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.
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).
In a study to test the hypothesis that defects in the metabolism of neuropeptides might be a contributing factor to human anterior pituitary tumor formation, the proenkephalin A, proopiomelanocortin (POMC), and tachykinin systems, which produce methionine enkephalin (ME), beta-endorphin (BE), and substance P (SP), respectively, were measured in patients who had a wide variety of pituitary tumors. Mass spectrometry was used to optimize the level of molecular specificity of the ME and BE analytical measurements, and radioimmunoassay was used to measure SP-like immunoreactivity (SP-li). Compared to data obtained from pituitaries from post-mortem controls, the non-secreting tumors contained a significantly lower amount of the POMC neuropeptide, BE. The lower ME level was not significant. However, two adrenocorticotrophic hormone (ACTH)-secreting tumors contained ME, BE, and SP-li amounts that were much higher than both the controls and nonsecreting tumors. These data suggest that a hypometabolism of the POMC precursor may be operating in non-secreting tumors, and that a hypermetabolism of the proenkephalin A, POMC, and tachykinin precursors may be operating in two ACTH-secreting tumors. These data demonstrate that mass spectrometry plays a critical role in the study of human pituitary tumors.
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