Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

Abstract

Gaining understanding of common complex diseases and their treatments are the main drivers for life sciences. As we show here, comprehensive protein set analyses offer new opportunities to decipher functional molecular networks of diseases and assess the efficacy and side-effects of treatments in vivo. Using mass spectrometry, we quantitatively detected several thousands of proteins and observed significant changes in protein pathways that were (dys-) regulated in diet-induced obesity mice. Analysis of the expression and post-translational modifications of proteins in various peripheral metabolic target tissues including adipose, heart, and liver tissue generated functional insights in the regulation of cell and tissue homeostasis during high-fat diet feeding and medication with two antidiabetic compounds. Protein set analyses singled out pathways for functional characterization, and indicated, for example, early-on potential cardiovascular complication of the diabetes drug rosiglitazone. In vivo protein set detection can provide new avenues for monitoring complex disease processes, and for evaluating preclinical drug candidates. Molecular & Cellular Proteomics 12:

Protein set analyses: how could this impact the clinic?

“…In drug-treated or non-treated insulin-resistant obese mice, we observed only a subtle variation of individual protein expression for most (rather abundant) proteins. Interestingly, we observed striking consistence of RNA and protein expression on the pathway but much less on the individual gene-protein level [14]. These results strengthened the idea that physiological outcomes arise from the context-specific interaction of various biomolecules, which can be grouped in functionally "…many biological or medical phenomena we are eventually interested in are primarily based on the emerging properties and functions of structured molecular networks, influenced by environmental and stochastic factors.…”

“…As we have shown recently, analyses of comprehensive sets of identified, well-characterized proteins can efficiently decipher functional molecular networks of complex diseases such as diet-induced insulin resistance and can help to assess drug treatments in vivo [14]. This protein set enrichment analysis approach [14][15][16] adapts the concept of gene set enrichment analysis [17], a popular bioinformatics tool to determine whether an a priori defined set of related biomolecules indicates statistically significant, concordant differences between various biological states (more information can be found at [101]). Gene or protein sets can be defined by the researcher, for example, by consulting various publicly available pathway databases.…”

“…Using nano-liquid chromatography coupled to electrospray ionization mass spectrometry we identified and quantitatively analyzed several thousands of proteins including posttranslational modifications such as phosphosites in peripheral metabolic target tissues [14]. Applying protein set enrichment and pathway analyses, we discovered striking changes in protein pathways that indicated differential regulation of cellular and tissue homeostasis during high fat diet and antidiabetic medication.…”

An Impaired Respiratory Electron Chain Triggers Down-regulation of the Energy Metabolism and De-ubiquitination of Solute Carrier Amino Acid Transporters

“…Lysates were sonicated for 1 min, boiled at 95°C for 5 min and precipitated with acetone at Ϫ20°C overnight. Lyophilized proteins were dissolved in 8 M urea, 10 mM Tris, pH 8, alkylated with a final concentration of 5.5 mM chloroacetamide for 30 min and Lys-C digested (1:2000) for 4 h at room temperature followed by a trypsin digestion (1:1000) overnight in 2 M urea at 37°C (23). Subsequent, the peptides were purified with C18 columns.…”

“…Therefore, samples were fractionated by strong cation exchange (SCX) chromatography. Five g of each fraction were again used for proteome profiling, whereas the remaining sample was used for TiO 2 (GL Sciences, Torrance, CA) enrichment of phosphorylated peptides, according to (23). All SAX and SCX fractions, as well as the PTM scans, were allocated to the corresponding replicate and analyzed jointly by MaxQuant (Fig.…”