Fast atom bombardment mass spectrometric quantitative analysis of methionine-enkephalin in human pituitary tissues

Abstract

Picomole amounts of endogenous methionine-enkephalin (ME = YGGFM) were quantified in 11 individual human pituitaries by fast atom bombardment mass spectrometry methods. Quantification was based either upon the comparison of the molecular ion (MH+) current of endogenous ME versus the current of a deuterated ME internal standard (d5-ME) or, similarly, upon the unimolecular decomposition MH+----YGGF-+ In the first field-free region to produce the unique tetrapeptide fragment ion. The latter method used the multiple reaction monitoring (MRM) mode. Native ME was purified with an octadecylsilyl (ODS) disposable cartridge and with multidimensional reversed-phase high-performance liquid chromatography. The amounts of ME determined were 18.26 +/- 19.98 ng of ME/mg of protein with the MH+ method and 15.28 +/- 16.59 ng of ME/mg of protein with the MRM method. A fraction (ca. 4%) of the total amount of ME from one pituitary was used to acquire these quantitative data, and ca. half of the remaining amount of a separate sample (no d5-ME added) was used to obtain a linked scan at constant B/E (B, magnetic field; E, electric field) of the ME MH+ at 574 u to produce the amino acid sequence determining fragment ions at m/z 297, 354, 411, 397, 278, and 425 u corresponding to Y2", Y3", Y4", A4, B3, and B4, respectively. That product ion spectrum was similar to a scan of 100 ng of synthetic ME. We calculated that the amount of pentapeptide for the MRM experiments corresponded to a total of 30 ng (52 pmol) of ME on the probe tip during quantification. On the other hand, we estimated that 3 times more, or 90 ng (156 pmol), ME was on the probe tip during acquisition of the product ion spectrum.