Publications by authors named "Christoph Roesli"

26 Publications

  • Page 1 of 1

Identification and Validation of Novel Subtype-Specific Protein Biomarkers in Pancreatic Ductal Adenocarcinoma.

Pancreas 2017 03;46(3):311-322

From the *German Cancer Research Center (DKFZ), Heidelberg, Germany; †Princess Margaret Cancer Center, Toronto, Ontario, Canada; ‡Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany; and §Novartis Pharma AG, Basel, Switzerland.

Objectives: Pancreatic ductal adenocarcinoma (PDAC) has been subclassified into 3 molecular subtypes: classical, quasi-mesenchymal, and exocrine-like. These subtypes exhibit differences in patient survival and drug resistance to conventional therapies. The aim of the current study is to identify novel subtype-specific protein biomarkers facilitating subtype stratification of patients with PDAC and novel therapy development.

Methods: A set of 12 human patient-derived primary cell lines was used as a starting material for an advanced label-free proteomics approach leading to the identification of novel cell surface and secreted biomarkers. Cell surface protein identification was achieved by in vitro biotinylation, followed by mass spectrometric analysis of purified biotin-tagged proteins. Proteins secreted into a chemically defined serum-free cell culture medium were analyzed by shotgun proteomics.

Results: Of 3288 identified proteins, 2 pan-PDAC (protocadherin-1 and lipocalin-2) and 2 exocrine-like-specific (cadherin-17 and galectin-4) biomarker candidates have been validated. Proximity ligation assay analysis of the 2 exocrine-like biomarkers revealed their co-localization on the surface of exocrine-like cells.

Conclusions: The study reports the identification and validation of novel PDAC biomarkers relevant for the development of patient stratification tools. In addition, cadherin-17 and galectin-4 may serve as targets for bispecific antibodies as novel therapeutics in PDAC.
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http://dx.doi.org/10.1097/MPA.0000000000000743DOI Listing
March 2017

MALDI-TOF and nESI Orbitrap MS/MS identify orthogonal parts of the phosphoproteome.

Proteomics 2016 05 2;16(10):1447-56. Epub 2016 May 2.

Chair of Proteomics and Bioanalytics, Technische Universität München, Freising, Germany.

Phosphorylation is a reversible posttranslational protein modification which plays a pivotal role in intracellular signaling. Despite extensive efforts, phosphorylation site mapping of proteomes is still incomplete motivating the exploration of alternative methods that complement existing workflows. In this study, we compared tandem mass spectrometry (MS/MS) on matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) and nano-electrospray ionization (nESI) Orbitrap instruments with respect to their ability to identify phosphopeptides from complex proteome digests. Phosphopeptides were enriched from tryptic digests of cell lines using Fe-IMAC column chromatography and subjected to LC-MS/MS analysis. We found that the two analytical workflows exhibited considerable orthogonality. For instance, MALDI-TOF MS/MS favored the identification of phosphopeptides encompassing clear motif signatures for acidic residue directed kinases. The extent of orthogonality of the two LC-MS/MS systems was comparable to that of using alternative proteases such as Asp-N, Arg-C, chymotrypsin, Glu-C and Lys-C on just one LC-MS/MS instrument. Notably, MALDI-TOF MS/MS identified an unexpectedly high number and percentage of phosphotyrosine sites (∼20% of all sites), possibly as a direct consequence of more efficient ionization. The data clearly show that LC-MALDI MS/MS can be a useful complement to LC-nESI MS/MS for phosphoproteome mapping and particularly so for acidic and phosphotyrosine containing peptides.
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http://dx.doi.org/10.1002/pmic.201500523DOI Listing
May 2016

Ion source-dependent performance of 4-vinylpyridine, iodoacetamide, and N-maleoyl derivatives for the detection of cysteine-containing peptides in complex proteomics.

Anal Bioanal Chem 2016 Mar 22;408(8):2055-67. Epub 2015 Oct 22.

German Cancer Research Center, Im Neuenheimer Feld 280, 69120, Heidelberg, Germany.

