Final Report Summary - GLYCOPROTEOMICS (Quantitative Glycomics and Glycoproteomics for Biomarker Discovery)
Understanding protein glycosylation in health and disease
Cell surface but also body fluid proteins are extensively decorated with specific sugar moieties, so called glycans, who together with proteins and lipids form the glycocalyx that is the first area of contact between cells, but also for numerous pathogens. These glycans build the basis for a universal language (glycome) that cells and proteins use to communicate and interact. In many diseases specific cells start talking different languages, resulting in different proteins and glycans being expressed on the cells surface. The glycomics & glycoproteomics tools developed in the course of the Marie Curie funded project "GLYCOPROTEOMICS" enable translation of the languages cells use to talk to each other in health and disease. It also is the basis to identify novel disease markers with the objective to identify diseases such as cancer at an earlier stage and thus initiate more effective countermeasures (Almeida and Kolarich 2016).
A Description of the Work Performed since the Beginning of the Project
In the project "GLYCOPROTEOMICS" we implemented a multidisciplinary approach combining analytical biochemistry with synthetic chemistry, glycobiology and bioinformatics to open novel opportunities for clinical glycomics and glycoproteomics.
We successfully developed a highly sensitive glycomics approach based on a technology that combines nano scale liquid chromatography with mass spectrometric detection that allows simultaneous acquisition of four different characterisation glycomics identifiers suitable to characterise N- and O-glycans. This 4-dimensional approach uses retention time, precursor mass, MS2 fragment fingerprint spectra and intensity as parameters that are all simultaneously determined within a single experiment. That allowed us to establish a glycan fragment fingerprint spectra database currently containing >350 different mostly human N- and O-glycans for automated qualitative LC-MS/MS spectra evaluation. This system has already been successfully applied in more than 14 interdisciplinary publications in the last 4 years with 8 additional ones to follow in the immediate future.
In the field of glycoproteomics we initiated and extended the expertise to produce synthetic N-glycopeptides and used these valuable compounds to systematically investigate instrumental parameters to improve glycoproteomics research in general, but also developed entirely novel methods for site specific glycan structure analysis using ion mobility mass spectrometry (Hinneburg, Hofmann et al. 2016). In cooperation with industry we also develop novel methodologies for the glycosylation site monitoring of chemically glycosylated proteins used for vaccination in humans have been developed and published (Moginger, Resemann et al. 2016).
As part of our bioinformatics initiatives we developed automated data handling workflows that are directly incorporated in the glycomics and glycoproteomics workflows. Using a combination of mass spectrometry vendor specific software tools and own developments based on scripts and programs written in "R" we have automated data analysis of PGC-LC ESI MS/MS data. We are currently working on further on glycosylation specific unsupervised clustering software tools to visualise the complex datasets in the best way possible.
A Description of the Main Results Achieved so Far
Major achievements have been made in four core research areas directly associated to glycomics and glycoproteomics methods:
I) Novel techniques for Glycomics & Glycoproteomics
Glycomics: My group has developed highly sensitive and selective glycomics approaches allowing in-depth glycome sequencing from minimal sample amounts, including complex clinical tissue specimens and body fluids (Everest-Dass, Jin et al. 2012, Jensen, Karlsson et al. 2012, Wongtrakul-Kish, Kolarich et al. 2013, Marco-Ramell, Miller et al. 2014, Kolarich, Windwarder et al. 2015, Carvalho, Catarino et al. 2016), cell culture material (Jensen, Karlsson et al. 2012, Ito, Kaji et al. 2015, Mereiter, Magalhaes et al. 2015, Carvalho, Catarino et al. 2016), or purified proteins (Jensen, Karlsson et al. 2012, Leymarie, Griffin et al. 2013, Carvalho, Catarino et al. 2016, Lohse, Meyer et al. 2016). We have also recently developed a method to extract and analyse N- and O-glycans from Formalin/Paraffin embedded (FFPE) histopathological tissue slides allowing us to sequence N- and O-glycans from as low as 1000 cells isolated by laser capture micro-dissection (Hinneburg et al, manuscript in preparation). We established a spectral library currently containing +350 assigned N- and O-glycan structures used in software assisted glycan identification.
