity of the amino acids.
12. A water-soluble peptide should be selected based on the hydrophobicity of the amino acids. Hydrophobic amino acids should comprise less than 40% of the peptide.
  1. The table is taken from Kamiie et al. [4] with some modification.