14.3.2013 1 New Developments in Capillary Electrophoresis with focus on Bioanalysis Lecture 4 Christian Nilsson Lecture Today • Focus on analysis of protein isoforms • Special focus on carbohydrate analysis • Applications in – Pharmaceutical industry – Diagnostics CE of proteins • Proteins are modified after formation – Post translational modifications (PTMs) • The proteins are modified during there lifetime • A protein often consist of many closely related isoforms – Heterogeneous mixture due to differential PTMs CE of proteins • 2 dimensional SDS-PAGE in combination with mass spectrometry is a common tool in proteomics • Separation techniques with high peak capacity is needed due to the huge amount of proteins. – High amount of proteins – Protein isoforms increase the complexity 2D Electrophoresis Usually isoelectric point (IEF) and molecular weight (SDS-PAGE) is the basis of separation Robots can be used to isolate protein spots for mass spectrometry analysis CE of proteins • One dimensional separation not enough for more complicated separation tasks. • Two ortogonal techniques increase the peak capacity. 14.3.2013 2 CE of proteins • High resolution mass spectrometry important for identification of proteins. – High resolution MS (for example Orbitrap) – Identification of complex molecules (for example intact glycoproteins) • MS can be combined with on capillary detection for quantification Post translational modifications • Not coded in the DNA. • Instead the state of the cell determines how the proteins are modified – Presence of enzymes – Presence of reactants. Post translational modification For example: • Phosphorylation • Glycosylation • Ubiquitination Protein phosphorylation • Regulation of kinases and phosphatases. – Regulate phosphorylation – Important for many signalling pathways • Phosphorylation can activate or deactivate enzymes • Important for regulation of the metabolism Protein phosphorylation Reversible step Ubiquitination • Important for degragration of proteins • Important for sorting in the endosomes 14.3.2013 3 Importance of PTMs Sorting of proteins in the endosomes Exosomes • 40-100 nm vesicles • Exosomes are released when multivesicular bodies fuse with the plasma membrane • Contain – Proteins – miRNA – mRNA Exosomes • The composition is dependent of – Type of Cell of origin – State of cell of origin • The content of the exosomes can be transported between cells – The content is active in the recieving cell Glycosylation of proteins • Significant amount of the mass of a glycoprotein • Most abundant form of PTM • Important for cell-cell communication Protein glycosylation • Alternation in glycosylation can change the biological activity of a glycoprotein • Glycosylation of insulin – might be important in diabetes Glycosylation Electron micrograph of a glycocalyx Thick carbohydrate coating that surround virtually all cells. Compsed of protein- and lipid-bound oligosaccharides. 14.3.2013 4 Glycosylation of proteins • N-linked – via the amines of asparagines • O-linked – via the hydroxyl group of serines and threonines – Blood groups – O-linked glycans Glycosylation of proteins Glycoproteins as recombinant drugs • Glycosylation patterns important for function • Important to control production procedure • Glycosylation profiles may vary based on the host system used for production • Host system not the same cell machinery as humans. • Analytical methods to analyze differences in glycosylation patterns Glycoproteins as recombinant drugs • Cell culture conditions may have a significant effect on glycosylation • Batch-to-batch consistency Monoclonal antibodies SDS-CGE • For recombinant monoclonal antibodies • Addition of N-linked carbohydrate chain to the heavy chain of IgG. • Reduced fragments of IgG can be analyzed to decrease complexity 14.3.2013 5 Recombinant monoclonal antibodies • Growing industry • High specificity makes antibodies excellent for human therapy • Immune system to fight diseases • Recombinant production is an alternative to production in mice Humanized mAb • The complementary regions, which are the responsible for antigen binding within the variable regions, have been transferred to human frameworks creating ‘‘humanized’’ antibodies. This is, in essence a human Ab with small segments containing mouse Ab genes. Monoclonal antibodies • Humanize monoclonal antibodies Applications of MABs • Diagnostic tests – Detect the presence of a substance – Useful for detecting a antigen in tissue section • Therapy – Specific binding to target cells or proteins – Stimulate the immune system to attack those targets. Applications of MABs • Treatment of cancer – MABs that bind to cancer cell-specific antigens and induce an immunological response against target cancer cells. • Treatment of autoimmune diseases Therapeutic proteins • During production and shelf life of therapeutic proteins, several PTMs can occur: – for example deamidation, oxidation and proteolytic cleavages 14.3.2013 6 Glycosylation relatively large in size. Imporatant to detect glycosylation at low levels. Recombinant monoclonal antibodies CE-SDS Detecting impurities Recombinant monoclonal antibodies CE-SDS CE-SDS • SDS can also be used to evaluate heterogenity, purity and manufacturing consistency • Linear or slightly branched polymers: polyacrylamide, polyethylene oxide, polyethylene glycol, dextran. • Add flexibility, water soluble, replaceable after each analysis • CE-SDS with fluorescence detection, to replace silver staining • However, less compatible with MS Separation of variants of human growth hormone Carbonhydrate analysis by CE-LIF • Glycosylation important for function of rMABs • Consistant glycosylation required by legislation • Chromophore introduced to carbonhydrate – Also add a charge Carbonhydrate analysis by CE-LIF • Procedure – Oligosaccharides on rMABs – Enzymatic removal of oligosaccharides – Derivatisation with APTS – Analyze 14.3.2013 7 Carbonhydrate analysis by CE-LIF Example of glycan analysis 9 sugar units 8 sugar units 10 sugar units CE-LIF Analysis of intact protein Recombinant human chrionic gonadotropin (rHCG) CE-MS 2D separation • CE a high-speed / high-efficiency technique • Ideal as the last dimension in a multidimension system • Reversed phase LC frequently coupled to CZE – Orthogonal in separation mechanism – Solvents in HPLC and buffers in CE reasonable compatable. • For separation of proteins and peptides • Can be used for studies of single cells • Fluorescent labeling of proteins/peptides 2D-CE Capillary isoelectric focusing • High resolution/peak capacity • Make it suitable for analysis of complex protein mixtures. • A good option as one dimension in 2D-CE 14.3.2013 8 2D separation