title: Mass spectrometric quantification of endogenous beta-endorphin.
abstract: Fast atom bombardment (FAB) mass spectrometry and multiple reaction monitoring (MRM) in the B/E linked-field scan mode were used to quantify endogenous beta-endorphin (BE) in individual human pituitary extracts. The experimental protocol includes the addition of a stable isotope-labeled internal standard ((2H4-Ile22)BE1-31, human) to the tissue homogenate before extraction, purification of the native BE by a combination of Sep-Pak chromatography and gradient high-performance liquid chromatography (HPLC), trypsin digestion to cleave BE into smaller peptides, and separation of the tryptic fragment BE20-24 (NAIIK) by isocratic reversed-phase HPLC. Mass spectrometric quantification is based upon recording either (a) the [M + H]+ ions of NAIIK and its deuterated analog ((2H4)NAIIK), or (b) the transitions ([NAIIK + H](+)----[NAI]+) and [((2H4)NAIIK + H](+)----[(2H4)NAI]+) using the B/E linked-field scan. Linear calibration curves were obtained using these two mass spectrometric techniques from standard solutions containing 1.25-20 micrograms of BE; each standard solution also contained 10 micrograms of (2H4)BE. The amounts (means +/- s.d.) of endogenous BE in five separate human pituitaries were found to be 156 +/- 84 [( M + H]+ method) and 169 +/- 99 pmol mg-1 protein (MRM method).
authors.affiliation: Charles B. Stout Neuroscience Mass Spectrometry Laboratory, University of Tennessee-Memphis 38163.
authors.forename: C
authors.initials: C
authors.lastname: Dass
authors.forename: J J
authors.initials: JJ
authors.lastname: Kusmierz
authors.forename: D M
authors.initials: DM
authors.lastname: Desiderio
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D010902:Pituitary Gland / Q000737:chemistry*; D016339:Spectrometry, Mass, Fast Atom Bombardment; D001615:beta-Endorphin / Q000737:chemistry*
chemical_list: D001615:beta-Endorphin