Isoform-specific quantification of endothelins in HUVEC culture supernatants by on-line high-performance liquid chromatography/electrospray mass spectrometry.
A method for the quantitative analysis of endothelin peptides in human umbilical vein endothelial cell (HUVEC) culture supernatants is reported. The analysis is isoform-specific and employs solid-phase extraction and subsequent HPLC fractionation followed by HPLC-ESIMS analysis. The peptide vasoactive-intestinal-contractor (VIC) was used as internal standard for the HPLC-ESIMS analysis. Linearity of calibration curves was from 50 fmol to 25 pmol. The limit of detection of the HPLC-ESIMS step using a buffer matrix was estimated at 50 fmol (S/N > 3). The overall limit of detection for supernatants of HUVEC was 500 fmol/mL. In HUVEC culture supernatants only ions of endothelin-1 (ET1) were observed. Basal levels were determined to be 1.8 +/- 0.3 pmol/mL. Quantitative results obtained for ET1 were in agreement with those obtained by using a standard addition method and by an ELISA method.
Structural and Biological Mass Spectrometry Unit, Department of Medical Bioanalysis, IIBB-CSIC, IDIBAPS, Rossello 161, 08036-Barcelona, Spain.
Montserrat
M
Carrascal
Klaus
K
Schneider
Carmen
C
Peralta
Gines
G
Escolar
Emilio
E
Gelpi
Joaquin
J
Abian
D000595:Amino Acid Sequence; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003470:Culture Media / Q000737:chemistry*; D016232:Endothelins / Q000032:analysis* / Q000737:chemistry; D004730:Endothelium, Vascular / Q000166:cytology*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008969:Molecular Sequence Data; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D003470:Culture Media; D016232:Endothelins