Research

Quantitative Proteomics

Within cells, proteins are the real effectors of all activities. The expression of certain proteins changes upon cell stimulation or stress, or when cells differentiate or turn into a disease state. Measuring the protein expression levels as well as characterizing their post-translational modifications gives us information on their cellular state. The goal of quantitative proteomics is to evaluate the relative expression of proteins between two cellular samples being compared. To achieve this, protein extracts from both cellular samples are usually tagged with isotope-labeled reagents, which differ in their isotopic composition. For example, the ICAT (Isotope-Coded Affinity Tag) labeling strategy, designed in Prof. Ruedi Aebersold’s lab, consists of alkylating the cysteines of proteins with either a light or a heavy tag, which respectively contains protons or deuteriums (H or D), or alternatively ¹²C or ¹³C. After digestion of the pooled protein samples with an enzyme, the proteolytic peptides are analyzed by mass spectrometry. The cysteine-containing peptides are detected as doublets, and their relative intensity reflects the relative abundance of the original protein in both samples.

Figure 1: Quantitative proteomics using ICAT reagents.
The ICAT reagent comprises a protein reactive group (such as a sulfhydryl-specific reactivity), a mass-encoded linker and an affinity tag (such as biotin). Variations of any of the three can be used to facilitate the quantification of many different modifications or activities. The general scheme used for this reagent is shown: first, the protein reactive groups (such as cysteine residues) are labeled separately with either light or heavy reagent, and then the proteins are mixed and digested by enzymes; second, the labeled peptides are captured and then quantified and identified by LC-MS/MS.

Signal Transduction Projects

Cells respond to environmental cues such as hormones or nutrients by activating distinct intracellular signal transduction pathways, typically involving cascades of protein phosphorylation, that adapt cellular parameters like metabolism and gene expression patterns to the altered conditions. Signal transduction modules are often extensively crosslinked and feedback regulated, so they are more dynamic networks than linear pathways. Quantitative proteomics techniques are especially well suited to study these signaling networks and learn more about their dynamic behavior. Our studies in this area of research mainly focus on clinically important signaling modules like the insulin/IGF and target of rapamycin (TOR) pathways. We use yeast, Drosophila or mammalian cells to study interaction partners and/or posttranslanslational modification of signaling proteins, and rely on the mass spectrometry and bioinformatics platforms in our lab to analyze our samples and evaluate the obtained data.

Biomarker Project

Protein biomarkers as diagnostic or prognostic indicators of disease are believed to play an increasing role in medicine as it transits into a predictive mode and are beginning to assume an even greater role in drug discovery and development. Due to its enormous complexity, the comprehensive analysis of the human proteome is an as-yet-unresolved technical challenge. However, biologically or clinically important information can be obtained if specific, information-rich protein classes, or sub-proteomes, are isolated and analyzed. We are working on a platform for this purpose where we combine solid phase extraction of glycopeptides from complex serum samples, the identification and quantification by tandem mass spectrometry (MS/MS), and the analysis by computational tools. The work done in Zurich is in close colaboration with the biomarker project ongoing at the Institute for Systems Biology in Seattle.

Figure 2. Schematic diagram of quantitative analysis of N-linked glycopeptides.
(a) Strategy for quantitative analysis of glycopeptides. Proteins from two biological samples are oxidized and coupled to hydrazide resin. Nonglycosylated peptides are removed by proteolysis and extensive washes. The nonglycosylated peptides are optionally collected and analyzed. The N-terminus of glycopeptides are isotope labeled by succinic anhydride carrying either d0 or d4. The beads are then combined and the isotopically tagged peptides are released by PNGase F and analyzed by MS. (b) Oxidation of a carbohydrate to an aldehyde followed by covalent coupling to hydrazide resin.