Discovering the N-Terminal Methylome by Repurposing of Proteomic Datasets.

Protein α-N-methylation is an underexplored post-translational modification involving the covalent addition of methyl groups to the free α-amino group at protein N-termini. To systematically explore the extent of α-N-terminal methylation in yeast and humans, we reanalyzed publicly accessible proteomic datasets to identify N-terminal peptides contributing to the α-N-terminal methylome. This repurposing approach found evidence of α-N-methylation of established and novel protein substrates with canonical N-terminal motifs of established α-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, α-N-methylation of noncanonical sequences was surprisingly prevalent, suggesting unappreciated and cryptic methylation events. Analysis of the amino acid frequencies of α-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 first position. We tested three potentially methylated proteins and confirmed the α-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 that proteomic searching for α-N-terminal methylation requires careful consideration of mass spectra. This study demonstrates the feasibility of reprocessing proteomic data for global α-N-terminal methylome investigations.

High-fat-diet induced obesity increases the proportion of linoleic acyl residues in dam serum and milk and in suckling neonate circulation.

Maternal obesity increases the risk of offspring to become obese and develop related pathologies. Exposure to maternal high-fat diet (HFD) only during lactation increases the risk of obesity-related diseases, suggesting that factors in milk affect long-term health. We hypothesized that prepregnancy obesity induced by HFD alters milk lipidome, and in turn, alterations may affect neonate serum lipidome. The objective of this study was to determine the effect of prepregnancy obesity induced by HFD on circulating lipids in dams and neonates and in milk. Female mice were fed an HFD (60% kcal fat) or control diet (CON, 10% kcal fat) beginning 4 weeks before breeding. On postnatal day 2 (PND2), pups were cross-fostered to create pup groups exposed to HFD during pregnancy, lactation, or both or exposed to CON. On PND12, dams were milked and then euthanized along with pups to collect blood. Serum and milk were processed for multiple reaction monitoring (MRM) lipidomics profiling to quantify the relative expression of lipid classes. Lipidome of HFD dam serum and milk had increased proportion of C18:2 free fatty acid and fatty acyl residues in all lipid classes. Lipidome of serum from pups exposed to maternal HFD during lactation was similarly affected. Thus, maternal HFD induced redistribution of fatty acyl residues in the dam's circulation, which was associated with modification in milk and suckling neonate's lipidome. Further studies are needed to determine if increased circulating levels of C18:2 in neonate affects development and predisposes offspring to obesity and metabolic syndrome.

Proteomic and metabolomic profiling reveals the involvement of apoptosis in meat quality characteristics of ovine M. longissimus from different callipyge genotypes.

Proteome and metabolome changes in muscles from callipyge mutation (+/C) and non-callipyge phenotype (+/+, C/+, and C/C) lambs were profiled to provide insight into the biochemical changes affecting meat quality attributes. M. longissimus thoracis from lambs with all four possible callipyge genotype (n = 4, C/+, C/C, +/C, and +/+) were collected after 3d aging and analyzed using mass-spectrometry based platforms. Among identified proteomes, cytochrome c (pro-apoptotic protein) was detected with significantly lower abundances in +/C. Anti-apoptotic HSP70, BAG3, and PARK7 were over-abundant in +/C, which could result in delayed apoptosis and possibly attributed to tougher meat in callipyge lambs. Eight glycolysis enzymes were overabundant in +/C lambs, whereas 3 enzymes involved in TCA cycle were overabundant in non-callipyge ones (C/C and/or C/+). Twenty-five metabolites were affected by genotypes (P < .05), including metabolic co-factors, polyphenols, and AA/short peptides. Our omics results provided insightful information for revealing the differences in biochemical attributes caused by callipyge mutation.

Analysis of Human Nuclear Protein Complexes by Quantitative Mass Spectrometry Profiling.

Analysis of protein complexes provides insights into how the ensemble of expressed proteome is organized into functional units. While there have been advances in techniques for proteome-wide profiling of cytoplasmic protein complexes, information about human nuclear protein complexes are very limited. To close this gap, we combined native size exclusion chromatography (SEC) with label-free quantitative MS profiling to characterize hundreds of nuclear protein complexes isolated from human glioblastoma multiforme T98G cells. We identified 1794 proteins that overlapped between two biological replicates of which 1244 proteins were characterized as existing within stably associated putative complexes. co-IP experiments confirmed the interaction of PARP1 with Ku70/Ku80 proteins and HDAC1 (histone deacetylase complex 1) and CHD4. HDAC1/2 also co-migrated with various SIN3A and nucleosome remodeling and deacetylase components in SEC fractionation including SIN3A, SAP30, RBBP4, RBBP7, and NCOR1. Co-elution of HDAC1/2/3 with both the KDM1A and RCOR1 further confirmed that these proteins are integral components of human deacetylase complexes. Our approach also demonstrated the ability to identify potential moonlighting complexes and novel complexes containing uncharacterized proteins. Overall, the results demonstrated the utility of SEC fractionation and LC-MS analysis for system-wide profiling of proteins to predict the existence of distinct forms of nuclear protein complexes.

Protein folding creates structure-based, noncontiguous consensus phosphorylation motifs recognized by kinases.

Linear consensus motifs are short contiguous sequences of residues within a protein that can form recognition modules for protein interaction or catalytic modification. Protein kinase specificity and the matching of kinases to substrates have been mostly defined by phosphorylation sites that occur in linear consensus motifs. However, phosphorylation can also occur within sequences that do not match known linear consensus motifs recognized by kinases and within flexible loops. We report the identification of Thr(253) in α-tubulin as a site that is phosphorylated by protein kinase C ßI (PKCßI). Thr(253) is not part of a linear PKC consensus motif. Instead, Thr(253) occurs within a region on the surface of α-tubulin that resembles a PKC phosphorylation site consensus motif formed by basic residues in different parts of the protein, which come together in the folded protein to form the recognition motif for PKCßI. Mutations of these basic residues decreased substrate phosphorylation, confirming the presence of this "structurally formed" consensus motif and its importance for the protein kinase-substrate interaction. Analysis of previously reported protein kinase A (PKA) and PKC substrates identified sites within structurally formed consensus motifs in many substrates of these two kinase families. Thus, the concept of consensus phosphorylation site motif needs to be expanded to include sites within these structurally formed consensus motifs.