• Homepage
• People
• Research
• Teaching
• Core facility
• Publications
• Open positions
• ERASMUS
• Contact
• Directions

• Workflows
• Instrumentation
• Research

Proteomics in systems biology

Systems biology relies on global analytical methodologies such as genomics, transcriptomics and proteomics to provide a quantitative description of the living cell. It is widely appreciated that system complexity grows in the direction genome-transcriptome-proteome, and that studying proteins, as well as their modifications and interactions provides the best measure of the gene function. Modern, gel-free and mass spectrometry (MS)-based quantitative proteomics is making a decisive impact across all life sciences and especially in the analysis of cellular homeostasis at the proteome level.  Relatively simple experimental set-ups based on high accuracy MS and powerful bioinformatics tools are capable of reliably identifying and quantifying expression levels of several thousand proteins in a single experiment, approaching the depth of message-based assays and reaching the analytical capacity to completely map the smaller proteomes, such as that of yeast. Likewise, recent progress in biochemical separation and enrichment protocols made it possible to detect dynamics of posttranslational modification sites upon a treatment, providing a wealth of specific and general clues about eukaryotic and prokaryotic signal transduction mechanisms that cannot be otherwise studied by genomics or transcriptomics. Finally, the newest MS approaches are capable of identifying, localizing and quantifying previously unknown modifications in an unbiased manner, providing an essential tool for investigating cross-talk between modifications in biological signal transduction.

 

Selected publications:

Phosphoproteomics - identification of kinase substrates

We are using a phosphoproteomics workflow based on SILAC labeling and phosphopeptide enrichment to identify kinase substrates in several eukatyotic and prokaryotic systems.

 

 

Selected publications:

 

1. Macek, B., Mann, M., Olsen, J.V. 2009. Global and site-specific quantitative phosphoproteomics: principles and applications. Ann Rev Pharmacol 49: 199-221

2. Olsen, J.V., Macek, B. 2009. High Accuracy Mass Spectrometry in Large-Scale Analysis of Protein Phosphorylation. Methods Mol Biol 492: 131-142

 

Microbial S/T/Y phosphoproteomics

Phosphorylation on serine, threonine, and tyrosine (Ser/Thr/Tyr) has long been considered  exclusive to eukaryotes, especially metazoans, and either not present or not functionally significant in bacteria. Instead, the two-component signaling system involving histidine and aspartate phosphorylation is the paradigm of bacterial signal transduction. We  recently applied the qualitative global peptide-based phosphoproteomics workflow to study Ser/Thr/Tyr protein phosphorylation in the model bacteria Bacillus subtilis, Escherichia coli,  Lactococcus lactis and in archaeon Halobacterium salinarum. This approach allowed us to analyze the bacterial phosphoproteome at the phosphorylation site level and to detect approximately 100 phosphorylation events in each analyzed bacterium. The number of phosphoproteins and phosphorylation sites detected in bacteria is much lower than in eukaryotes, where there is evidence for more than 10,000 phosphosites. However, essential proteins and enzymes involved in carbon metabolism and sugar transport were found to be significantly over-represented among detected phosphoproteins, supporting the emerging concept of Ser/Thr/Tyr phosphorylation as an important regulatory mechanism in the bacterial cell. Almost all glycolytic and tricarboxylic acid (TCA) cycle enzymes were found to be phosphorylated, and regulation of some of these enzymes by phosphorylation is already known. Interestingly, bacterial phosphoproteins and phosphorylated residues are significantly more conserved than their non-phosphorylated counterparts. A number of potential phosphorylation sites are conserved from Archaea to humans, pointing to the likely presence of this regulatory modification since the earliest stages of cellular life. Given the rapid increase of antibiotic resistance among pathogenic species, there is an urgent need for identification of alternative regulatory pathways in microorganisms and Ser/Thr/Tyr phosphorylation should be considered as a potential avenue to disrupt bacterial growth. 

 

Selected publications:

 

 

 

Proteogenomics (refinement of genomics data using shot-gun proteomics)

The ongoing efforts in genome sequencing have to date resulted in numerous completed genomes with little or no gene annotation. Available annotations usually rely on computational predictions of protein coding genes. Peptides from proteome lysates identified by mass spectrometry can be mapped directly onto the raw genome sequence, thus enabling the verification, re-annotation as well as the identification of unpredicted genes in a straightforward manner. We are developing proteogenomics strategies based on high accuracy mass spectometry and use them to refine the genome annotation of several model organisms.

 

 Selected publications:

Development of mass spectrometry-based methods for biomarker discovery and quantitation

Multiple reaction monitoring (MRM) analyses rely on detection of precursor/fragment ion combinations (transitions) of protein-specific (proteotypic) peptides. To improve specificity and quantitation, proteotypic peptides are often synthesized, stable isotope-labeled and mixed with the sample, which requires processing of thousands of peptides for a comprehensive proteome analysis. Among several ways of protein labeling, SILAC stands out as a simple method of introducing a defined label into all proteins of a cell/organism. Consequently, a digest of SILAC-labeled cells/tissue contains all labeled proteotypic peptides of proteins expressed at the time of harvesting and presents a generic source of labeled proteotypic peptides which can be mixed with the sample of interest. We are currently assessing the feasibility of SILAC in MRM-based biomarker discovery and quantitation.

 

 

Selected publications: