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Characterization of pig liver glutathione S-transferases using HPLC-electrospray-ionization mass spectrometry.

ABSTRACT: We have characterized 11 porcine liver cytosolic glutathione S-transferase (GST) subunits from their precise molecular mass, immunoreactivity and partial amino acid sequence. Four Alpha-, six Mu- and one unexpected Pi-class GST subunits were found with average molecular masses of 24.984-25.228 kDa, 25.039-25.657 kDa and 23.510 kDa respectively. Molecular masses were established using electrospray-ionization mass spectrometry, with a precision of +/- 3-4 mass units. Glutathione (GSH) and S-hexylglutathione (ShGSH) were tested as affinity ligands in the purification procedure. The binding selectivity of GSH was better than that of ShGSH, although non-GST proteins were retained on both matrices. As already described in other studies, a number of non-GST proteins bound to the affinity resins. Two of them were tentatively identified as mevalonate kinase and carbonyl reductase. The characterization of pig liver cytosolic GST subunits pattern achieved in this work should constitute a useful tool for rapid evaluation of these enzymes' expression in modulation studies.

SUBMITTER: Rouimi P

PROVIDER: S-EPMC1217567 | biostudies-other | 1996 Aug

REPOSITORIES: biostudies-other

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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(?)-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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Characterization of pig liver glutathione S-transferases using HPLC-electrospray-ionization mass spectrometry.

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