MASS-SPECTROMETRIC QUANTIFICATION OF ENDOGENOUS BETA-ENDORPHIN

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 ((H-2(4)-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 ((H-2(4))NAIIK), or (b) the transitions {[NAIIK + H]+ --> [NAI]+} and {[(H-2(4))NAIIK + H]+ --> [(H-2(4))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-mu-g of BE; each standard solution also contained 10-mu-g of (H-2(4))BE. The amounts (x-BAR +/- 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).