Cysteine is unique among the proteinogenic amino acids due to its ability to form disulfide bonds. While this property is of vital importance for protein structures and biological processes, it causes difficulties for the mass spectrometric identification of cysteine-containing peptides. A common approach to overcome these problems in bottom-up proteomics is the reduction and covalent modification of sulfhydryl groups prior to enzymatic digestion. In this study, established alkylating agents and N-maleoyl amino acids with variable hydrophobicity were characterized with respect to a variety of relevant parameters and subsequently evaluated in a large-scale analysis using different ion sources. Depending on the compound, the ion source had a profound impact on the relative and absolute identification of cysteine-containing peptides. The best results were obtained by derivatization of the cysteine residues with 4-vinylpyridine and subsequent matrix-assisted laser desorption ionization (MALDI). Modification with 4-vinylpyridine increased the number of cysteine-containing peptides identified with any other compound using LC-MALDI/MS at least by a factor of 2. This experimental observation is mirrored by differences in the gas-phase basicities, which were computed for methyl thiolate derivatives of the compounds using density functional theory. With electrospray ionization (ESI), complementary use of reagents from three different compound classes, e.g., iodoacetamide, 4-vinylpyridine, and N-maleoyl beta-alanine, was beneficial compared to the application of a single reagent.
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http://dx.doi.org/10.1007/s00216-015-9113-4DOI Listing
March 2016

Recent advances in proteomically subtyping pancreatic ductal adenocarcinomas and their potential clinical impact.

Authors:
Christoph Roesli

Expert Rev Proteomics 2015 Feb 19;12(1):5-8. Epub 2014 Nov 19.

Junior Research Group Biomarker Discovery, German Cancer Research Center, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a median overall survival of 6 months. Late diagnosis due to the absence of specific symptoms during disease development, in addition to extensive metastatic potential and resistance to chemotherapy and radiotherapy, are the most important reasons for short survival. Research efforts have therefore been focused on the development of early disease detection. However, the only US FDA-approved clinical biomarker, CA19-9, is considered inapplicable for screening and/or early detection of PDAC. The following editorial provides the reader with a short introduction to the topic of PDAC and gives focus to the current state of proteomic research in the field of PDAC biomarker discovery. This editorial also highlights the efforts made to subdivide this tumor entity and the potential clinical impact of patient stratification. Finally, the author provides opinions on the impact of proteomics to PDAC subtype stratification over the next 5 years.
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http://dx.doi.org/10.1586/14789450.2015.983478DOI Listing
February 2015

A comprehensive surface proteome analysis of myeloid leukemia cell lines for therapeutic antibody development.

J Proteomics 2014 Mar 30;99:138-51. Epub 2014 Jan 30.

ETH Zurich, Department of Chemistry and Applied Biosciences, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland. Electronic address:

Unlabelled: A detailed characterization of the cell surface proteome facilitates the identification of target antigens, which can be used for the development of antibody-based therapeutics for the treatment of hematological malignancies. We have performed cell surface biotinylation of five human myeloid leukemia cell lines and normal human granulocytes, which was used for mass spectrometric analysis and allowed the identification and label-free, relative quantification of 320 membrane proteins. Several proteins exhibited a pronounced difference in expression between leukemia cell lines and granulocytes. We focused our attention on CD166/ALCAM, as this protein was strongly up-regulated on all AML cell lines and AML blasts of some patients. A human monoclonal antibody specific to CD166 (named H8) was generated using phage display technology. H8 specifically recognized AML cells in FACS analysis while demonstrating tumor targeting properties in vivo. After in vitro screening of five potent cytotoxic agents, a duocarmycin derivative was used for the preparation of an antibody-drug conjugate, which was able to kill AML cells in vitro with an IC50 of 8nM. The presented atlas of surface proteins in myeloid leukemia provides an experimental basis for the choice of target antigens, which may be used for the development of anti-AML therapeutic antibodies.

Biological Significance: The ability to discriminate between malignant and healthy, essential cells represents an important requirement for the development of armed antibodies for the therapy of hematological malignancies. Our proteomic study is, to our knowledge, the first large scale comparison of the accessible cell surface proteome of leukemia cells and normal blood cells, facilitating the choice of a suitable target for the treatment of acute myeloid leukemia (AML). An antibody drug conjugate was generated recognizing the CD166 antigen which was found to be strongly up-regulated in all AML cell lines and AML blasts of some patients. This antibody drug conjugate SIP(H8)-Duo might be further characterized in therapy experiments and might lead to a new targeted treatment option for AML.
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http://dx.doi.org/10.1016/j.jprot.2014.01.022DOI Listing
March 2014

Label-free quantification using MALDI mass spectrometry: considerations and perspectives.

Anal Bioanal Chem 2012 Sep 23;404(4):1039-56. Epub 2012 Feb 23.

Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HI-STEM gGmbH), Heidelberg, Germany.

