Combining [13C6]-phenylisothiocyanate and the Edman degradation reaction: a possible breakthrough for absolute...

This manuscript describes the results of a preliminary experiment performed as 'proof of concept' of a novel approach to absolute quantitation of proteins without the use of standard proteins. Absolute quantitation remains a challenging issue in the proteomics field. Therefore, we propose a combination of [(13)C(6)]-phenylisothiocyanate (PITC) and the Edman degradation reaction as a possible breakthrough. [(13)C(6)]-PITC was synthesized from [(13)C(6)]-aniline with O,O'-di-2-pyridyl thiocarbonate to prepare [(13)C(6)]-phenylthiohydantoin (PTH)-amino acids as internal standards. Upon the Edman degradation reaction, it has been confirmed that a model protein, bovine serum albumin (BSA), releases the N-terminal amino acid quantitatively as PTH-Asp. The standard curve of PTH-Asp against [(13)C(6)]-PTH-Asp showed good linearity (r(2) = 0.9977). BSA could be quantified as PTH-Asp using the standard curve. In addition, the residual des-Asp(1)-BSA provided sufficient information for further protein identification.