Fibrinogen and hemoglobin predict near future cardiovascular events in asymptomatic individuals.

To identify circulating proteins predictive of acute cardiovascular disease events in the general population, we performed a proteomic screen in plasma from asymptomatic individuals. A "Discovery cohort" of 25 individuals who subsequently incurred a cardiovascular event within 3 years (median age = 70 years, 80% male) was matched to 25 controls remaining event-free for > 5 years (median age = 72 years, 80% male). Plasma proteins were assessed by data independent acquisition mass spectrometry (DIA-MS). Associations with cardiovascular events were tested using Cox regression, adjusted for the New Zealand Cardiovascular Risk Score. Concentrations of leading protein candidates were subsequently measured with ELISAs in a larger (n = 151) independent subset. In the Discovery cohort, 76 plasma proteins were robustly quantified by DIA-MS, with 8 independently associated with cardiovascular events. These included (HR = hazard ratio [95% confidence interval] above vs below median): fibrinogen alpha chain (HR = 1.84 [1.19-2.84]); alpha-2-HS-glycoprotein (also called fetuin A) (HR = 1.86 [1.19-2.93]); clusterin isoform 2 (HR = 1.59 [1.06-2.38]); fibrinogen beta chain (HR = 1.55 [1.04-2.30]); hemoglobin subunit beta (HR = 1.49 [1.04-2.15]); complement component C9 (HR = 1.62 [1.01-2.59]), fibronectin isoform 3 (HR = 0.60 [0.37-0.99]); and lipopolysaccharide-binding protein (HR = 1.58 [1.00-2.49]). The proteins for which DIA-MS and ELISA data were correlated, fibrinogen and hemoglobin, were analyzed in an Extended cohort, with broader inclusion criteria and longer time to events, in which these two proteins were not associated with incident cardiovascular events. We have identified eight candidate proteins that may independently predict cardiovascular events occurring within three years in asymptomatic, low-to-moderate risk individuals, although these appear not to predict events beyond three years.

The Arabidopsis thaliana chloroplast proteome reveals pathway abundance and novel protein functions.

BACKGROUND: Chloroplasts are plant cell organelles of cyanobacterial origin. They perform essential metabolic and biosynthetic functions of global significance, including photosynthesis and amino acid biosynthesis. Most of the proteins that constitute the functional chloroplast are encoded in the nuclear genome and imported into the chloroplast after translation in the cytosol. Since protein targeting is difficult to predict, many nuclear-encoded plastid proteins are still to be discovered. RESULTS: By tandem mass spectrometry, we identified 690 different proteins from purified Arabidopsis chloroplasts. Most proteins could be assigned to known protein complexes and metabolic pathways, but more than 30% of the proteins have unknown functions, and many are not predicted to localize to the chloroplast. Novel structure and function prediction methods provided more informative annotations for proteins of unknown functions. While near-complete protein coverage was accomplished for key chloroplast pathways such as carbon fixation and photosynthesis, fewer proteins were identified from pathways that are downregulated in the light. Parallel RNA profiling revealed a pathway-dependent correlation between transcript and relative protein abundance, suggesting gene regulation at different levels. CONCLUSIONS: The chloroplast proteome contains many proteins that are of unknown function and not predicted to localize to the chloroplast. Expression of nuclear-encoded chloroplast genes is regulated at multiple levels in a pathway-dependent context. The combined shotgun proteomics and RNA profiling approach is of high potential value to predict metabolic pathway prevalence and to define regulatory levels of gene expression on a pathway scale.

Absolute quantification of apolipoproteins and associated proteins on human plasma lipoproteins.

Lipoprotein-associated proteins form an intrinsic part of the major plasma lipoprotein classes. There is increasing evidence that the quantity of these proteins per lipoprotein particle determines lipoprotein function including redox, inflammatory and thrombotic properties and may impact on lipoprotein-related risks for developing heart disease. However, only limited information on the relative quantity of these proteins has been published and no comprehensive absolute quantitative data providing the stoichiometry of proteins associated with lipoproteins is available to date. To address this, we performed extensive absolute quantification by mass spectrometry of 17 lipoprotein-associated proteins on VLDL, LDL, Lp(a) and HDL from healthy subjects. For the first time we show the exact stoichiometry of apolipoproteins on different lipoprotein classes. The most distinct differences were seen in the abundance of all apoCs, apoE and apoF. We further revealed strong variations between individual samples, which indicates the complexity of the protein complement of lipoproteins and can provide additional insights into lipoprotein-related risk factors. This approach has the potential to determine alterations in the protein profiles of lipoproteins in disease states such as CVD or diabetes and, if performed on large cohorts, to translate into a tool for identifying new candidate biomarkers for risk of disease. BIOLOGICAL SIGNIFICANCE: A more comprehensive picture about the protein complement on individual lipoprotein classes is the goal of lipoprotein proteomics analyses. Despite many such studies, there is a lack of absolute quantitative data on lipoproteins isolated from individual subjects. The stoichiometry of lipoprotein-associated proteins rather than their presence or absence could provide insights into an individual's predisposition for disease such as heart disease or diabetes. Our study provides a comprehensive overview of the absolute quantity of proteins on the major apolipoprotein classes VLDL, LDL, Lp(a) and HDL.

Proteomics of Lipoprotein(a) identifies a protein complement associated with response to wounding.

