Proteomics in experimental 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; 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 bacteria and yeasts. 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. We are currently developing and applying methods that are capable of comprehensive and accurate identification and quantitation of proteomes in various model systems.

Selected publications:

Semanjski M, Gratani FL, Englert T, Nashier P, Beke V, Nalpas N, Germain E, George S, Wolz C, Gerdes K, Macek B. 2021. Proteome Dynamics during Antibiotic Persistence and Resuscitation. mSystems 6(4):e0054921

Cvetesic, N., Semanjski, M., Soufi, B., Krug, K., Gruic-Sovulj, I., Macek, B. 2016. Proteome-wide measurement of non-canonical bacterial mistranslation by quantitative mass spectrometry of protein modifications. Sci Reports 6:28631.

Carpy, A., Krug, K., Graf, S., Koch, A., Popic, S., Hauf, S., Macek,B. 2014. Absolute proteome and phosphoproteome dynamics during the cell cycle of fission yeast. Mol Cell Proteomics13(8):1925-36.

Soares, N.C., Spät, P., Krug, K., Macek, B. 2013.Global dynamics of the Escherichia coli proteome and phosphoproteome during growth in minimal medium. J Prot Res. 12(6):2611-21.

Schütz, W., Hausmann, N., Krug, K., Hampp, R., Macek, B. 2011. Extending SILAC to proteomics of plant cell lines. Plant Cell. 23(5):1701-5.

Soufi, B., Kumar, C., Gnad, F., Mann, M., Mijakovic, I., Macek, B. 2010. Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) applied to quantitative proteomics of Bacillus subtilis. J Prot Res 9(7):3638-46.


PTM analysis, phosphoproteomics and identification of kinase substrates

Increasing number of publications demonstrate that the proteomics has “matured” enough to comprehensively detect and quantify phosphoproteomes of eukaryotic and prokaryotic organisms. The challenge and the emphasis in the fields of proteomics and cell biology have now shifted to the next stage - detection of kinase and phosphatase substrates and their integration into regulatory networks in the cell. 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:

Gratani FL, Englert T, Nashier P, Sass P, Mann P, Czech L, Neumann N, Doello S, Blobelt R, Alberti S, Forchhammer K, Bange G, Höfer K, Macek B. 2023. E. coli toxin YjjJ (HipH) is a Ser/Thr protein kinase that impacts cell division, carbon metabolism and ribosome assembly. mSystems, 8(1):e0104322

Zittlau KI, Terradas AL, Nalpas N, Geisler S, Kahle PJ, Macek B. 2022. Temporal Analysis of Protein Ubiquitylation and Phosphorylation During Parkin-dependent Mitophagy. Mol Cell Proteomics, 21(2):100191

Semanjski, M., Germain, E., Bratl, K., Kiessling, A., Gerdes K., Macek, B. 2018. The kinases HipA and HipA7 phosphorylate different substrate pools in Escherichia coli to promote multidrug tolerance. Science Signaling 11, eaat5750

Kliza, K., Taumer, C., Pinzuti, I., Franz-Wachtel, M., Kunzelmann, S., Stieglitz, B., Macek, B., Husnjak, K. 2017. Internally tagged ubiquitin: a tool to identify linear polyubiquitin-modified proteins by mass spectrometry. Nat Methods, doi: 10.1038/nmeth.4228. [Epub ahead of print]

Ravikumar, V., Shi, L., Krug, K., Derouiche, A., Jers, C., Cousin, C., Kobir, A., Mijakovic, I., Macek, B. 2014.Quantitative phosphoproteome analysis of Bacillus subtilis reveals novel substrates of the kinase PrkC and phosphatase PrpC. Mol Cell Proteomics 13(8):1965-78.

Franz-Wachtel, M., Eisler, S., Krug, K., Wahl, S., Carpy, A., Nordheim, A., Pfizenmaier, K., Hausser, A., Macek, B. 2012. Global detection of Protein Kinase D-dependent phosphorylation events in nocodazole-treated human cells. Mol Cell Proteomics. 11(5):160-70.

Koch, A., Krug, K., Pengelley, S.,Macek, B#., Hauf, S#. 2011. Mitotic substrates of the kinase Aurora with roles in chromatin regulation identified through quantitative phosphoproteomics of fission yeast. Science Signaling. 4 (179): rs6.

