Combination of Selective Immunoassays and Mass Spectrometry to Characterize Preproghrelin-Derived Peptides in Mouse Tissues

Abstract

Preproghrelin is a prohormone producing several preproghrelin-derived peptides with structural and functional heterogeneity: acyl ghrelin (AG), desacyl ghrelin (DAG), and obestatin. The absence of selective and reliable assays to measure these peptides simultaneously in biological samples has been a limitation to assess their real proportions in tissues and plasma in physiological and pathological conditions. We aimed at reliably measure the ratio between the different preproghrelin-derived peptides in murine tissues using selective immunoassays combined with a highly sensitive mass spectrometry method. AG-, DAG-, and obestatin-immunopositive fractions from the gastrointestinal tract of adult wild-type and ghrelin-deficient mice were processed for analysis by mass spectrometry (MS) with a Triple Quadrupole mass spectrometer. We found that DAG was predominant in mouse plasma, however it only represented 50% of total ghrelin (AG+DAG) production in the stomach and duodenum. Obestatin plasma levels accounted for about 30% of all circulating preproghrelin-derived peptides, however, it represented <1% of total preproghrelin-derived peptides production (AG+DAG+Obestatin) in the stomach. Assays were validated in ghrelin-deficient mice since neither ghrelin nor obestatin immunoreactivities were detected in their stomach, duodenum nor plasma. MS analyses confirmed that obestatin-immunoreactivity in stomach corresponded to the C-terminal amidated form of the peptide but not to des(1-10)-obestatin, nor to obestatin-Gly. In conclusion, specificity of ghrelin and obestatin immunoreactivities in gastrointestinal tissues using selective immunoassays was validated by MS. Obestatin was less abundant than AG or DAG in these tissues. Whether this is due to inefficient processing rate of preproghrelin into mature obestatin in gastrointestinal mouse tissues remains elusive.

Keywords: acyl ghrelin; desacyl ghrelin; immunoreactivity; mass spectrometry; obestatin.

Figure 1

Chromatographic profiles of SRM analyses related to preproghrelin-derived peptides in the stomach and duodenum from 60% chromatographic fractions in ghrl+/+ and ghrl−/− mice. SRM analyses of the synthetic peptide (standard) and tissue samples obtained from the fraction eluted with 60% acetonitrile: (A) standard, (B) acyl ghrelin, (C) des-acyl ghrelin, (D) obestatin, (E) obestatin-Gly, (F) des(1–10)-obestatin. Acyl ghrelin and obestatin-NH₂ were present in both the stomach and duodenum of ghrl+/+ mice but absent in the tissues of ghrl−/− mice. Des-acyl ghrelin was present in the stomach but not in the duodenum of ghrl+/+ mice and absent in the tissues of ghrl−/− mice. The star (^*) denotes a peak in ghrelin chromatogram that is related to the “in source” neutral loss of the acyl group of acyl ghrelin converted partially acyl ghrelin to desacyl ghrelin after LC separation. Obestatin-Gly and des(1–10)-obestatin were absent in both the stomach and duodenum of ghrl+/+ and ghrl−/− mice. See experimental section for SRM method design.