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 [(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.
Oe
Tomoyuki
T
Department of Bio-analytical Chemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Aobayama, Aoba-ku, Sendai 980-8578, Japan. t-oe@mail.pharm.tohoku.ac.jp
Maekawa
Masamitsu
M
Satoh
Ryo
R
Lee
Seon Hwa
SH
Goto
Takaaki
T
D000818:Animals; D002138:Calibration; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D017879:Isothiocyanates / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics / Q000379:methods*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D017879:Isothiocyanates; D011506:Proteins; C005441:phenylisothiocyanate; D012710:Serum Albumin, Bovine