Lipoprotein(a) [Lp(a)] is a major independent risk factor for cardiovascular disease. Twenty percent of the general population exhibit levels above the risk threshold highlighting the importance for clinical and basic research. Comprehensive proteomics of human Lp(a) will provide significant insights into Lp(a) physiology and pathogenicity. Using liquid chromatography-coupled mass spectrometry, we established a high confidence Lp(a) proteome of 35 proteins from highly purified particles. Protein interaction network analysis and functional clustering revealed proteins assigned to the two major biological processes of lipid metabolism and response to wounding. The latter includes the processes of coagulation, complement activation and inflammatory response. Furthermore, absolute protein quantification of apoB-100, apo(a), apoA1, complement C3 and PON1 gave insights into the compositional stoichiometry of associated proteins per particle. Our proteomics study has identified Lp(a)-associated proteins that support a suggested role of Lp(a) in response to wounding which points to mechanisms of Lp(a) pathogenicity at sites of vascular injury and atherosclerotic lesions. This study has identified a high confidence Lp(a) proteome and provides an important basis for further comparative and quantitative analyses of Lp(a) isolated from greater numbers of plasma samples to investigate the significance of associated proteins and their dynamics for Lp(a) pathogenicity.

Dimyristoylphosphotidylcholine induces conformational changes in apoB that lowers lipoprotein(a).

Lipoprotein(a) [Lp(a)] is assembled by the binding of apolipoprotein B (apoB) lysine residues on LDL to lysine binding sites in apolipoprotein(a) [apo(a)] and the subsequent formation of a disulphide bond between apoB and apo(a). In this study, we induced changes in apoB conformation by adding phospholipids to LDL and tested the effect of the altered apoB conformation on Lp(a) assembly. The addition of dimyristoylphosphatidylcholine (DMPC) to isolated LDL induced a decrease in the alpha-helical content of apoB and increased the immunoreactivity of the apoB C terminus toward monoclonal antibodies in the region. These conformational changes were associated with a reduction in the ability of the DMPC-modified LDL to form Lp(a) in in vitro assays. Furthermore, administration of DMPC to Lp(a) transgenic mice lead to a significant but transient decrease in Lp(a) levels (18.6% decrease at 2 h, P < 0.001) which coincided with the association of DMPC with LDL in plasma. Our study shows that changes in apoB conformation in the C-terminal region alter the exposure of sequences required for Lp(a) assembly and reduce the formation of Lp(a) both in vitro and in vivo. We conclude that manipulation of LDL surface phospholipids alters Lp(a) levels.

Proteome dynamics during plastid differentiation in rice.

We have analyzed proteome dynamics during light-induced development of rice (Oryza sativa) chloroplasts from etioplasts using quantitative two-dimensional gel electrophoresis and tandem mass spectrometry protein identification. In the dark, the etioplast allocates the main proportion of total protein mass to carbohydrate and amino acid metabolism and a surprisingly high number of proteins to the regulation and expression of plastid genes. Chaperones, proteins for photosynthetic energy metabolism, and enzymes of the tetrapyrrole pathway were identified among the most abundant etioplast proteins. The detection of 13 N-terminal acetylated peptides allowed us to map the exact localization of the transit peptide cleavage site, demonstrating good agreement with the prediction for most proteins. Based on the quantitative etioplast proteome map, we examined early light-induced changes during chloroplast development. The transition from heterotrophic metabolism to photosynthesis-supported autotrophic metabolism was already detectable 2 h after illumination and affected most essential metabolic modules. Enzymes in carbohydrate metabolism, photosynthesis, and gene expression were up-regulated, whereas enzymes in amino acid and fatty acid metabolism were significantly decreased in relative abundance. Enzymes involved in nucleotide metabolism, tetrapyrrole biosynthesis, and redox regulation remained unchanged. Phosphoprotein-specific staining at different time points during chloroplast development revealed light-induced phosphorylation of a nuclear-encoded plastid RNA-binding protein, consistent with changes in plastid RNA metabolism. Quantitative information about all identified proteins and their regulation by light is available in plprot, the plastid proteome database (http://www.plprot.ethz.ch).

Analysis of shotgun proteomics and RNA profiling data from Arabidopsis thaliana chloroplasts.

The integration of data from transcriptional profiling and shotgun proteomics experiments provides additional information about the identified proteins that goes beyond their plain detection. We have analyzed results from MS/MS shotgun detection of 426 Arabidopsis chloroplast proteins and genome-wide RNA profiling to identify correlations between gene expression, protein abundance and protein characteristics that influence their detection in high-throughput proteome analyses. The integrated data analysis revealed a significant molecular mass bias for the detection of proteins that were expressed at low transcript levels. Overall, the sequence coverage of most of the identified proteins increases with transcript levels indicating a positive correlation between transcript and relative protein abundance. This does not apply to a subset of the identified proteins suggesting specific properties that alter their detection in shotgun proteomics. This integrative comparison is a suitable strategy to validate large scale proteomics data and offers an assessment of the depth of the proteome analysis and the confidence in protein identification.

Proteome analysis of the rice etioplast: metabolic and regulatory networks and novel protein functions.

We report an extensive proteome analysis of rice etioplasts, which were highly purified from dark-grown leaves by a novel protocol using Nycodenz density gradient centrifugation. Comparative protein profiling of different cell compartments from leaf tissue demonstrated the purity of the etioplast preparation by the absence of diagnostic marker proteins of other cell compartments. Systematic analysis of the etioplast proteome identified 240 unique proteins that provide new insights into heterotrophic plant metabolism and control of gene expression. They include several new proteins that were not previously known to localize to plastids. The etioplast proteins were compared with proteomes from Arabidopsis chloroplasts and plastid from tobacco Bright Yellow 2 cells. Together with computational structure analyses of proteins without functional annotations, this comparative proteome analysis revealed novel etioplast-specific proteins. These include components of the plastid gene expression machinery such as two RNA helicases, an RNase II-like hydrolytic exonuclease, and a site 2 protease-like metalloprotease all of which were not known previously to localize to the plastid and are indicative for so far unknown regulatory mechanisms of plastid gene expression. All etioplast protein identifications and related data were integrated into a data base that is freely available upon request.