Although plasma proteins have important roles in biological processes and are the direct targets of many drugs, the genetic factors that control inter-individual variation in plasma protein levels are not well understood. Here we characterize the genetic architecture of the human plasma proteome in healthy blood donors from the INTERVAL study. We identify 1,927 genetic associations with 1,478 proteins, a fourfold increase on existing knowledge, including trans associations for 1,104 proteins. To understand the consequences of perturbations in plasma protein levels, we apply an integrated approach that links genetic variation with biological pathway, disease, and drug databases. We show that protein quantitative trait loci overlap with gene expression quantitative trait loci, as well as with disease-associated loci, and find evidence that protein biomarkers have causal roles in disease using Mendelian randomization analysis. By linking genetic factors to diseases via specific proteins, our analyses highlight potential therapeutic targets, opportunities for matching existing drugs with new disease indications, and potential safety concerns for drugs under development.

We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection, gas phase ion/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites. From a 30-g (Ϸ600 pmol) sample of total yeast protein, we identify 1,252 phosphorylation sites on 629 proteins. Identified phosphoproteins have expression levels that range from <50 to 1,200,000 copies per cell and are encoded by genes involved in a wide variety of cellular processes. We identify a consensus site that likely represents a motif for one or more uncharacterized kinases and show that yeast kinases, themselves, contain a disproportionately large number of phosphorylation sites. Detection of a pHis containing peptide from the yeast protein, Cdc10, suggests an unexpected role for histidine phosphorylation in septin biology. From diverse functional genomics data, we show that phosphoproteins have a higher number of interactions than an average protein and interact with each other more than with a random protein. They are also likely to be conserved across large evolutionary distances.yeast phosphoproteome ͉ network analysis I n an earlier study of the yeast phosphoproteome (1), we digested proteins from a whole cell lysate with trypsin, used immobilized metal-affinity chromatography (IMAC) to enrich the sample for phosphopeptides, and analyzed the resulting mixture by nano-flow HPLC interfaced to electrospray ionization tandem mass spectrometry (MS/MS). Low-energy collision-activated dissociation (CAD) was used to fragment the peptide backbone and to produce ions of types b and y (Fig. 1) required for successful sequence analysis and identification of phosphorylation sites. We detected Ͼ1,000 phosphopeptides but defined only 383 sites of phosphorylation, largely because the CAD process often promoted elimination of phosphoric acid from Ser and Thr residues without breaking the amide bonds along the peptide backbone. The resulting MS/MS spectra were essentially devoid of sequence information.To circumvent this problem, we have modified the LTQ mass spectrometer for ion/ion chemistry and now fragment both peptides (2) and intact proteins (3) by electron transfer dissociation (ETD). In this process, fluoranthene radical-anions are generated in a chemical ionization source and used as reagents to transfer an electron to a multiply charged peptide generated by electrospray ionization. This reaction is highly exothermic, reduces the peptide charge by one, and triggers fragmentation of the peptide backbone to produce a homologous series of complementary fragment ions of type c and z⅐ (Fig. 1). Subtraction of m/z values for fragments within a given ion series that differ by a single amino acid affords the mass...

Mass spectrometry has played an integral role in the identification of proteins and their posttranslational modifications (PTM). However, analysis of some PTMs, such as phosphorylation, sulfonation, and glycosylation, is difficult with collision-activated dissociation (CAD) since the modification is labile and preferentially lost over peptide backbone fragmentation, resulting in little to no peptide sequence information. The presence of multiple basic residues also makes peptides exceptionally difficult to sequence by conventional CAD mass spectrometry. Here we review the utility of electron transfer dissociation (ETD) mass spectrometry for sequence analysis of posttranslationally modified and/or highly basic peptides. Phosphorylated, sulfonated, glycosylated, nitrosylated, disulfide bonded, methylated, acetylated, and highly basic peptides have been analyzed by CAD and ETD mass spectrometry. CAD fragmentation typically produced spectra showing limited peptide backbone fragmentation. However, when these peptides were fragmented using ETD, peptide backbone fragmentation produced a complete or almost complete series of ions and thus extensive peptide sequence information. In addition, labile PTMs remained intact. These examples illustrate the utility of ETD as an advantageous tool in proteomic research by readily identifying peptides resistant to analysis by CAD. A further benefit is the ability to analyze larger, non-tryptic peptides, allowing for the detection of multiple PTMs within the context of one another.

Phase II metabolic enzymes are a battery of critical proteins that detoxify xenobiotics by increasing their hydrophilicity and enhancing their disposal. These enzymes have long been studied for their preventative and protective effects against mutagens and carcinogens and for their regulation via the Keap1 (Kelch-like ECH associated protein 1) / Nrf2 (Nuclear factor erythroid 2 related factor 2) / ARE (antioxidant response elements) pathway. Recently, a series of studies have reported the altered expression of phase II genes in postmortem tissue of patients with various neurological diseases. These observations hint at a role for phase II enzymes in the evolution of such conditions. Furthermore, promising findings reveal that overexpression of phase II genes, either by genetic or chemical approaches, confers neuroprotection in vitro and in vivo. Therefore, there is a need to summarize the current literature on phase II genes in the central nervous system (CNS). This should help guide future studies on phase II genes as therapeutic targets in neurological diseases. In this review, we first briefly introduce the concept of phase I, II and III enzymes, with a special focus on phase II enzymes. We then discuss their expression regulation, their inducers and executors. Following this background, we expand our discussion to the neuroprotective effects of phase II enzymes and the potential application of Nrf2 inducers to the treatment of neurological diseases.

Brain, liver, kidney, heart, and skeletal muscle from fatty liver dystrophy (fld/fld) mice, which do not express lipin 1 (lipin), contained much less Mg 2؉ -dependent phosphatidic acid phosphatase (PAP) activity than tissues from wild type mice. Lipin harboring the fld 2j (Gly 84 3 Arg) mutation exhibited relatively little PAP activity. These results indicate that lipin is a major PAP in vivo and that the loss of PAP activity contributes to the fld phenotype. PAP activity was readily detected in immune complexes of lipin from 3T3-L1 adipocytes, where the protein was found both as a microsomal form and a soluble, more highly phosphorylated, form. Fifteen phosphorylation sites were identified by mass spectrometric analyses. Insulin increased the phosphorylation of multiple sites and promoted a gel shift that was due in part to phosphorylation of Ser 106 . In contrast, epinephrine and oleic acid promoted dephosphorylation of lipin. The PAP-specific activity of lipin was not affected by the hormones or by dephosphorylation of lipin with protein phosphatase 1. However, the ratio of soluble to microsomal lipin was markedly increased in response to insulin and decreased in response to epinephrine and oleic acid. The results suggest that insulin and epinephrine control lipin primarily by changing localization rather than intrinsic PAP activity.