Ikeda F, Deribe YL, Skånland SS, Stieglitz B, Grabbe C, Franz-Wachtel M, van Wijk SJ, Goswami P, Nagy V, Terzic J, Tokunaga F, Androulidaki A, Nakagawa T, Pasparakis M, Iwai K, Sundberg JP, Schaefer L, Rittinger K, Macek B, Dikic I. SHARPIN forms a linear ubiquitin ligase complex regulating NF-kappaB activity and apoptosis. Nature. 2011 Mar 31;471(7340):637-41.


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:

Macek B, Forchhammer K, Hardouin J, Weber-Ban E, Grangeasse C, Mijakovic I. 2019. Protein post-translational modifications in bacteria. Nat Rev Microbiol 17(11):651-664

Rosenberg, A., Soufi, B., Ravikumar, V., Soares, N.C., Krug, K., Smith, Y, Macek, B., Ben-Yehuda, S. 2015. Phosphoproteome Dynamics Mediate Revival of Bacterial Spores. BMC Biology 13:76.

Soufi, B., Soares, N.C., Ravikumar, V., Macek, B. 2012. Proteomics reveals evidence of cross-talk between protein modifications in bacteria: Focus on protein acetylation and phosphorylation. Curr Opinion Microbiology 15(3):357-63.

Mijakovic, I., Macek, B. 2012. Impact of phosphoproteomics on studies of bacterial physiology. FEMS Microbiology Reviews 36(4):877-92.

Macek, B., Gnad, F., Soufi, B., Kumar, C., Olsen, J.V., Mijakovic, I., Mann, M. 2008. Phosphoproteome analysis of E. coli reveals evolutionary conservation of bacterial Ser/Thr/Tyr phosphorylation. Mol Cell Proteomics 7(2):299-307

Macek, B., Mijakovic, I., Olsen, J.V., Gnad, F., Kumar, C., Jensen, P.R., Mann, M. 2007. The Serine/Threonine/ Tyrosine phosphoproteome of the model bacterium Bacillus subtilis. Mol Cell Proteomics 6 (4): 697-707


Proteogenomics, Metaproteomics and Individualized Proteomics

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:

Spät P, Krauspe V, Hess WR, Macek B*, Nalpas N*. 2023. Deep proteogenomics of a photosynthetic cyanobacterium. J Prot Res, 22(6):1969-1983

Nalpas N, Hoyles L, Anselm V, Ganief T, Martinez-Gili L, Grau C, Droste-Borel I, Davidovic L, Altafaj X, Dumas M, Macek B. 2021. An Integrated Workflow for Enhanced Taxonomic and Functional Coverage of the Mouse Faecal Metaproteome. Gut Microbes 13:1, 199483

Schmitt M, Sinnberg T, Niessner H, Forschner A, Garbe C, Macek B*, Nalpas NC*. 2021. Individualized proteogenomics reveals mutational landscape of melanoma patients in response to immunotherapy. Cancers 13(21), 5411

Schmitt M, Sinnberg T, Bratl K, Zittlau K,Garbe C, Macek B*, Nalpas NC*. 2021. Proteogenomics reveals perturbed signalling networks in Malignant Melanoma Cells Resistant to BRAF inhibition. Mol Cell Proteomics 20:100163

Ravikumar V, Nalpas NC, Anselm V, Krug K, Lenuzzi M, Šestak MS, Domazet-Lošo T, Mijakovic I, Macek B. 2018. In-depth analysis of Bacillus subtilis proteome identifies new ORFs and traces the evolutionary history of modified proteins. Sci Reports 8(1):17246.

Krug, K., Popic, S., Carpy, A., Täumer, C.,Macek, B. 2014. Construction and assessment of individualized proteogenomic databases for large-scale analysis of non-synonymous single nucleotide variants. Proteomics 14(23-24):2699-708.

Krug, K., Carpy, A., Behrends, G., Matic, K., Soares, N.C., Macek, B. 2013. Deep coverage of the Escherichia coli proteome enables the assessment of database search strategies in bacterial proteogenomics experiments. Mol Cell Proteomics 12(11):3420-30.

Krug, K., Nahnsen, S., Macek, B. 2011. Mass spectrometry at the interface of genomics and proteomics. Mol Biosystems 7(2):284-91.

Borchert N, Dieterich C, Krug K, Schütz W, Jung S, Nordheim A, Sommer RJ, Macek B. 2010. Proteogenomics of Pristionchus pacificus reveals distinct proteome structure of nematode models. Genome Res. 20(6):837-46