Direct links between proteomic and genomic/transcriptomic data are not frequently made, partly because of lack of appropriate bioinformatics tools. To help address this, we have developed the PG Nexus pipeline. The PG Nexus allows users to covisualize peptides in the context of genomes or genomic contigs, along with RNA-seq reads. This is done in the Integrated Genome Viewer (IGV). A Results Analyzer reports the precise base position where LC-MS/MS-derived peptides cover genes or gene isoforms, on the chromosomes or contigs where this occurs. In prokaryotes, the PG Nexus pipeline facilitates the validation of genes, where annotation or gene prediction is available, or the discovery of genes using a "virtual protein"-based unbiased approach. We illustrate this with a comprehensive proteogenomics analysis of two strains of Campylobacter concisus . For higher eukaryotes, the PG Nexus facilitates gene validation and supports the identification of mRNA splice junction boundaries and splice variants that are protein-coding. This is illustrated with an analysis of splice junctions covered by human phosphopeptides, and other examples of relevance to the Chromosome-Centric Human Proteome Project. The PG Nexus is open-source and available from https://github.com/IntersectAustralia/ap11_Samifier. It has been integrated into Galaxy and made available in the Galaxy tool shed.

In recent years, proteomic data have contributed to genome annotation efforts, most notably in humans and mice, and spawned a field termed "proteogenomics". Yeast, in contrast with higher eukaryotes, has a small genome, which has lent itself to simpler ORF prediction. Despite this, continual advances in mass spectrometry suggest that proteomics should be able to improve genome annotation even in this well-characterized species. Here we applied a proteogenomics workflow to yeast to identify novel protein-coding genes. Specific databases were generated, from intergenic regions of the genome, which were then queried with MS/MS data. This suggested the existence of several putative novel ORFs of <100 codons, one of which we chose to validate. Synthetic peptides, RNA-Seq analysis, and evidence of evolutionary conservation allowed for the unequivocal definition of a new protein of 78 amino acids encoded on chromosome X, which we dub YJR107C-A. It encodes a new type of domain, which ab initio modeling suggests as predominantly α-helical. We show that this gene is nonessential for growth; however, deletion increases sensitivity to osmotic stress. Finally, from the above discovery process, we discuss a generalizable strategy for the identification of short ORFs and small proteins, many of which are likely to be undiscovered.

Post-translational lysine methylation is well established as a regulator of histone activity; however, it is emerging that these modifications are also likely to play extensive roles outside of the histone code. Here we obtain new insights into non-histone lysine methylation and protein lysine methyltransferase (PKMT) activity by elucidating absolute stoichiometries of lysine methylation, using mass spectrometry and absolute quantification (AQUA), in wild-type and 5 PKMT gene deletion strains of Saccharomyces cerevisiae. By analyzing 8 sites of methylation in 3 non-histone proteins, elongation factor 1-α (EF1α), elongation factor 2 (EF2), and 60S ribosomal protein L42-A/B (Rpl42ab), we find that production of preferred methylation states on individual lysine residues is commonplace and likely occurs through processive PKMT activity, Class I PKMTs can be associated with processive methylation, lysine residues are selectively methylated by specific PKMTs, and lysine methylation exists over a broad range of stoichiometries. Together these findings suggest that specific sites and forms of lysine methylation may play specialized roles in the regulation of non-histone protein activity. We also uncover new relationships between two proteins previously characterized as PKMTs, SEE1 and EFM1, in EF1α methylation and show that past characterizations of EFM1 as having direct PKMT activity may require reinterpretation.

Hmt1p is the predominant arginine methyltransferase in Its substrate proteins are involved in transcription, transcriptional regulation, nucleocytoplasmic transport and RNA splicing. Hmt1p-catalyzed methylation can also modulate protein-protein interactions. Hmt1p is conserved from unicellular eukaryotes through to mammals where its ortholog, PRMT1, is lethal upon knockout. In yeast, however, the effect of knockout on the transcriptome and proteome has not been described. Transcriptome analysis revealed downregulation of phosphate-responsive genes inΔ, including acid phosphatases ,, and , phosphate transporters and and the vacuolar transporter chaperone Analysis of the Δ proteome revealed decreased abundance of phosphate-associated proteins including phosphate transporter Pho84p, vacuolar alkaline phosphatase Pho8p, acid phosphatase Pho3p and subunits of the vacuolar transporter chaperone complex Vtc1p, Vtc3p and Vtc4p. Consistent with this, phosphate homeostasis was dysregulated inΔ cells, showing decreased extracellular phosphatase levels and decreased total P in phosphate-depleted medium. , we showed that transcription factor Pho4p can be methylated at Arg-241, which could explain phosphate dysregulation inΔ if interplay exists with phosphorylation at Ser-242 or Ser-243, or if Arg-241 methylation affects the capacity of Pho4p to homodimerize or interact with Pho2p. However, the Arg-241 methylation site was not validated and the localization of a Pho4p-GFP fusion inΔ was not different from wild type. To our knowledge, this is the first study to reveal an association between Hmt1p and phosphate homeostasis and one which suggests a regulatory link between S-adenosyl methionine and intracellular phosphate.