Glycoproteomics: We developed hitherto unprecedented, novel technologies to elucidate specific glyco-epitope features directly from glycopeptides using ion-mobility mass spectrometry (Hinneburg, Hofmann et al. 2016), providing a unique opportunity for differentiating sialic acid linkages directly on individual proteins and glycosylation sites. Our synthetic N-glycopeptide library was also used to significantly improve mass spectrometry based glycopeptide sequencing approaches (Hinneburg, Stavenhagen et al. 2016). Novel protein tailored approaches for in-depth characterisation of chemically glycosylated vaccine candidates were developed and are currently being translated into an industrial setting (Moginger, Resemann et al. 2016). We were also the first to systematically investigate glycopeptide ionisation for label free quantitative glycoproteomics (Stavenhagen, Hinneburg et al. 2013).
II) Application of these to investigate disease & functional glycoconjugate aspects
The glycomics methods are currently applied in various clinical projects investigating protein glycosylation from a diverse disease spectrum such as hepatocellular carcinoma, basal cell carcinoma, inflammatory bowel diseases, colon and gastric cancer (Mereiter, Magalhaes et al. 2015, Carvalho, Catarino et al. 2016). This allowed us to identify promising colon cancer glycan signatures and identify hitherto not described human N-glycan sequences on an important gastrointestinal tumour marker. We are currently evaluating the potential of this glycan signature to improve colon cancer diagnosis. Our methods allowed us to map the majority of the human Immunoglobulome (Hinneburg, Stavenhagen et al. 2016) within a simple workflow, but are also heavily applied in various past (Kolarich, Jensen et al. 2012, Klatt, Rohe et al. 2013, Janesch, Schirmeister et al. 2015) and ongoing projects.
III) In vitro and in vivo functional studies of individual glycoproteins and glyco-epitopes
In cooperation with other groups we investigated the role glycosylation plays for important tumour associated proteins (Carvalho, Catarino et al. 2016) and novel anti-tumour agents (Lohse, Meyer et al. 2016). Assays to elucidate specific glycan structure-function relationships have just recently been established in my group.
IV) Synthetic N- glycopeptide libraries for analytical and functional glycoproteomics
As synthetic N-glycopeptides are not commercially available, we established and improved the production of Fmoc protected, complex N-glycosylated Asparagine building blocks for solid phase glycopeptide synthesis allowing us to obtain >500 mg of the fully protected building block within two weeks. We applied these valuable compounds already in a variety of development projects (Stavenhagen, Hinneburg et al. 2013, Hinneburg, Hofmann et al. 2016, Hinneburg, Stavenhagen et al. 2016) and with industrial and academic partners and this research area is currently further expanded.
The combination of the different proficiencies in particular in glycome and glycoproteome sequencing has made us a world-leading group to successfully address glycomics and glycoproteomics questions in health and disease.
Potential Impact and Use and Socio-Economic Impact of the Project
The achievements made in the project Glycoproteomics currently provide novel opportunities in basic and applied clinical research. In collaboration with clinical partners and other glycobiologists we are currently systematically investigating disease specific glycosylation signatures using the methods developed in the course of this project. Our mid-term aims are to provide novel insights into diseases such as cancer and chronic inflammatory diseases which present a major health burden for patients and society likewise. These insights will provide novel therapeutic targets, support precision medicine and will lead to detection of novel, better biomarkers that allow identification of the onset of specific diseases to enable medical professionals to set countermeasures at an earlier stage (Almeida and Kolarich 2016). Thus we expect that on long term the achievements made in the course of this project will make a major contribution to current societal and economical challenges in particular in the health sector, but also provide novel opportunities in the industrial sector as emphasised by the numerous industrial collaborations that this project fostered.