Profound knowledge of protein abundances in healthy tissues and their changes in disease is crucial for understanding biological processes in basic science and for the development of novel diagnostics and therapeutics. Mass spectrometrybased label-free protein quantification is used increasingly often to gain insights into physiological changes observed in perturbed systems. Although the soft ionization techniques electrospray ionization and matrix-assisted laser desorption/ionization have both been used for protein quantification, this article focuses on instrumental setups with a MALDI ion source. Beside reviewing current bioinformatic data-processing tools for label-free quantification and elaborating on the technical benefits of combining UHPLC and MALDI-MS, we outline the potential of state-of-the-art instruments by reporting unpublished results obtained from twenty-four complex biological samples. This review points out that the capabilities of LC-MALDI MS systems have not yet been fully utilized because of a lack of suitable software tools.
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http://dx.doi.org/10.1007/s00216-012-5832-yDOI Listing
September 2012

Proteomic identification of vanin-1 as a marker of kidney damage in a rat model of type 1 diabetic nephropathy.

Kidney Int 2011 Aug 4;80(3):272-81. Epub 2011 May 4.

Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.

At present, the urinary albumin excretion rate is the best noninvasive predictor for diabetic nephropathy (DN) but major limitations are associated with this marker. Here, we used in vivo perfusion technology to establish disease progression markers in an animal model of DN. Rats were perfused with a reactive ester derivative of biotin at various times after streptozotocin treatment. Following homogenization of kidney tissue and affinity purification of biotinylated proteins, a label-free mass spectrometry-based proteomic analysis of tryptic digests identified and relatively quantified 396 proteins. Of these proteins, 24 and 11 were found to be more than 10-fold up- or downregulated, respectively, compared with the same procedure in vehicle-treated rats. Changes in the expression of selected differentially regulated proteins were validated by immunofluorescence detection in kidney tissue from control and diabetic rats. Immunoblot analysis of pooled human urine found that concentrations of vanin-1, an ectoenzyme pantetheinase, distinguished diabetic patients with macroalbuminuria from those with normal albuminuria. Uromodulin was elevated in the urine pools of the diabetic patients, regardless of the degree of albuminuria, compared with healthy controls. Thus, in vivo biotinylation facilitates the detection of disease-specific changes in the abundance of potential biomarker proteins for disease monitoring and/or pharmacodelivery applications.
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http://dx.doi.org/10.1038/ki.2011.116DOI Listing
August 2011

The accessible cerebral vascular proteome in a mouse model of cerebral β-amyloidosis.

J Proteomics 2011 Apr 22;74(4):539-46. Epub 2011 Jan 22.

Department of Chemistry and Applied Biosciences, ETH Zürich, Zurich, Switzerland.

Assessing protein changes in the cerebral vasculature of brain disorders may increase our understanding of disease pathogenesis and facilitate diagnostic and therapeutic intervention. By combining perfusion of mice with a charged reactive biotin derivative and subsequent quantification of the biotinylated proteins, the proteome accessible from the vasculature in an APPPS1 transgenic mouse model of cerebral β-amyloidosis was identified and compared to that in non-transgenic control mice. Our results provide proof-of-concept of this technology for the identification of new targets for antibody-based therapy or pharmacodelivery, and for neuroimaging in neurodegenerative diseases.
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http://dx.doi.org/10.1016/j.jprot.2011.01.010DOI Listing
April 2011

A novel reactive ester derivative of biotin with reduced membrane permeability for in vivo biotinylation experiments.

Proteomics 2010 Oct;10(19):3544-8

Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

The in vivo perfusion of rodent models of disease with biotin derivatives and the subsequent comparative proteomic analysis of healthy and diseased tissues represent a promising methodology for the identification of vascular accessible biomarkers. A novel, triply charged biotinylation reagent, NHS-β-Ala-(L-Asp)(3)-biotin, was synthesized and validated in terms of its applicability for in vivo protein biotinylation. Compared to sulfo-NHS-LC-biotin, NHS-β-Ala-(L-Asp)(3)-biotin exhibited a reduced membrane permeability and a preferential labeling of proteins localized in compartments readily accessible in vivo from the vasculature.
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http://dx.doi.org/10.1002/pmic.201000308DOI Listing
October 2010

Expression, engineering and characterization of the tumor-targeting heterodimeric immunocytokine F8-IL12.

Protein Eng Des Sel 2010 Aug 15;23(8):653-61. Epub 2010 Jun 15.

Philochem AG, c/o ETH Zürich, Institute of Pharmaceutical Sciences, Wolfgang-Pauli-Strasse 10, HCI E520, CH-8093 Zürich, Switzerland.

