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Combining [ 13 C 6 ]‐phenylisothiocyanate and the Edman degradation reaction: a possible breakthrough for absolute quantitative proteomics together with protein identification
Author(s) -
Oe Tomoyuki,
Maekawa Masamitsu,
Satoh Ryo,
Lee Seon Hwa,
Goto Takaaki
Publication year - 2009
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.4372
Subject(s) - edman degradation , chemistry , bovine serum albumin , chromatography , degradation (telecommunications) , amino acid , quantitative proteomics , albumin , proteomics , peptide sequence , biochemistry , gene , telecommunications , computer science
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.