Analysis of methionine enkephalin in human pituitary by multi-dimensional reversed-phase high-performance liquid chromatography, radioreceptor assay, radioimmunoassay, fast atom bombardment mass spectrometry, and mass spectrometry-mass spectrometry.
Methionine enkephalin (ME = YGGFM) was measured in five individual human post-mortem pituitaries using four different analytical methods, with the objective of comparing the molecular specificities of the methods. Radioreceptor assay (RRA) used a receptor-rich preparation from brain and [3H]etorphine as radioligand to determine ME-like receptoractivity (ME-LR). Radioimmunoassay (RIA) measured ME-like immunoreactivity (ME-LI). Pituitary samples analyzed by RRA and RIA were purified first with a high-performance liquid chromatography (HPLC) gradient on a polymer analytical column. Fast atom bombardment mass spectrometry (FAB-MS) in two different detection modes quantified ME using the protonated molecular ion MH+ of ME at 574 a.m.u. and B/E linked-field selected reaction monitoring (SRM) to monitor the specific unimolecular metastable transition that produced the unique amino acid sequence-determining tetrapeptide fragment ion YGGFA+ from the MH+ precursor ion. Both FAB-MS methods used the deuterated internal standard YGG[2H5-F]M. Samples analyzed with FAB-MS were purified first with multi-dimensional reversed-phase HPLC. The first dimension was an ODS gradient, and the second dimension was a polymer isocratic elution. The following ME amounts were measured (mean +/- standard error of the mean): ME-LR, 7.0 +/- 1.9 micrograms g-1 tissue; ME-LI, 1.8 +/- 0.7 micrograms g-1 tissue; MH+, 2.7 +/- 0.6 micrograms g-1 tissue; SRM, 3.0 +/- 0.8 micrograms g-1 tissue. The FAB SRM method provided the highest level of molecular specificity amount these four analytical methods used to measure picomole amounts of endogenous ME in a human pituitary.
Lovelace
J L
JL
Department of Biochemistry, University of Tennessee, Memphis 38163.
Kusmierz
J J
JJ
Desiderio
D M
DM
D000328:Adult; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004744:Enkephalin, Methionine / Q000032:analysis*; D006801:Humans; D008032:Limbic System / Q000737:chemistry; D013058:Mass Spectrometry; D010902:Pituitary Gland / Q000737:chemistry*; D011863:Radioimmunoassay; D011869:Radioligand Assay; D016339:Spectrometry, Mass, Fast Atom Bombardment
D004744:Enkephalin, Methionine