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Table 6 Reagents used for QTAP

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

Reagent Composition Storage temp. Note (how to prepare, store and use)
Denaturing buffer 500 mM Tris–HCl (pH 8.5), 7 M guanidine HCl, 10 mM EDTA R.T. Stir solution at 50°C to dissolve guanidine HCl.
DTT solution 50 μg/μL dithiothreitol (DTT) - Should be freshly prepared and used within 1 h.
IAA solution 50 μg/μL iodoacetamide (IAA) in denaturing buffer - Should be freshly prepared in denaturing buffer, protected from light and used within 1 h.
Tris–HCl buffer 0.1 M Tris–HCl (pH 8.5) R.T. -
Urea solution 6 M urea, 0.1 M Tris–HCl (pH 8.5) - Should be freshly prepared.
LysC solution 0.5 μg/μL lysyl endopeptidase (LysC; Wako 129–02541) -80°C Should be divided into single-use aliquots (e.g., 5 μL/tube) and stored at -80°C until use.
ProteaseMax solution 1% w/v ProteaseMax (Promega V2072) -20°C Should be divided into single-use aliquots (e.g., 10 μL/tube) and stored at -20°C until use.
Trypsin solution 0.5 μg/μL TPCK-trypsin (Promega V5113) -80°C Should be divided into single-use aliquots (e.g., 5 μL/tube) and stored at -80°C until use.
Peptide mixture 100 nM St or IS peptide mixture -80°C Mix stock solutions of different peptides so that each peptide concentration is 100 nM. Should be divided into single-use aliquots (e.g., 50 μL/tube) and stored at -80°C until use.
  1. Abbreviations: R.T. room temperature; St standard; IS internal standard.