Absolute quantification of semicarbazide-sensitive amine oxidase in human umbilical artery by single-reaction monitoring with electrospray tandem mass spectrometry.
Semicarbazide-sensitive amine oxidase (SSAO; E.C.1.4.3.6.) is widely distributed in different tissues, particularly in vascular smooth muscle and adipose tissue. Its physiological function remains unclear. Up to now, the common method to determine SSAO is based on enzymatic activity measurement. However, enzymatic activity could be easily influenced by the temperature, pH, and circumstance. In the present study, we have developed the single-reaction monitoring (SRM) approach for measuring the absolute amount of SSAO expression in human umbilical artery based on LC-ESI-MS/MS. The measurement of protein was converted to the measurement of a unique peptide of SSAO from Homo sapiens. The peptide (YQLAVTQR) was confirmed to be unique to the SSAO in human using the ExPasy blast tools, and thus the synthetic peptide was used as the standard which can produce abundant parent ion (m/z = 490.0) and daughter ion (m/z = 687.4) in the mass spectrometry. Trap drive and fragmentation energy of MS/MS of the unique peptide was 60 V and 0.6 V, respectively. The calibration curve was linear over the range of 1.99-127.8 fmol/microL, with 1.99 fmol/microl of the lower limit of quantification. The inter- and intra-day precisions and recoveries for all samples were satisfactory. The results demonstrated SSAO protein concentration was 7.75 fmol/g wet weight. It proved that the novel assay was sensitive and selective to measure the amount of SSAO protein originated from H. sapiens.
Zhang
Yongqian
Y
School of Life Science, Beijing Institute of Technology, Beijing, 100081, China.
Xiao
Shengyuan
S
Wang
Lin
L
Wang
Hongbin
H
Zhu
Yong
Y
Li
Yujuan
Y
Deng
Yulin
Y
D000273:Adipose Tissue / Q000201:enzymology; D006631:Amine Oxidase (Copper-Containing) / Q000032:analysis* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D006801:Humans; D010455:Peptides / Q000032:analysis; D051381:Rats; D012015:Reference Standards; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D014469:Umbilical Arteries / Q000201:enzymology*
D010455:Peptides; D006631:Amine Oxidase (Copper-Containing)