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HPLC-electrospray ionization-mass spectrometry optimization by high-performance design of experiments for astrocyte glutamine measurement.

ABSTRACT: The amino acid glutamine (Gln) is a likely source of energy in the brain during neuroglucopenia. Effects of glucose deficiency on astrocyte Gln homeostasis remain unclear, as analytical tools of requisite sensitivity for quantification of intracellular levels of this molecule are not currently available. Here, a primary hypothalamic astrocyte culture model was used in conjunction with design of experiments (DOE)-refined high-performance liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) methodology to investigate the hypothesis that glucoprivation alters astrocyte Gln content in a sex-specific manner. Critical mass spectrometric parameters for Gln derivative chromatographic response were identified by comparing the performance of central composite design, Box-Behnken design, and Optimal Design (OD)-A, -D, -I, -Distance, and -Modified Distance DOE models. The outcomes showed that the OD-A-generated response was superior relative to other design outcomes. Forecasted surface plot critical mass spectrometric parameters were maximized by OD-A, OD-Distance, and OD-Modified Distance designs. OD-A produced a high-performance method that yielded experimental run and forecasted surface plot maximal responses. Optimized mass spectrometric analysis of male versus female astrocyte Gln content provides novel evidence that glucoprivation significantly depletes this amino acid in female, but not in male, and that this sex-specific response may involve differential sensitivity to estrogen receptor signaling. This technological advance will facilitate efforts to ascertain how distinctive physiological and pathophysiological stimuli impact astrocyte Gln metabolism in each sex.

SUBMITTER: Bheemanapally K

PROVIDER: S-EPMC7874509 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:We present the first comprehensive capillary electrophoresis electrospray ionization mass spectrometry (CESI-MS) analysis of post-translational modifications derived from H1 and core histones. Using a capillary electrophoresis system equipped with a sheathless high-sensitivity porous sprayer and nano-liquid chromatography electrospray ionization mass spectrometry (nano-LC-ESI-MS) as two complementary techniques, we characterized H1 histones isolated from rat testis. Without any pre-separation of the perchloric acid extraction, a total of 70 different modified peptides, including 50 phosphopeptides, were identified in the rat linker histones H1.0, H1a-H1e, and H1t. Out of the 70 modified H1 histone peptides, 27 peptides could be identified with CESI-MS only, and 11 solely with LC-ESI-MS. Immobilized metal-affinity chromatography enrichment prior to MS analysis yielded a total of 55 phosphopeptides; 22 of these peptides could be identified only by CESI-MS, and 19 only by LC-ESI-MS, showing the complementarity of the two techniques. We mapped 42 H1 modification sites, including 31 phosphorylation sites, of which 8 were novel sites. For the analysis of core histones, we chose a different strategy. In a first step, the sulfuric-acid-extracted core histones were pre-separated using reverse-phase high-performance liquid chromatography. Individual rat testis core histone fractions obtained in this way were digested and analyzed via bottom-up CESI-MS. This approach yielded the identification of 42 different modification sites including acetylation (lysine and N(α)-terminal); mono-, di-, and trimethylation; and phosphorylation. When we applied CESI-MS for the analysis of intact core histone subtypes from butyrate-treated mouse tumor cells, we were able to rapidly detect their degree of modification, and we found this method very useful for the separation of isobaric trimethyl and acetyl modifications. Taken together, our results highlight the need for additional techniques for the comprehensive analysis of post-translational modifications. CESI-MS is a promising new proteomics tool as demonstrated by this, the first comprehensive analysis of histone modifications, using rat testis as an example.

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HPLC-electrospray ionization-mass spectrometry optimization by high-performance design of experiments for astrocyte glutamine measurement.

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