Cell surface but also body fluid proteins are extensively decorated with specific sugar moieties, so called glycans, who together with proteins and lipids form the glycocalyx that is the first area of contact between cells, but also for numerous pathogens. These glycans build the basis for a universal language (glycome) that cells and proteins use to communicate and interact. In many diseases specific cells start talking different languages, resulting in different proteins and glycans being expressed on the cells surface. The glycomics & glycoproteomics tools developed in the course of the Marie Curie funded project "GLYCOPROTEOMICS" enable translation of the languages cells use to talk to each other in health and disease. It also is the basis to identify novel disease markers with the objective to identify diseases such as cancer at an earlier stage and thus initiate more effective countermeasures (Almeida and Kolarich 2016).
A Description of the Work Performed since the Beginning of the Project
In the project "GLYCOPROTEOMICS" we implemented a multidisciplinary approach combining analytical biochemistry with synthetic chemistry, glycobiology and bioinformatics to open novel opportunities for clinical glycomics and glycoproteomics.
We successfully developed a highly sensitive glycomics approach based on a technology that combines nano scale liquid chromatography with mass spectrometric detection that allows simultaneous acquisition of four different characterisation glycomics identifiers suitable to characterise N- and O-glycans. This 4-dimensional approach uses retention time, precursor mass, MS2 fragment fingerprint spectra and intensity as parameters that are all simultaneously determined within a single experiment. That allowed us to establish a glycan fragment fingerprint spectra database currently containing >350 different mostly human N- and O-glycans for automated qualitative LC-MS/MS spectra evaluation. This system has already been successfully applied in more than 14 interdisciplinary publications in the last 4 years with 8 additional ones to follow in the immediate future.
In the field of glycoproteomics we initiated and extended the expertise to produce synthetic N-glycopeptides and used these valuable compounds to systematically investigate instrumental parameters to improve glycoproteomics research in general, but also developed entirely novel methods for site specific glycan structure analysis using ion mobility mass spectrometry (Hinneburg, Hofmann et al. 2016). In cooperation with industry we also develop novel methodologies for the glycosylation site monitoring of chemically glycosylated proteins used for vaccination in humans have been developed and published (Moginger, Resemann et al. 2016).
As part of our bioinformatics initiatives we developed automated data handling workflows that are directly incorporated in the glycomics and glycoproteomics workflows. Using a combination of mass spectrometry vendor specific software tools and own developments based on scripts and programs written in "R" we have automated data analysis of PGC-LC ESI MS/MS data. We are currently working on further on glycosylation specific unsupervised clustering software tools to visualise the complex datasets in the best way possible.
A Description of the Main Results Achieved so Far
Major achievements have been made in four core research areas directly associated to glycomics and glycoproteomics methods:
I) Novel techniques for Glycomics & Glycoproteomics
Glycomics: My group has developed highly sensitive and selective glycomics approaches allowing in-depth glycome sequencing from minimal sample amounts, including complex clinical tissue specimens and body fluids (Everest-Dass, Jin et al. 2012, Jensen, Karlsson et al. 2012, Wongtrakul-Kish, Kolarich et al. 2013, Marco-Ramell, Miller et al. 2014, Kolarich, Windwarder et al. 2015, Carvalho, Catarino et al. 2016), cell culture material (Jensen, Karlsson et al. 2012, Ito, Kaji et al. 2015, Mereiter, Magalhaes et al. 2015, Carvalho, Catarino et al. 2016), or purified proteins (Jensen, Karlsson et al. 2012, Leymarie, Griffin et al. 2013, Carvalho, Catarino et al. 2016, Lohse, Meyer et al. 2016). We have also recently developed a method to extract and analyse N- and O-glycans from Formalin/Paraffin embedded (FFPE) histopathological tissue slides allowing us to sequence N- and O-glycans from as low as 1000 cells isolated by laser capture micro-dissection (Hinneburg et al, manuscript in preparation). We established a spectral library currently containing +350 assigned N- and O-glycan structures used in software assisted glycan identification.
