Combining [¹³C₆]-phenylisothiocyanate and the edman degradation reaction: A possible breakthrough for absolute quantitative proteomics together with protein identification

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 [¹³C₆]-phenylisothiocyanate (PITC) and the Edman degradation reaction as a possible breakthrough. [¹³C₆]-PITC was synthesized from [¹³C₆]-aniline with O,O(-di-2-pyridyl thiocarbonate to prepare [¹³C₆]-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 [¹³C₆]-PTH-Asp showed good linearity (r² 1/40. 9977). BSA could be quantified as PTH-Asp using the standard curve. In addition, the residual des-Asp¹-BSA provided sufficient information for further protein identification.