Electrospray mass spectrometry for the analysis of opioid peptides and for the quantification of endogenous methionine enkephalin and β-endorphin

Electrospray ionization mass spectrometry was used to characterize several different neuropeptides, whose molecular weights ranged from 555 to 3463 Da, and to quantify endogenous methionine enkephalin (ME) and β -endorphin (β E) extracted from a human pituitary gland. Methionine enkephalin and leucine enkephalin both yield only an [M + H] + ion with electrospray mass spectrometry; the other peptides produce a series of multiply charged even-electron molecular ions of the general nature [M + nH]n+ in proportion to the number of basic amino acid units present, with no evidence of fragmentation. The electrospray mass spectra are characterized by low background noise. The quantiftcation of ME is based on a comliarison of the ion current due to the [M + H] + ion of native and of a deuterated ME ([2H5 s−4Phe]−ME) internal standard. The calibration curve is linear in the range of ca. 1-35 pmol synthetic ME. The amounts of ME determined in three separate human pituitary extracts were 9.1, 8.2, and 4.7 pmol/mg protein. The corresponding amount of ME in a canine pituitary was 39.8 pmol/mg protein. To quantify β E, the ion current due to the [M + 5H]5 + ion was monitored and compared to an external calibration curve obtained by analyzing solutions of synthetic β E in the range 5 μmol-50 pmol. The analysis of a human pituitary yielded 660 fmol β E/mg protein.