Glycoproteomics: We developed hitherto unprecedented, novel technologies to elucidate specific glyco-epitope features directly from glycopeptides using ion-mobility mass spectrometry (Hinneburg, Hofmann et al. 2016), providing a unique opportunity for differentiating sialic acid linkages directly on individual proteins and glycosylation sites. Our synthetic N-glycopeptide library was also used to significantly improve mass spectrometry based glycopeptide sequencing approaches (Hinneburg, Stavenhagen et al. 2016). Novel protein tailored approaches for in-depth characterisation of chemically glycosylated vaccine candidates were developed and are currently being translated into an industrial setting (Moginger, Resemann et al. 2016). We were also the first to systematically investigate glycopeptide ionisation for label free quantitative glycoproteomics (Stavenhagen, Hinneburg et al. 2013).
II) Application of these to investigate disease & functional glycoconjugate aspects
The glycomics methods are currently applied in various clinical projects investigating protein glycosylation from a diverse disease spectrum such as hepatocellular carcinoma, basal cell carcinoma, inflammatory bowel diseases, colon and gastric cancer (Mereiter, Magalhaes et al. 2015, Carvalho, Catarino et al. 2016). This allowed us to identify promising colon cancer glycan signatures and identify hitherto not described human N-glycan sequences on an important gastrointestinal tumour marker. We are currently evaluating the potential of this glycan signature to improve colon cancer diagnosis. Our methods allowed us to map the majority of the human Immunoglobulome (Hinneburg, Stavenhagen et al. 2016) within a simple workflow, but are also heavily applied in various past (Kolarich, Jensen et al. 2012, Klatt, Rohe et al. 2013, Janesch, Schirmeister et al. 2015) and ongoing projects.
III) In vitro and in vivo functional studies of individual glycoproteins and glyco-epitopes
In cooperation with other groups we investigated the role glycosylation plays for important tumour associated proteins (Carvalho, Catarino et al. 2016) and novel anti-tumour agents (Lohse, Meyer et al. 2016). Assays to elucidate specific glycan structure-function relationships have just recently been established in my group.
IV) Synthetic N- glycopeptide libraries for analytical and functional glycoproteomics
As synthetic N-glycopeptides are not commercially available, we established and improved the production of Fmoc protected, complex N-glycosylated Asparagine building blocks for solid phase glycopeptide synthesis allowing us to obtain >500 mg of the fully protected building block within two weeks. We applied these valuable compounds already in a variety of development projects (Stavenhagen, Hinneburg et al. 2013, Hinneburg, Hofmann et al. 2016, Hinneburg, Stavenhagen et al. 2016) and with industrial and academic partners and this research area is currently further expanded.
The combination of the different proficiencies in particular in glycome and glycoproteome sequencing has made us a world-leading group to successfully address glycomics and glycoproteomics questions in health and disease.
Potential Impact and Use and Socio-Economic Impact of the Project
The achievements made in the project Glycoproteomics currently provide novel opportunities in basic and applied clinical research. In collaboration with clinical partners and other glycobiologists we are currently systematically investigating disease specific glycosylation signatures using the methods developed in the course of this project. Our mid-term aims are to provide novel insights into diseases such as cancer and chronic inflammatory diseases which present a major health burden for patients and society likewise. These insights will provide novel therapeutic targets, support precision medicine and will lead to detection of novel, better biomarkers that allow identification of the onset of specific diseases to enable medical professionals to set countermeasures at an earlier stage (Almeida and Kolarich 2016). Thus we expect that on long term the achievements made in the course of this project will make a major contribution to current societal and economical challenges in particular in the health sector, but also provide novel opportunities in the industrial sector as emphasised by the numerous industrial collaborations that this project fostered.