title: Quantification of peptides using N-terminal isotope coding and C-terminal derivatization for sensitive analysis by micro liquid chromatography-tandem mass spectrometry.
abstract: Stable isotope-coding coupled with mass spectrometry is a popular method for quantitative proteomics and peptide quantification. However, the efficiency of the derivatization reaction at a particular functional group, especially in complex structures, can affect accuracy. Here, we present a dual functional-group derivatization of bioactive peptides followed by micro liquid chromatography-tandem mass spectrometry (LC-MS/MS). By separating the sensitivity-enhancement and isotope-coding derivatization reactions, suitable chemistries can be chosen. The peptide amino groups were reductively alkylated with acetaldehyde or acetaldehyde-d4to afford N-alkylated products with different masses. This process is simple, quick and high-yield, and accurate comparative analysis can be achieved for the mass-differentiated peptides. Then, the carboxyl groups were derivatized with 1-(2-pyrimidinyl)piperazine to increase MS/MS sensitivity. Angiotensins I-IV, bradykinin and neurotensin were analyzed after online solid phase extraction by micro LC-MS/MS. In all instances, a greater than 17-fold increase in sensitivity was achieved, compared with the analyses of the underivatized peptides. Furthermore, the values obtained from the present method were in agreement with the result from isotope dilution quantification using isotopically labeled angiotensin I [Asp-Arg-(Val-d8)-Tyr-Ile-His-Pro-(Phe-d8)-His-Leu]. Copyright © 2016 John Wiley & Sons, Ltd.
authors: Bio-medical Standards Group, Research Institute for Material and Chemical Measurement, National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), C-3, 1-1-1, Umezomo, Tsukuba, Ibaraki, 305-8563, Japan.
authors: Yohei
authors: Y
authors: Sakaguchi
authors: Bio-medical Standards Group, Research Institute for Material and Chemical Measurement, National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), C-3, 1-1-1, Umezomo, Tsukuba, Ibaraki, 305-8563, Japan.
authors: Tomoya
authors: T
authors: Kinumi
authors: Bio-medical Standards Group, Research Institute for Material and Chemical Measurement, National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), C-3, 1-1-1, Umezomo, Tsukuba, Ibaraki, 305-8563, Japan.
authors: Akiko
authors: A
authors: Takatsu
mesh_terms: D000079:Acetaldehyde / Q000032:analysis / Q000737:chemistry; D002065:Buspirone / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods*; D003903:Deuterium / Q000032:analysis / Q000737:chemistry; D006801:Humans; D057230:Limit of Detection; D016014:Linear Models; D009479:Neuropeptides / Q000097:blood* / Q000737:chemistry* / Q000378:metabolism; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D009479:Neuropeptides; D003903:Deuterium; D000079:Acetaldehyde; C037921:1-(2-pyrimidinyl)piperazine; D002065:Buspirone
keywords: LC-MS/MS; amine; carboxylic acid; derivatization; isotope coding; peptide