Proinflammatory cytokines have been used for several years in patients with advanced cancer but their administration is typically associated with severe toxicity hampering their application to therapeutically active regimens. This problem can be overcome by using immunocytokines (cytokines fused to antibody or antibody fragments) which selectively deliver the active cytokine to the tumor environment. Preclinical and recent clinical results confirmed that this approach is a very promising avenue to go. We designed an immunocytokine consisting of the scFv(F8) specific to extra-domain A of fibronectin and the very potent human cytokine interleukin-12 (IL12). The heterodimeric nature of IL12 allows the engineering of various immunocytokine formats, based on different combinations of the two subunits (p35 and p40) together with the scFv. In comparison to monomeric or homodimeric cytokines, the construction of a heterodimeric immunocytokine poses many challenges, e.g. gene dosing, stable high-yield expression as well as good manufacture practice (GMP) purification and characterization. In this paper, we describe the successful construction, characterization and production of the heterodimeric immunocytokine F8-IL12. The positive outcome of this feasibility study leads now to GMP production of F8-IL12, which will soon enter clinical trials.
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http://dx.doi.org/10.1093/protein/gzq038DOI Listing
August 2010

Methods for the identification of vascular markers in health and disease: from the bench to the clinic.

J Proteomics 2010 Oct 10;73(11):2219-29. Epub 2010 Jun 10.

Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

Several diseases are characterized by changes in the molecular composition of vascular structures, thus offering the opportunity to use specific ligands (e.g., monoclonal antibodies) for imaging and therapy application. This novel pharmaceutical strategy, often referred to as "vascular targeting", promises to facilitate the discovery and development of selective biopharmaceuticals for the management of angiogenesis-related diseases. This article reviews novel biomedical applications based on vascular targeting strategies, as well as methodologies which have been used for the discovery of vascular markers of pathology.
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http://dx.doi.org/10.1016/j.jprot.2010.05.017DOI Listing
October 2010

Chemical proteomic and bioinformatic strategies for the identification and quantification of vascular antigens in cancer.

J Proteomics 2010 Sep 9;73(10):1954-73. Epub 2010 Jun 9.

Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland.

One avenue towards the development of more selective anti-cancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. In this context, the targeted delivery of therapeutic agents to newly-formed blood vessels ("vascular targeting") is particularly attractive, because of the dependence of tumors on new blood vessels to sustain growth and invasion, and because of the accessibility of neo-vascular structures for therapeutic agents injected intravenously. Ligand-based vascular targeting strategies crucially rely on good-quality vascular tumor markers. Here we describe a number of established technologies for the enrichment of accessible vascular proteins based on the isolation of glycoproteins, the in vivo coating of accessible cell surfaces with colloidal silica and the in vivo perfusion with reactive ester derivatives of biotin. Label-free as well as isotopic labeling based strategies for the subsequent MS-based protein quantification are outlined. Finally, bioinformatic workflows for protein quantification are depicted aiming at assisting in the evaluation of appropriate strategies for individual projects. This review gives an overview of current chemical proteomic strategies for the enrichment and quantification of the accessible vascular proteome and helps in selecting bioinformatic strategies for data analysis and validation.
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http://dx.doi.org/10.1016/j.jprot.2010.05.018DOI Listing
September 2010

DeepQuanTR: MALDI-MS-based label-free quantification of proteins in complex biological samples.

Proteomics 2010 Jul;10(14):2631-43

Institute of Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.

The quantification of changes in protein abundance in complex biological specimens is essential for proteomic studies in basic and applied research. Here we report on the development and validation of the DeepQuanTR software for identification and quantification of differentially expressed proteins using LC-MALDI-MS. Following enzymatic digestion, HPLC peptide separation and normalization of MALDI-MS signal intensities to the ones of internal standards, the software extracts peptide features, adjusts differences in HPLC retention times and performs a relative quantification of features. The annotation of multiple peptides to the corresponding parent protein allows the definition of a Protein Quant Value, which is related to protein abundance and which allows inter-sample comparisons. The performance of DeepQuanTR was evaluated by analyzing 24 samples deriving from human serum spiked with different amounts of four proteins and eight complex samples of vascular proteins, derived from surgically resected human kidneys with cancer following ex vivo perfusion with a reactive ester biotin derivative. The identification and experimental validation of proteins, which were differentially regulated in cancerous lesions as compared with normal kidney, was used to demonstrate the power of DeepQuanTR. This software, which can easily be used with established proteomic methodologies, facilitates the relative quantification of proteins derived from a wide variety of different samples.
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http://dx.doi.org/10.1002/pmic.200900634DOI Listing
July 2010

Tumour-targeting properties of antibodies specific to MMP-1A, MMP-2 and MMP-3.

