Epigenetic underpinnings of freeze avoidance in the goldenrod gall moth, Epiblema scudderiana.

The goldenrod gall moth (Epiblema scudderiana) is a cold hardy insect that survives subzero temperatures during the winter by supercooling bodily fluids to approximately -40 °C, allowing the insect to remain unfrozen despite the freezing temperatures. This is characterized by a drastic increase of cryoprotectant glycerol along with widespread downregulation of non-essential genes and processes to conserve cellular energy. This study examined the role of epigenetic enzymes in regulating this freeze-avoidant process across a range of freezing temperatures experienced in nature. Cold and subzero temperature exposure in E. scudderiana resulted in upregulation of select DNA methyltransferase (DNMT) enzymes with concurrent decreases in DNMT activity and no change in activity of the Ten-Eleven Translocation (TET) demethylation enzyme activities. Levels of histone acetyltransferase (HAT) and histone deacetylase (HDAC) activity decreased during cold exposures. The increase in DNMT expression and concurrent decrease in HAT activity suggests a role for DNA methylation to assist with transcriptional suppression. These findings propose that epigenetic regulation of genes and histones underpin the winter survival strategies of this insect.

Online porous graphic carbon chromatography coupled with tandem mass spectrometry for post-translational modification analysis.

RATIONALE: Porous graphic carbon chromatography (PGC) has a different mechanism in the retention of tryptic peptides compared with reversed-phase chromatography and in this study we show that coupling PGC with tandem mass spectrometry offer advantages for the quantitation of phosphorylation stoichiometry and characterization of site-specific glycosylation. METHODS: Digests of protein standards (horse myoglobin, bovine fetuin and ß-casein) were analyzed with a capillary liquid chromatography/tandem mass spectrometry (LC/MS/MS) system by coupling an Agilent 1100 HPLC system to a Synapt G2-Si HDMS (Waters). Peptides were separated using a HyperCarb PGC column (300 µm i.d. × 100 mm) packed with 3 µm particles. MS/MS data were collected in data-dependent mode and three MS/MS scans were acquired after the full MS scan. RAW data were transformed to .mgf by PLGS (Waters) and searched against the Swissprot database by Mascot. Chromatograms and MS/MS spectra of identified compounds were extracted with Masslynx (Waters) and imported to Origin for analysis. Glycan composition and peptide sequence were manually annotated. RESULTS: PGC/MS/MS enabled accurate quantitation of the stoichiometry of specific phosphorylation sites from ß-casein by efficient separation of the phosphopeptide and its non-phosphorylated counterpart, which cannot be achieved by reversed-phase chromatography. PGC/MS/MS also enabled comprehensive characterization of protein sialoglycosylation as isomeric glycopeptides with different combinations of α2-3- and α2-6-linked sialic acids can be separated and the ratios of each combination were verified by exoglycosidase digestion. CONCLUSIONS: PGC has demonstrated superior separation of peptides with phosphorylation and glycosylation and can be used as an alternative in the proteomic characterization of post-translational modifications (PTMs) by polar groups.