2018 Apr 13. doi: 10.1021/acs.jproteome.7b00825. [Epub ahead of print]

Labeling Carboxyl Groups of Surface Exposed Proteins Provides an Orthogonal Approach for Cell Surface Isolation.



Quantitative profiling of cell surface proteins is critically important for the understanding of cell-cell communication, signaling, tissue development and homeostasis. Traditional proteomics methods are challenging for cell surface proteins due to their hydrophobic nature and low abundance, necessitating alternative methods to efficiently identify and quantify this protein group. Here, we established carboxyl reactive biotinylation for selective and efficient biotinylation and isolation of surface-exposed proteins of living cells. We assessed the efficiency of carboxyl reactive biotinylation for plasma membrane proteins by comparing with a well-established protocol, amine reactive biotinylation using SILAC (Stable Isotope Labeling in Cell Culture). Our results show that carboxyl reactive biotinylation of cell surface proteins is both more selective and more efficient than amine reactive biotinylation. We conclude that it is a useful approach, which is partially orthogonal to amine reactive biotinylation, allowing to cast a wider net for a comprehensive profiling of cell surface proteins.



 2018 Feb 2;8(1):2269. doi: 10.1038/s41598-018-20231-5.

Comparative phosphoproteomic analysis reveals signaling networks regulating monopolar and bipolar cytokinesis.

Author information

Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey.
Bioinformatics Division (MF1), Robert Koch Institute, Berlin, Germany.
Chair of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany.
Department of Genetics, Stanford University, School of Medicine, CA, USA.
Graduate School of Informatics, Department of Health Informatics, METU, Ankara, Turkey.
Cancer Systems Biology Laboratory (CanSyL), METU, Ankara, Turkey.
Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey. nozlu@ku.edu.tr.
Koç University Research Center for Translational Medicine (KUTTAM), Istanbul, Turkey. nozlu@ku.edu.tr.


The successful completion of cytokinesis requires the coordinated activities of diverse cellular components including membranes, cytoskeletal elements and chromosomes that together form partly redundant pathways, depending on the cell type. The biochemical analysisof this process is challenging due to its dynamic and rapid nature. Here, we systematically compared monopolar and bipolar cytokinesis and demonstrated that monopolar cytokinesis is a good surrogate for cytokinesis and it is a well-suited system for global biochemical analysis in mammalian cells. Based on this, we established a phosphoproteomic signature of cytokinesis. More than 10,000 phosphorylation sites were systematically monitored; around 800 of those were up-regulated during cytokinesis. Reconstructing the kinase-substrate interaction network revealed 31 potentially active kinases during cytokinesis. The kinase-substrate network connects proteins between cytoskeleton, membrane and cell cycle machinery. We also found consensus motifs of phosphorylation sites that can serve as biochemical markers specific to cytokinesis. Beyond the kinase-substrate network, our reconstructed signaling network suggests that combination of sumoylation and phosphorylation may regulate monopolar cytokinesis specific signaling pathways. Our analysis provides a systematic approach to the comparison of different cytokinesis types to reveal alternative ways and a global overview, in which conserved genes work together and organize chromatin and cytoplasm during cytokinesis.



 2017 Oct;17(20). doi: 10.1002/pmic.201600100. Epub 2017 Sep 4.

Proteomics in Cell Division.

Author information

Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey.
Biomedical Sciences and Engineering, Koç University, Istanbul, Turkey.


Cell division requires a coordinated action of the cell cycle machinery, cytoskeletal elements, chromosomes, and membranes. Cell division studies have greatly benefitted from the mass spectrometry (MS)-based proteomic approaches for probing the biochemistry of highly dynamic complexes and their coordination with each other as a cell progresses into division. In this review, the authors first summarize a wide-range of proteomic studies that focus on the identification of sub-cellular components/protein complexes of the cell division machinery including kinetochores, mitotic spindle, midzone, and centrosomes. The authors also highlight MS-based large-scale analyses of the cellular components that are largely understudied during cell division such as the cell surface and lipids. Then, the authors focus on posttranslational modification analyses, especially phosphorylation and the resulting crosstalk with other modifications as a cell undergoes cell division. Combining proteomic approaches that probe the biochemistry of cell division components with functional genomic assays will lead to breakthroughs toward a systems-level understanding of cell division.


Cell division; Cytokinesis; Mass spectrometry; Phosphorylation; Proteomics



Author information

Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey.
Department of Chemical and Biological Engineering, Koç University, Istanbul, Turkey.
Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey. nozlu@ku.edu.tr.