Eur J Nucl Med Mol Imaging 2010 Aug 20;37(8):1559-65. Epub 2010 Apr 20.

Department of Chemistry and Applied Biosciences, ETH Zürich, Institute of Pharmaceutical Sciences, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland.

Purpose: Matrix metalloproteinases (MMPs), a group of more than 20 zinc-containing endopeptidases, are upregulated in many diseases, but several attempts to use radiolabelled MMP inhibitors for imaging tumours have proved unsuccessful in mouse models, possibly due to the limited specificity of these agents or their unfavourable pharmacokinetic profiles. In principle, radiolabelled monoclonal antibodies could be considered for the selective targeting and imaging of individual MMPs.

Methods: We cloned, produced and characterized high-affinity monoclonal antibodies specific to murine MMP-1A, MMP-2 and MMP-3 in SIP (small immunoprotein) miniantibody format using biochemical and immunochemical methods. We also performed comparative biodistribution analysis of their tumour-targeting properties at three time points (3 h, 24 h, 48 h) in mice bearing subcutaneous F9 tumours using radioiodinated protein preparations. The clinical stage L19 antibody, specific to the alternatively spliced EDB domain of fibronectin, was used as reference tumour-targeting agent for in vivo studies.

Results: All anti-MMP antibodies and SIP(L19) strongly stained sections of F9 tumours when assessed by immunofluorescence methods. In biodistribution experiments, SIP(SP3), specific to MMP-3, selectively accumulated at the tumour site 24 and 48 h after intravenous injection, but was rapidly cleared from other organs. By contrast, SIP(SP1) and SIP(SP2), specific to MMP-1A and MMP-2, showed no preferential accumulation at the tumour site.

Conclusion: Antibodies specific to MMP-3 may serve as vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to sites of disease.
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http://dx.doi.org/10.1007/s00259-010-1446-9DOI Listing
August 2010

A proteomic approach for the identification of vascular markers of liver metastasis.

Cancer Res 2010 Jan 8;70(1):309-18. Epub 2009 Dec 8.

Department of Chemistry and Applied Biosciences, ETH Zürich, Zurich, Switzerland.

Vascular proteins expressed at liver metastasis sites could serve as prognostic markers or as targets for pharmacodelivery applications. We employed a proteomic approach to define such proteins in three syngeneic mouse models of liver metastasis. Vascular structures were biotinylated in vivo by a terminal perfusion technique, followed by mass spectrometric analysis of accessible biotinylated proteins. In this manner, we identified 12 proteins for which expression was selectively associated with liver metastasis, confirming this association by tissue immunofluorescence or in vivo localization with radiolabeled antibodies. In summary, our findings identify vascular proteins that may have prognostic or drug-targeting use in addressing liver metastases, a common issue in many advanced cancers.
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http://dx.doi.org/10.1158/0008-5472.CAN-09-2939DOI Listing
January 2010

In vivo biotinylation of the vasculature in B-cell lymphoma identifies BST-2 as a target for antibody-based therapy.

Blood 2010 Jan 10;115(3):736-44. Epub 2009 Nov 10.

Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

The discovery of accessible markers of lymphoma may facilitate the development of antibody-based therapeutic strategies. Here, we describe the results of a chemical proteomic study, based on the in vivo biotinylation of vascular proteins in lymphoma-bearing mice followed by mass spectrometric and bioinformatic analysis, to discover proteins expressed at the tissue-blood border of disseminated B-cell lymphoma. From a list of 58 proteins, which were more than 10-fold up-regulated in nodal and extranodal lymphoma lesions compared with their levels in the corresponding normal host organs, we validated BST-2 as a novel vascular marker of B-cell lymphoma, using immunochemical techniques and in vivo biodistribution studies. Furthermore, targeting BST-2 with 2 independent monoclonal antibodies delayed lymphoma growth in a syngeneic mouse model of the disease. The results of this study delineate a strategy for the treatment of systemic B-cell lymphoma in humans and suggest that anti-BST-2 antibodies may facilitate pharmacodelivery approaches that target the tumor-stroma interface.
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http://dx.doi.org/10.1182/blood-2009-08-239004DOI Listing
January 2010

Comparative analysis of the membrane proteome of closely related metastatic and nonmetastatic tumor cells.

Cancer Res 2009 Jul 2;69(13):5406-14. Epub 2009 Jun 2.

Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

The identification of proteins that are preferentially expressed on the membrane of metastatic tumor cells is of fundamental importance in cancer research. Here, we report the systematic comparison of the membrane proteome of two closely related murine teratocarcinoma cell lines (F9B9 and F9DR), of which only one (F9DR) is capable of forming liver metastases in vivo. The proteomic methodology used in this study featured the surface protein biotinylation on tumor cells followed by protein purification on streptavidin resin and relative quantification of corresponding tryptic peptides by mass spectrometric procedures. The study allowed the identification of 998 proteins and the determination of their relative abundance. Proteins previously known to be associated with metastatic spread were found to be either up-regulated (e.g., synaptojanin-2) or down-regulated (e.g., Ceacam1) in F9DR cells. A dramatic increase in abundance at the cell membrane was observed for a broad variety of proteins (e.g., high-mobility group protein B1), which were mainly thought to reside in intracellular compartments, a finding that was confirmed using confocal laser scanning microscopy and immunochemical analysis of cell cultures. Furthermore, we showed by microautoradiographic analysis that certain target proteins can readily be reached by intravenously administered radiolabeled antibodies. Finally, we showed that the most promising antigens for antibody-based pharmacodelivery approaches are strongly and selectively expressed on the surface of tumor cells in three different syngeneic mouse models of liver metastases. Taken together, our results indicate that the expression of intracellular proteins on the membrane of metastatic cells is a feature much more common than previously expected.
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http://dx.doi.org/10.1158/0008-5472.CAN-08-0999DOI Listing
July 2009

Improved protein sequence coverage by on resin deglycosylation and cysteine modification for biomarker discovery.

Proteomics 2009 Feb;9(3):783-7

Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

Membrane proteins and secreted factors (soluble proteins or extracellular matrix components) are the targets of most monoclonal antibodies, which are currently in clinical development. These proteins are frequently post-translationally modified, e.g. by the formation of disulfide bonds or by glycosylation, which complicates their identification using proteomics technologies. Here, we describe a novel methodology for the on resin deglycosylation and cysteine modification of proteins after in vitro, in vivo or ex vivo biotinylation. Biotinylated proteins are captured on streptavidin resin and all subsequent modifications, as well as the proteolytic digestion, which yields peptides for MS analysis, are performed on resin. Using biotinylated bovine fetuin-A as a test protein, an improvement in sequence coverage from 7.9 to 58.7% could be shown, including the identification of all three glycosylation sites. Furthermore, a complex mixture derived from the ex vivo biotinylation of vascular structures in human kidney with cancer obtained by perfusion after surgical resection revealed almost a doubling of sequence coverage for all checked proteins when analyzed by LC-MALDI TOF/TOF.
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http://dx.doi.org/10.1002/pmic.200800466DOI Listing
February 2009

Identification of new accessible tumor antigens in human colon cancer by ex vivo protein biotinylation and comparative mass spectrometry analysis.

Int J Cancer 2008 Dec;123(12):2856-64

Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

One of the most promising new strategies for the development of efficacious cancer therapies relies on the targeted delivery of biopharmaceutical to the tumor environment by the use of selective and specific antibodies. The identification of accessible perivascular proteins selectively overexpressed in cancer tissue may facilitate the development of antibody-based biopharmaceutical administration. This approach is potentially highly selective and specific, combining the presence of tumor biomarkers readily accessible from the blood vessels and the high rate of angiogenesis characteristic of cancer tissues. We performed ex vivo perfusions of surgically resected human colon cancer using a reactive ester derivative of biotin, thus achieving a selective covalent modification of accessible proteins in vascular structures and stroma. After extraction and purification, biotinylated proteins were digested and the resulting peptides submitted to a comparative mass spectrometry-based proteomic analysis, revealing quantitative differences between normal and cancer colon. Sixty-seven of the total 367 proteins identified were found to be preferentially expressed at the tumor site. We generated human monoclonal antibodies against 2 potential tumor targets, NGAL and GW112, and we proved their selective expression in cancer colon and not or barely in healthy tissues. This article presents the first proteomic analysis of human colorectal cancer structures readily accessible from the tumor vasculature, revealing the overexpression of novel tumor antigens which may serve as selective targets for antibody-based imaging and therapeutic biomolecular strategies.
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http://dx.doi.org/10.1002/ijc.23861DOI Listing
December 2008

Identification of the surface-accessible, lineage-specific vascular proteome by two-dimensional peptide mapping.

FASEB J 2008 Jun 7;22(6):1933-44. Epub 2008 Jan 7.

Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology, ETH Zurich, Wolfgang-Pauli-Str. 10, HCI H303, CH-8093 Zurich, Switzerland.

