Combining [ 13 C 6 ]‐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 [ 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. Copyright © 2009 John Wiley & Sons, Ltd.