Aurora B is a serine/threonine kinase that has a central role in the regulation of mitosis. The observation of Aurora B overexpression in cancer makes it a promising target to develop antitumoral inhibitors. We describe a new potential inhibitor that exclusively targets the interaction site of Aurora B and its activator INCENP. We performed a structure-based virtual screening and determined five potential candidates of 200 000 compounds, which selectively bind to the Aurora B::INCENP interaction site, but not to the ATP-binding site (kinase pocket) of Aurora B or other related kinases. Further characterization in vivo validated the inhibitory role of one of these five compounds in Aurora B::INCENP complex formation and exhibited hallmarks of Aurora inhibition such as chromosome congression and segregation defects that interfere with the progression into cytokinesis and result in multinuclear cells. Our results provide an alternative approach on the way of exploring specific kinase inhibitors.



 2015 Aug 13. 

Phosphoproteomic analysis of Aurora kinase inhibition in Monopolar Cytokinesis.

1Department of Molecular Biology and Genetics, Koç University, Istanbul, Turkey
2 Research Group Bioinformatics (NG 4), Robert Koch-Institute, Berlin, Germany.
3 Department of Genetics, Stanford University, School of Medicine, CA.
4 Department of Bioanalytics, Institute of Biotechnology, Technische Universität Berlin, Berlin Germany


Cytokinesis is the last step of the cell cycle which requires coordinated activities of the microtubule cytoskeleton, actin cytoskeleton and membrane compartments. Aurora B kinase is one of the master regulatory kinases that orchestrate multiple events during cytokinesis. To reveal targets of the Aurora B kinase, we combined quantitative mass spectrometry with chemical genetics. Using the quantitative proteomic approach, SILAC (Stable Isotope Labeling with Amino acids in Cell culture), we analyzed the phosphoproteome of monopolar cytokinesis upon VX680 or AZD1152 mediated Aurora kinase inhibition. In total our analysis quantified over twenty thousand phosphopeptides in response to the Aurora-B kinase inhibition; 246 unique phosphopeptides were significantly down-regulated and 74 were up-regulated. Our data provide a broad analysis of downstream effectors of Aurora kinase and offer insights into how Aurora kinase regulates cytokinesis.



 2014 Dec 4. pii: e201385162. 

Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.


1Department of Molecular Biology and GeneticsKoç UniversityIstanbul, Turkey

 2Max Planck Institute for Molecular Cell Biology and GeneticsDresden, Germany

 3Research Group Bioinformatics (NG 4)Robert Koch‐InstituteBerlin, Germany

 4Proteomics Center at Children's Hospital BostonBostonMA, USA

5Department of Systems BiologyHarvard Medical SchoolBostonMA, USA

 6Department of NeurobiologyHarvard Medical School and Children's Hospital Boston,BostonMA, USA 



The cell surface is the cellular compartment responsible for communication with the environment. The interior of mammalian cells undergoes dramatic reorganization when cells enter mitosis. These changes are triggered by activation of the CDK1 kinase and have been studied extensively. In contrast, very little is known of the cell surface changes during cell division. We undertook a quantitative proteomic comparison of cell surface-exposed proteins in human cancer cells that were tightly synchronized in mitosis or interphase. Six hundred and twenty-eight surface and surface-associated proteins in HeLa cells were identified; of these, 27 were significantly enriched at the cell surface in mitosis and 37 in interphase. Using imaging techniques, we confirmed the mitosis-selective cell surface localization of protocadherin PCDH7, a member of a family with anti-adhesive roles in embryos. We show that PCDH7 is required for development of full mitotic rounding pressure at the onset of mitosis. Our analysis provided basic information on how cell cycle progression affects the cell surface. It also provides potential pharmacodynamic biomarkers for anti-mitotic cancer chemotherapy.

© 2014 The Authors.


SILAC ; PCDH7; cell cycle; cell rounding; cell surface



Towards single-cell LC-MS phosphoproteomics.

Polat AN1Özlü N.

1Department of Molecular Biology and Genetics, Science Faculty, Koç University, Istanbul, Turkey. anpolat@ku.edu.tr nozlu@ku.edu.tr.


Protein phosphorylation is a ubiquitous posttranslational modification, which is heavily involved in signal transduction. Misregulation of protein phosphorylation is often associated with a decrease in cell viability and complex diseases such as cancer. The dynamic and low abundant nature of phosphorylated proteins makes studying phosphoproteome a challenging task. In this review, we summarize state of the art proteomic techniques to study and quantify peptide phosphorylation in biological systems and discuss their limitations. Due to its short-lived nature, the phosphorylation event cannot be precisely traced in a heterogonous cell population, which highlights the importance of analyzing phosphorylation events at the single cell level. Mainly, we focus on the methodical and instrumental developments in proteomics and nanotechnology, which will help to build more accurate and robust systems for the feasibility of phosphorylation analysis at the single cell level. We propose that an automated and miniaturized construction of analytical systems holds the key to the future of phosphoproteomics; therefore, we highlight the benchmark studies in this direction. Having advanced and automated microfluidic chip LC systems will allow us to analyze single-cell phosphoproteomics and quantitatively compare it with others. The progress in the microfluidic chip LC systems and feasibility of the single-cell phosphoproteomics will be beneficial for early diagnosis and detection of the treatment response of many crucial diseases.




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