Table 6 Reagents used for QTAP

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.