The formation of blood vessels (angiogenesis) and of lymphatic vessels (lymphangiogenesis) actively contributes to cancer progression and inflammation. Thus, there has been a quest for identifying the molecular mechanisms that control lymphatic and blood vessel formation and function. Membrane and extracellular matrix proteins can serve as suitable targets for imaging and/or therapeutic targeting; however, conventional proteomic technologies often fail to identify them systematically due to insolubility in water and low abundance of membrane proteins. To circumvent this problem, we applied a gel-free proteomics methodology termed two-dimensional peptide mapping (2D-PM) to cultured blood vascular (BECs) and lymphatic (LECs) endothelial cells. 2D-PM comprises biotinylation of surface-accessible proteins, their selective enrichment, separation by HPLC, and analysis by mass spectrometry. We identified 184 proteins that were specifically or predominantly expressed by LECs and 185 proteins specifically expressed by BECs, whereas 377 additional proteins were equally detected in both cell types. For representative proteins, the differential, lineage-specific expression was confirmed by Western analyses of cultured cells and by differential immunofluorescence analyses of tissue samples. Our results identify the surface-accessible, vascular lineage-specific proteome, and they also reveal 2D-PM as a powerful technology for the large-scale screening of lineage-specific protein expression.
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http://dx.doi.org/10.1096/fj.07-100529DOI Listing
June 2008

The extra-domain A of fibronectin is a vascular marker of solid tumors and metastases.

Cancer Res 2007 Nov;67(22):10948-57

Department of Chemistry and Applied Biosciences, ETH Zurich, Zurich, Switzerland.

One of the most promising new avenues for the development of more selective and efficacious cancer therapies relies on the antibody-mediated targeted delivery of bioactive agents (e.g., cytokines) to the tumor environment. The identification of quantitative differences in the expression of accessible vascular proteins in metastatic lesions and host organs facilitate the development of antibody-based strategies, which should be highly efficient and selective, considering the fact that an over-exuberant neovasculature is a characteristic feature of aggressive cancers, and that tumor blood vessels are readily accessible for i.v. administered therapeutic agents. Metastasis is the main cause of death in cancer. The availability of metastasis-specific antigens accessible from the bloodstream will allow a selective delivery of therapeutic agents to metastatic lesions using antibodies as vehicles. Using a combination of vascular biotinylation of 129Sv mice bearing F9 liver metastases and mass spectrometry, we have identified 435 accessible proteins in metastasis and host organ specimens, of which 117 were exclusively detected in metastases. In particular, we found that the alternatively spliced extra-domain A (EDA) of fibronectin is strongly expressed in the neovasculature of liver metastases, while being undetectable in most normal organs. A human antibody to EDA was used to show EDA expression in the neovasculature of metastases and primary tumors of human cancer patients and to target mouse liver metastases and subcutaneous tumors in vivo. Human antibody fragments specific to the EDA domain of fibronectin promise to serve as general vehicles for the efficient and selective delivery of imaging agents or therapeutic molecules to metastatic sites.
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http://dx.doi.org/10.1158/0008-5472.CAN-07-1436DOI Listing
November 2007

In vivo protein biotinylation and sample preparation for the proteomic identification of organ- and disease-specific antigens accessible from the vasculature.

Nat Protoc 2006 ;1(1):192-9

Institute of Pharmaceutical Sciences, ETH Zurich, 8093 Zurich, Switzerland.

Targeted delivery of bioactive molecules to diseased organs or tissues by means of binding molecules specific to markers of diseases represents a promising area of pharmaceutical intervention. The availability of markers of pathology, ideally accessible from the vasculature, is crucial for such strategies. To this aim, here we present a protocol based on terminal perfusion of mice with a reactive ester derivate of biotin that enables the covalent modification of proteins readily accessible from the bloodstream. Biotinylated proteins from total organ or tissue extracts are (i) purified on streptavidin resin in the presence of strong detergents, (ii) digested on the resin and (iii) subjected to proteomic analysis. This technology is applicable to comparative proteomic investigations of differentially expressed, accessible proteins in numerous animal models having different physiological and pathological processes.
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http://dx.doi.org/10.1038/nprot.2006.29DOI Listing
June 2007

A chemical proteomics approach for the identification of accessible antigens expressed in human kidney cancer.

Mol Cell Proteomics 2006 Nov 21;5(11):2083-91. Epub 2006 Jul 21.

Metastasis Research Laboratory, Center of Experimental Cancer Research, University of Liège, 4000 Liège, Belgium.

