Characterization of β‐endorphin in human pituitary by fast atom bombardment mass spectrometry of trypsin‐generated fragments*

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Abstract
A novel mass spectrometric method possessing a high level of structural specificity is described for characterization in biological fluids and tissues of endogenous β-endorphin of the human amino acid sequence (βh-EP). The method is based upon purification of tissue extracts by an RP-HPLC gradient, followed by trypsinolysis of that particular HPLC fraction corresponding to the elution time of synthetic βh-EP. The tryptic digest of that endogenous βh-EP fraction was purified further by a second RP-HPLC gradient. A unique tryptic fragment selected from the second gradient was analyzed by fast atom bombardment mass spectrometry and B/E linked-field scan MS/MS techniques to provide molecular weight and amino acid sequence-determIntng fragment ion information, respectively, of that fragment. Collectively, these independent analytical methodologies provided unequivocal structure evidence for the presence of endogenous βh-EP in human pituitary. The method was established first by utilizing synthetic Ph-EP to optimize experimental parameters, and then applied to the analysis of βh-EP in post-mortem human pituitary extracts. The suitability of the present method for semiquantitation of tissue extracts is also demonstrated. The corresponding detection limit of the synthetic βh-EP was 90fmol, and human pituitary contained 1.5pmol of βh-EPmg−1 protein. The method can be extended readily to the analysis of β-endorphin derived from other species and tissues.

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