Labelling of proteins by use of iodination and detection by ICP-MS

Abstract

The labelling of three different proteins, bovine serum albumin (BSA), chicken egg white lysozyme and porcine gastric mucosa pepsin, with iodine using a commercially available reaction kit has been investigated for total protein amounts of 500 μg. The assay described here has already often been applied but with radioactive iodine isotopes and is extended in this work for the use of the stable isotope ¹²⁷I and detection by ICP-MS. The reaction conditions are investigated and optimised for proteins separated by SDS-PAGE and electroblotted onto NC membranes. Detection has been performed by laser ablation ICP-MS on the membranes. Long reaction times result in degradation of proteins, whereas for short reaction times (4 min) integrated sensitivities of even 10⁸ cps pmol⁻¹ of protein have been realized. Total amounts of proteins have been investigated in the range from 0.015 (BSA) to 105 pmol (lysozyme). A calibration was performed for BSA from 0.015 to 15 pmol but the limit of detection was reached already at about 150 fmol due to high iodine blank values, only. The labelling procedure described here does not affect protein mobility in SDS-PAGE, because the change of the molecular weight is very moderate for the proteins investigated here. This is quite opposite to those procedures where chelating compounds are applied. ESI-FTICR-MS has been used to identify histidine and tyrosine as the target amino acids for iodination. Additionally, oxidation of methionine residues has been observed.