A promising avenue toward the development of more selective anticancer drugs consists in the targeted delivery of bioactive molecules to the tumor environment by means of binding molecules specific to tumor-associated markers. We have used a chemical proteomics approach based on the ex vivo perfusion and biotinylation of accessible structures within surgically resected human kidneys with tumor to gain information about accessible and abundant antigens that are overexpressed in human cancer. This procedure led to the selective labeling with biotin of vascular structures. Biotinylated proteins were purified on streptavidin resin and identified using mass spectrometric methodologies, revealing 637 proteins, 184 of which were only found in tumor specimens and 223 of which were only found in portions of normal kidneys. Immunohistochemical and PCR analysis confirmed that several of the putative cancer antigens identified in this study are indeed preferentially expressed in tumors. In conclusion, we have developed a methodology that allows the identification of accessible biomarkers in human tissues. The tumor-associated antigens identified in this study may be suitable targets for antibody-based anticancer therapies. The experimental approach described here should be applicable to other surgical specimens and to other pathologies as well as to the study of basic physiological and immunological processes.
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http://dx.doi.org/10.1074/mcp.M600164-MCP200DOI Listing
November 2006

Two-dimensional mass spectrometric mapping.

Curr Opin Chem Biol 2006 Feb 6;10(1):35-41. Epub 2006 Jan 6.

Institute of Pharmaceutical Sciences, Department of Chemistry and Applied Biosciences, Swiss Federal Institute of Technology, Zürich, Wolfgang-Pauli-Str. 10, ETH-Hönggerberg, 8093 Zürich, Switzerland.

The identification and relative quantification of proteins in closely related biological samples is the backbone for many investigations in systems biology and for the discovery of biomarkers. While two-dimensional gel-based methodologies are still widely used for comparative proteomic studies, the recent advent of gel-free methodologies may allow the analysis of a larger number of samples in an automated fashion. Most of the technologies presented in this review require a chemical modification of proteins before analysis, and rely on the relative intensities of mass spectrometry signals for protein quantification. In particular, two-dimensional mass spectrometric mapping methodologies provide a visual representation of mass spectrometric data, thus facilitating the identification of differences in relative protein abundance.
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http://dx.doi.org/10.1016/j.cbpa.2005.12.017DOI Listing
February 2006

A comparison of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques for 2-D peptide mapping of membrane proteins.

Proteomics 2005 Aug;5(12):3035-9

Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology-Zurich, Wolfgang-Pauli-Str. 10, CH-8093 Zurich, Switzerland.

2-D peptide mapping is a novel technique for the relative quantification of membrane proteins (Scheurer S. et al., Proteomics 2005, in press). Using closely related metastatic and nonmetastatic teratocarcinoma cell lines as a model system, we have performed a comparative analysis of different biotinylation reagents, tryptic digestion procedures, and mass spectrometric techniques, with the aim to increase the number of proteins identified by 2-D peptide mapping. Our experience indicates that the LC-MALDI TOF/TOF technique is superior to LC-ESI MS/MS in terms of the number of proteins identified and confidence in protein identification. Furthermore, the best results were obtained by tryptic digestion of proteins eluted from a streptavidin column using a cleavable biotin derivative.
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http://dx.doi.org/10.1002/pmic.200402069DOI Listing
August 2005

Identification and relative quantification of membrane proteins by surface biotinylation and two-dimensional peptide mapping.

Proteomics 2005 Jul;5(11):2718-28

Institute of Pharmaceutical Sciences, Swiss Federal Institute of Technology Zurich, ETH-Hönggerberg, Zurich, Switzerland.

Membrane proteins play a central role in biological processes, but their separation and quantification using two-dimensional gel electrophoresis is often limited by their poor solubility and relatively low abundance. We now present a method for the simultaneous recovery, separation, identification, and relative quantification of membrane proteins, following their selective covalent modification with a cleavable biotin derivative. After cell lysis, biotinylated proteins are purified on streptavidin-coated resin and proteolytically digested. The resulting peptides are analyzed by high-pressure liquid chromatography and mass spectrometry, thus yielding a two-dimensional peptide map. Matrix assisted laser desorption/ionization-time of flight signal intensity of peptides, in the presence of internal standards, is used to quantify the relative abundance of membrane proteins from cells treated in different experimental conditions. As experimental examples, we present (i) an analysis of a BSA-spiked human embryonic kidney membrane protein extract, and (ii) an analysis of membrane proteins of human umbilical vein endothelial cells cultured in normoxic and hypoxic conditions. This last study allowed the recovery of the vascular endothelial-cadherin/actin/catenin complex, revealing an increased accumulation of beta-catenin at 2% O(2) concentration.
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http://dx.doi.org/10.1002/pmic.200401163DOI Listing
July 2005