Architecture and dynamics of protein complexes
and interaction networks
It is a widely accepted concept in biology that proteins exert their function in cells while being part of larger functional complexes. Our research focuses on the characterization of protein complexes and their dynamics during cell differentiation, cell cycle and upon stimulation by external stimuli like UV irradiation, and how this influences protein functioning. Usually, the protein of interest is immunoprecipitated either by using in vivo protein tagging or by highly specific antibodies under relatively mild conditions. Subsequently, the complex partners are separated by SDS-PAGE, in-gel digested (or directly digested from the beads) and identified by mass spectrometry. One of the major challenges is to differentiate between bona fide interaction partners of your protein of interest that make up the core protein complex and 'second-shell' interactors and non-specifically binding proteins. By gradually changing the conditions under which the protein complexes are isolated from cellular extracts, we are able to get a rough idea about the relative strength of protein-protein interactions.
Analysis of posttranslational modifications
After translation, the posttranslational modification of particular amino acid residues extends the range of functions of the protein by attaching to it other biochemical functional groups such as acetate, phosphate, methyl, ubiquitin, various lipids and carbohydrates, by changing the chemical nature of an amino acid or by making structural changes, like the formation of disulfide bridges. We try to answer questions such as how amino acid modifications can affect protein complex composition and how they are involved in cellular functioning. Mass spectrometry has proven to be an excellent tool for the characterization of posttranslational modifications such as acetylation, methylation, phosphorylation, ubiquitylation, ISGylation, and so on. In order to analyze such modifications by mass spectrometry, it is often necessary to enrich for proteolytic peptides bearing the modification of interest. For example, phosphorylated peptides can be enriched for using a protocol that involves trapping such peptides on TiO2 chromatographic column material. Furthermore, we are developing tools for the quantitative analysis of dynamic posttranslational modifications and try to answer questions such as how the phosphorylation state of a protein complex depends on its position in the cell cycle and how modifications on histones and nucleosome associated proteins change after DNA damage. Recently, we have characterized by mass spectrometry multiple sites of ubiquitylation on Tramtrack, a protein which was found to be the major substrate of the ubiquitin specific protease UBP64 in Drosophila.
18O and SILAC labeling for quantitative proteomics
We have optimized and implemented a method that uses
18O labeling of tryptic peptides to account for quantitative differences in protein levels between different samples. When all proteolytic peptides in e.g. a control sample are labeled with
18O, we can easily differentiate between real interactors of our protein of interest and non-specific background proteins: in the case of background proteins, both heavily-labeled and non-labeled peptides will appear in the same mass spectrum. In contrast, if the protein is specific for the sample, only the light variant will be observed. We have successfully applied this method for quickly subtracting background proteins from real interactors in co-immunoprecipitations.
Another way to study changes in the proteome in a quantitative manner is by using Stable Isotope Labeling of Amino acids in Cell culture (SILAC). In this approach, one sample originates from cells that were grown under normal conditions and the other sample is from cells that were grown in media containing amino acids that were labeled with heavy carbon (13C), nitrogen (15N) and/or hydrogen (D) isotopes. One of our research projects involves investigation of changes in histone modifications after DNA damage and we use SILAC to identify and quantify such changes.
Characterization of complex protein mixtures
For highly complex mixtures like complete cellular lysates, nuclear extracts or body fluids, multi-dimensional separations of proteins and/or proteolytic peptides should be applied to get the highest number of protein identifications. Before reversed-phase separation in a typical nanoflow LC-MS/MS setup that we use in the lab, peptides can be separated and fractionated by either strong cation exchange (SCX), strong anion exchange (SAX), iso-electric focusing (IEF) or hydrophilic interaction liquid chromatography (HILIC). Usually, 5 to 30 fractions for each preparation are collected and subjected to nanoflow LC-MS/MS. SDS-PAGE separation of proteins is also used as a first fractionation step. Generally, for complex biological mixtures, multiple separation steps are required to obtain as many protein identifications as possible. We are applying combinations of fractionation steps in our attempts to study proteomes or body fluids such as human follicular fluid, urine, etc.