Phosphorylation is a key regulator of protein function under (patho)physiological conditions, and defining site‐specific phosphorylation is essential to understand basic and disease biology. In vertebrates, the investigative focus has primarily been on serine, threonine and tyrosine phosphorylation, but mounting evidence suggests that phosphorylation of other “non‐canonical” amino acids also regulates critical aspects of cell biology. However, standard methods of phosphoprotein characterisation are largely unsuitable for the analysis of non‐canonical phosphorylation due to their relative instability under acidic conditions and/or elevated temperature. Consequently, the complete landscape of phosphorylation remains unexplored. Here, we report an unbiased phosphopeptide enrichment strategy based on strong anion exchange (SAX) chromatography (UPAX), which permits identification of histidine (His), arginine (Arg), lysine (Lys), aspartate (Asp), glutamate (Glu) and cysteine (Cys) phosphorylation sites on human proteins by mass spectrometry‐based phosphoproteomics. Remarkably, under basal conditions, and having accounted for false site localisation probabilities, the number of unique non‐canonical phosphosites is approximately one‐third of the number of observed canonical phosphosites. Our resource reveals the previously unappreciated diversity of protein phosphorylation in human cells, and opens up avenues for high‐throughput exploration of non‐canonical phosphorylation in all organisms.

Salmonella Typhimurium sequence type (ST) 313 causes invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa, targeting susceptible HIV+, malarial, or malnourished individuals. An in-depth genomic comparison between the ST313 isolate D23580 and the well-characterized ST19 isolate 4/74 that causes gastroenteritis across the globe revealed extensive synteny. To understand how the 856 nucleotide variations generated phenotypic differences, we devised a large-scale experimental approach that involved the global gene expression analysis of strains D23580 and 4/74 grown in 16 infection-relevant growth conditions. Comparison of transcriptional patterns identified virulence and metabolic genes that were differentially expressed between D23580 versus 4/74, many of which were validated by proteomics. We also uncovered the S. Typhimurium D23580 and 4/74 genes that showed expression differences during infection of murine macrophages. Our comparative transcriptomic data are presented in a new enhanced version of the Salmonella expression compendium, SalComD23580: http://bioinf.gen.tcd.ie/cgi-bin/salcom_v2.pl. We discovered that the ablation of melibiose utilization was caused by three independent SNP mutations in D23580 that are shared across ST313 lineage 2, suggesting that the ability to catabolize this carbon source has been negatively selected during ST313 evolution. The data revealed a novel, to our knowledge, plasmid maintenance system involving a plasmid-encoded CysS cysteinyl-tRNA synthetase, highlighting the power of large-scale comparative multicondition analyses to pinpoint key phenotypic differences between bacterial pathovariants.

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In this paper, we discuss the challenge of large-scale quantification of a proteome, referring to our programme that aims to define the absolute quantity, in copies per cell, of at least 4000 proteins in the yeast Saccharomyces cerevisiae. We have based our strategy on the well-established method of stable isotope dilution, generating isotopically labelled peptides using QconCAT technology, in which artificial genes, encoding concatenations of tryptic fragments as surrogate quantification standards, are designed, synthesised de novo and expressed in bacteria using stable isotopically enriched media. A known quantity of QconCAT is then co-digested with analyte proteins and the heavy:light isotopologues are analysed by mass spectrometry to yield absolute quantification. This workflow brings issues of optimal selection of quantotypic peptides, their assembly into QconCATs, expression, purification and deployment.