Non-labeled P02-C01 peptide (RDYHPRDHTATWGGG) and labeled P02-C01 peptide (RDYH*PRDHTATWGGG) were ordered from Biomatik, Wilmington, DE, USA. *P in labeled P02-C01 peptide stands for [13C5, 15N1]-Pro amino acid residue. Both peptides were 97% pure based on HPLC analysis.
Samples of 0.5 nmol of NISTmAb were mixed with 50 nmol of non-labeled P02-C01 in 40 μL of 25 mmol/L His-buffer (pH 6.0), incubated on ice for 30 min, and loaded on protein A HP SpinTrap columns (GE Healthcare) pre-equilibrated with cold 25 mmol/L His-buffer (pH 6.0). After a quick wash with 0.6 mL of cold His-buffer (150 g, 30 sec), the columns were eluted with 0.4 mL of 0.5% TFA (150 g, 30 sec). The eluates were supplemented with 200 pmol of labeled P02-C01 peptide and dried. Supplementation with labeled P02-C01 provides an internal standard for future LC-MS/MS quantification.
For saturation binding, samples of 0.5 nmol of NISTmAb stressed by agitation were mixed with increasing amounts of non-labeled P02-C01 (150, 200, 300, 600, 1500, 3000, 4500, and 6000 nmol). The binding assay was then performed as described above.
Samples of 0.5 nmol of NISTmAb were mixed with 50 nmol of non-labeled P02-C01 in 40 μL of 25 mmol/L His-buffer (pH 6.0), incubated on ice for 30 min, and loaded on protein A HP SpinTrap columns (GE Healthcare) pre-equilibrated with cold 25 mmol/L His-buffer (pH 6.0). After a quick wash with 0.6 mL of cold His-buffer (150 g, 30 sec), the columns were eluted with 0.4 mL of 0.5% TFA (150 g, 30 sec). The eluates were supplemented with 200 pmol of labeled P02-C01 peptide and dried. Supplementation with labeled P02-C01 provides an internal standard for future LC-MS/MS quantification.
For saturation binding, samples of 0.5 nmol of NISTmAb stressed by agitation were mixed with increasing amounts of non-labeled P02-C01 (150, 200, 300, 600, 1500, 3000, 4500, and 6000 nmol). The binding assay was then performed as described above.