Sobreira, T. J. P. | bioRxiv Science

bioRxiv2021

Discovering the N-terminal Methylome by Repurposing of Proteomic Datasets

Protein -N-methylation is an underexplored post-translational modification involving the covalent addition of methyl groups to the free -amino group at protein N-termini. To systematically explore the extent of -N-terminal methylation in yeast and humans, we reanalyzed publicly accessible proteomic datasets to identify N-terminal peptides contributing to the -N-terminal methylome. This repurposing approach found evidence of -N-methylation of established and novel protein substrates with canonical N-terminal motifs of established -N-terminal methyltransferases, including human NTMT1/2 and yeast Tae1. NTMT1/2 are implicated in cancer and aging processes but have unclear and context-dependent roles. Moreover, -N-methylation of non-canonical sequences was surprisingly prevalent, suggesting unappreciated and cryptic methylation events. Analysis of the amino acid frequencies of -N-methylated peptides revealed a [S]1-[S/A/Q]2 pattern in yeast and [A/N/G]1-[A/S/V]2-[A/G]3 in humans, which differs from the canonical motif. We delineated the distribution of the two types of prevalent N-terminal modifications, acetylation, and methylation, on amino acids at the 1st position. We tested three potentially methylated proteins and confirmed the -N-terminal methylation of Hsp31 by additional proteomic analysis and immunoblotting. The other two proteins, Vma1 and Ssa3, were found to be predominantly acetylated, indicating proteomic searching for -N-terminal methylation requires careful consideration of mass spectra. This study demonstrates the feasibility of reprocessing proteomic data for global -N-terminal methylome investigations. The raw MS data that supports the findings of this study were deposited with PRIDE identifier: PXD022833. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/439552v3_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@124e5b9org.highwire.dtl.DTLVardef@1660eb7org.highwire.dtl.DTLVardef@15027aaorg.highwire.dtl.DTLVardef@15c0da6_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract (For TOC only). C_FIG

bioinformatics↗

bioRxiv2019

Lipidome profiles of postnatal day 2 vaginal swabs reflect fat composition of gilts postnatal diet

We hypothesized that postnatal development of the vagina is impacted by early nutritional environment. Our objective was to determine if lipid profiles of vaginal swabs were different between gilts suckled by sow or fed milk replacer the first 48 h postpartum, with and without a lard-based fat supplement. Gilts (>1.3 kg) were selected at birth across 8 litters and assigned to treatments: colostrum suckled (S, n=8); S plus fat supplement (SF, n=5); bottle-fed milk replacer (B, n=8); or B plus fat supplement (BF, n=7). At 48 h postnatal, vaginal swabs were taken with a cytology brush, immersed in ultrapure water to burst cells, and lipids extracted for analysis using multiple reaction monitoring (MRM)-profiling. Lipids extracted from serum collected at 48 h from gilts and milk collected from sows at 24 h were also analyzed with MRM-profiling. Receiver operating characteristic curve analysis found 18 lipids highly distinguished [area-under-the-curve (AUC) > 0.9] between S and B gilts, including phosphatidylethanolamine with 34 carbon and four unsaturations in the fatty acyl residues [PE(34:4)]. Twelve lipids from vaginal swabs highly correlated (r > 0.6; p < 0.01) with nutrition source. Lipids more abundant in milk replacer drove association. For example, mean intensity of PE (34:4) was 149-fold higher in milk replacer than colostrum, with 1.6- and 2.12-fold higher levels in serum and vaginal swab samples (p < 0.001), respectively, of B versus S gilts. Findings support that vaginal swabs can be used to noninvasively study effects of perinatal nutrition on tissue composition.\n\nSummary sentenceVaginal swab lipidome profiles at 48 h reflect the fat composition of neonatal diet during first two days postnatal.

developmental biology↗