MeCAT peptide labeling for the absolute quantification of proteins by 2D-LC-ICP-MS

Metal-Coded Affinity Tags (MeCAT) reagents were devised for the absolute quantification of labeled proteins and peptides using inductively coupled plasma mass spectrometry (ICP-MS). After the recent publication of quantification approaches for digested proteins, this work presents a multidimensional strategy for the application of MeCAT to samples which require higher chromatographic resolution. Two-dimensional separations based on strong cation exchange (SCX) and reversed-phase (RP) chromatography, were used for the quantification of lysozyme, bovine serum albumin and transferrin after tryptic digestion. The elution protocols were optimized to improve the resolution of the MeCAT-labeled peptides which led to faster elutions in SCX and longer retention times in RP compared with unlabeled peptides. The optimized method provided enough resolution for the samples analyzed. Peptides losses during the whole procedure were studied. Although recoveries of greater than 90% were found in the RP dimension, important global losses in the two-dimensional offline approach forced us to use specific internal standards, in this case MeCAT-labeled standard peptides. External calibration and label-specific isotope dilution analysis (IDA) were tested and compared as possible quantification techniques. While both techniques showed accurate and precise determinations, the label-specific IDA technique resulted in more straightforward measurements and more affordable external calibrations. Finally, simultaneous quantification of three different samples labeled with different lanthanides was successfully performed demonstrating the potential of MeCAT combined with ICP-MS for multiplexing. Electrospray ionization mass spectrometry techniques provided the structural information needed for the identification of the labeled species.