Development of combined micro -preparation, separation, and mass spectrometry methods and application in the microdialysis study of neuropeptide metabolism

Two sets of mass spectrometry-based methods were developed specifically for the in vivo study of extracellular neuropeptide biochemistry. First, an integrated micro-concentration/desalting/matrix-addition device was constructed for matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) to achieve attomole sensitivity for microdialysis samples. Second, capillary electrophoresis (CE) was incorporated into the above micro-liquid chromatography (LC) and MALDI MS system to provide two-dimensional separation and identification (i.e. electrophoretic mobility and molecular mass) for the analysis of complex mixtures. The latter technique includes two parts of instrumentation: (1) the coupling of a preconcentration LC column to the inlet of a CE capillary, and (2) the utilization of a matrix-precoated membrane target for continuous CE effluent deposition and for automatic MALDI MS analysis (imaging) of the CE track.^ Initial in vivo data reveals a carboxypeptidase A (CPA) activity in rat brain involved in extracellular neurotensin metabolism. Benzylsuccinic acid, a CPA inhibitor, inhibited neurotensin metabolite NT1-12 formation by 70%, while inhibitors of other major extracellular peptide metabolizing enzymes increased NT1-12 formation. CPA activity has not been observed in previous in vitro experiments. Next, the validity of the methodology was demonstrated in the detection and structural elucidation of an endogenous neuropeptide, (L)VV-hemorphin-7, in rat brain upon ATP stimulation. Finally, the combined micro-LC/CE/MALDI MS was used in the in vivo metabolic study of peptide E, a mu-selective opioid peptide with 25 amino acid residues. Profiles of 88 metabolites were obtained, their identity being determined by their mass-to-charge ratio and electrophoretic mobility. The results indicate that there are several primary cleavage sites in vivo for peptide E in the release of its enkephalin-containing fragments. ^

Pollen records and age determinations from 2 profiles at Lake Nikolay, Lena Delta

Radiocarbon-dated pollen, rhizopod, chironomid and total organic carbon (TOC) records from Nikolay Lake (73°20'N, 124°12'E) and a pollen record from a nearby peat sequence are used for a detailed environmental reconstruction of the Holocene in the Lena Delta area. Shrubby Alnus fruticosa and Betula exilis tundra existed during 10,300-4800 cal. yr BP and gradually disappeared after that time. Climate reconstructions based on the pollen and chironomid records suggest that the climate during ca. 10,300-9200 cal. yr BP was up to 2-3 °C warmer than the present day. Pollen-based reconstructions show that the climate was relatively warm during 9200-6000 cal. yr BP and rather unstable between ca. 5800-3700 cal. yr BP. Both the qualitative interpretation of pollen data and the results of quantitative reconstruction indicate that climate and vegetation became similar to modern-day conditions after ca. 3600 cal. yr BP. The chironomid-based temperature reconstruction suggests a relatively warm period between ca. 2300 and 1400 cal. yr BP, which corresponds to the slightly warmer climate conditions reconstructed from the pollen. Modern chironomid and rhizopod assemblages were established after ca. 1400 cal. yr BP.