Antibody Epitope and Affinity Determination of the Myocardial Infarction Marker Myoglobin by SPR-Biosensor Mass Spectrometry

Abstract

Myoglobin (MG) is a biomarker for heart muscle injury, making it a potential target protein for early detection of myocardial infarction. Elevated myoglobin levels alone have low specificity for acute myocardial infarction (AMI) but in combination with cardiac troponin T have been considered highly efficient diagnostic biomarkers. Myoglobin is a monomeric heme protein with a molecular weight of 17 kDa that is found in skeletal and cardiac tissue as an intracellular storage unit of oxygen. MG consists of eight αhelices connected by loops and a heme group responsible for oxygen-binding. Monoclonal antibodies are widely used analytical tools in biomedical research and have been employed for immunoanalytical detection of MG. However, the epitope(s) recognized by MG antibodies have been hitherto unknown. Precise molecular identification of the epitope(s) recognized by antibodies is of key importance for the development of MG as a diagnostic biomarker. The epitope of a monoclonal MG antibody was identified by proteolytic epitope extraction mass spectrometry in combination with surface plasmon resonance (SPR) biosensor analysis. The MG antibody was immobilized both on an affinity microcolumn and a gold SPR chip. The SPR kinetic analysis provided an affinity-binding constant K D of 270 nM for MG. Binding of a tryptic peptide mixture followed by elution of the epitope from the SPR-MS affinity interface by mild acidification provided a single-epitope peptide located at the C-terminus [146−153] [YKELGFQG] of MG. The specificity and affinity of the epitope were ascertained by synthesis and affinity-mass spectrometric characterization of the epitope peptide.

Identification and Affinity Determination of Protein-Antibody and Protein-Aptamer Epitopes by Biosensor-Mass Spectrometry Combination

“…The proteolytic epitope extraction-MS approach has been successfully applied to a number of epitope identifications of monoclonal, as well as polyclonal antibodies [ 6 , 7 , 38 ]. Two antibody immobilization methods were explored: (i), random immobilization on activated surface-assembled monolayer (SAM) chips; and (ii), oriented antibody immobilization using the F c binding of protein G [ 38 ] ( Figure S3 ).…”

“…The proteolytic epitope extraction-MS approach has been successfully applied to a number of epitope identifications of monoclonal, as well as polyclonal antibodies [ 6 , 7 , 38 ]. Two antibody immobilization methods were explored: (i), random immobilization on activated surface-assembled monolayer (SAM) chips; and (ii), oriented antibody immobilization using the F c binding of protein G [ 38 ] ( Figure S3 ). The comparison of both methods clearly showed that immobilization using covalent crosslinking at the Fc region to protein G was superior to standard immobilization with regard to long-term antibody stabilities; reproducibility; antibody consumption for immobilization; and non-specific binding to the surface [ 32 , 38 ] ( Figure S3 ).…”

“…In bioanalytical applications, immobilization using protein G from preactivated Sepharose bead columns, and from SPR chips was highly suitable to provide epitope and affinity determinations with high sensitivity [ 38 ] ( Figure 2 ; Table 1 ). Using EDC/NHS activation on the SPR chip, protein G coupling can be performed in volatile aqueous buffer (ammonium-acetate or -bicarbonate) and provides stable crosslinking of the antibody to the Fc region.…”

“…The oxygen binding Myoglobin (MG) is one of the major proteins expressed in skeletal and cardiac muscle as a single cytoplasmic heme protein of 153 amino acids [ 38 , 39 , 40 , 41 ] ( Figure S4 ). Due to its functional similarity to hemoglobin, MG reversibly binds oxygen and facilitates oxygen transport from red blood cells to mitochondria, with a single O 2 -binding site and a hyperbolic saturation curve.…”

“…Due to its functional similarity to hemoglobin, MG reversibly binds oxygen and facilitates oxygen transport from red blood cells to mitochondria, with a single O 2 -binding site and a hyperbolic saturation curve. The three-dimensional structure of MG has been described in detail [ 38 ], with 75% of the main chain being folded in an α-helical conformation ( Figure 3 ). MG has recently gained increased interest due to its potential role as a biomarker for acute myocardial infarct (AMI), in comparison to corresponding alternative biomarkers [ 41 ].…”