Epitope characterization of proteins and aptamers with mass spectrometry.

The way in which professor Michael Przybylski has combined the spirit of research with entrepreneurship has set an example for any and all scientists. He has made significant achievements in the fields of mass spectrometry, biochemistry and medicine, and has initiated important technological developments in the area of protein analysis. Between 2016 and 2023 professor Przybylski's scientific focus shifted on protein interactions with emphasis on aptamer-protein and antibody-protein analysis. This review focuses on professor Przybylski's achievements in the last few years highlighting his impact on the scientific community, on his students and colleagues.

Michael Przybylski (1948-2023) Devoted Half a Century to Mass Spectrometry.

Michael Przybylski (1948-2023) was a Polymer Chemist by training and devoted nearly his entire scientific life, almost 50 years, to mass spectrometry and its biomedical applications. After earning his PhD in Chemistry, there followed a Postdoc stay at the National Cancer Institute, Bethesda, MD, USA, and his habilitation at the University of Mainz, Germany. Soon thereafter, Michael Przybylski took the Chair for Analytical Chemistry at the University of Konstanz, Germany, where he served as Director of the Analytical Chemistry and Biopolymer Structure Analysis Laboratory. As Emeritus Michael Przybylski moved the Steinbeis Centre for Biopolymer Analytics and Biomedical Mass Spectrometry to Rüsselsheim, Germany. Michael Przybylski's research was from the beginning interdisciplinary-oriented and in many ways groundbreaking: leading to over 400 scientific papers published in internationally renowned journals and to about 25 patents. Michael Przybylski gave approximately 150 invited lectures and was awarded several scientific prizes. In recognition of his outstanding achievements and fruitful collaboration, he received the Doctorate of honor from the "Alexandru Ioan Cuza" University of Iasi, Romania. Michael Przybylski was the Director of the by him founded "Biopolymer Analytics and Biomedical Mass Spectrometry" research center until his sudden and unexpected death.

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

Analytical methods for molecular characterization of diagnostic or therapeutic targets have recently gained high interest. This review summarizes the combination of mass spectrometry and surface plasmon resonance (SPR) biosensor analysis for identification and affinity determination of protein interactions with antibodies and DNA-aptamers. The binding constant (KD) of a protein-antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip. A proteolytic peptide mixture is then applied to the chip, and following removal of unbound material by washing, the epitope(s) peptide(s) are eluted and identified by MALDI-MS. The SPR-MS combination was applied to a wide range of affinity pairs. Distinct epitope peptides were identified for the cardiac biomarker myoglobin (MG) both from monoclonal and polyclonal antibodies, and binding constants determined for equine and human MG provided molecular assessment of cross immunoreactivities. Mass spectrometric epitope identifications were obtained for linear, as well as for assembled ("conformational") antibody epitopes, e.g., for the polypeptide chemokine Interleukin-8. Immobilization using protein G substantially improved surface fixation and antibody stabilities for epitope identification and affinity determination. Moreover, epitopes were successfully determined for polyclonal antibodies from biological material, such as from patient antisera upon enzyme replacement therapy of lysosomal diseases. The SPR-MS combination was also successfully applied to identify linear and assembled epitopes for DNA-aptamer interaction complexes of the tumor diagnostic protein C-Met. In summary, the SPR-MS combination has been established as a powerful molecular tool for identification of protein interaction epitopes.

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

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 α-helices 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 KD 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.

Epitope Identification and Affinity Determination of an Inhibiting Human Antibody to Interleukin IL8 (CXCL8) by SPR- Biosensor-Mass Spectrometry Combination.

The polypeptide chemokine Interleukin-8 (IL8) plays a crucial role in inflammatory processes in humans. IL8 is involved in chronic inflammatory lung diseases, rheumatoid arthritis, and cancer. Previous studies have shown that the interaction of IL8 with its natural receptors CXCR1 and CXCR2 is critical in these diseases. Antibodies have been used to study the receptor interaction of IL8; however, the binding epitopes were hitherto unknown. Identification of the antibody epitope(s) could lead to a molecular understanding of the inhibiting mechanism and development of improved inhibitors. Here, we report the epitope identification and the affinity characterization of IL8 to a monoclonal anti-human IL8 antibody inhibiting the receptor binding by a combination of surface plasmon resonance (SPR) biosensor analysis and MALDI-mass spectrometry. SPR determination of IL8 with the immobilized antibody revealed high affinity (KD, 82.2 nM). Epitope identification of IL-8 was obtained by proteolytic epitope-extraction mass spectrometry of the peptide fragments upon high pressure trypsin digestion, using an affinity microcolumn with immobilized anti-IL-8 antibody. MALDI-MS of the affinity-bound peptide elution fraction revealed an assembled (discontinuous) epitope comprising two specific peptides, IL8 [12-20] and IL8 [55-60]. Identical epitope peptides were identified by direct MALDI-MS of the eluted epitope fraction from the immobilized anti-IL8 antibody on the SPR chip. SPR determination of the synthetic epitope peptides provided high affinities confirming their binding specificity. The previously reported finding that the anti-Il8 antibody is inhibiting the IL8-CXCR1 interaction is well consistent with the overlapping region of epitope interactions identified in the present study.

Unveiling the molecular mechanisms underpinning biorecognition of early-glycated human serum albumin and receptor for advanced glycation end products.

Serum levels of early-glycated albumin are significantly increased in patients with diabetes mellitus and may play a role in worsening inflammatory status and sustaining diabetes-related complications. To investigate possible pathological recognition involving early-glycated albumin and the receptor for advanced glycation end products (RAGE), an early-glycated human serum albumin (HSAgly), with a glycation pattern representative of the glycated HSA form abundant in diabetic patients, and the recombinant human RAGE ectodomain (VC1) were used. Biorecognition between the two interactants was investigated by combining surface plasmon resonance (SPR) analysis and affinity chromatography coupled with mass spectrometry (affinity-MS) for peptide extraction and identification. SPR analysis proved early-glycated albumin could interact with the RAGE ectodomain with a steady-state affinity constant of 6.05 ± 0.96 × 10-7 M. Such interaction was shown to be specific, as confirmed by a displacement assay with chondroitin sulfate, a known RAGE binder. Affinity-MS studies were performed to map the surface area involved in the recognition. These studies highlighted that a region surrounding Lys525 and part of subdomain IA were involved in VC1 recognition. Finally, an in silico analysis highlighted (i) a key role for glycation at Lys525 (the most commonly glycated residue in HSA in diabetic patients) through a triggering mechanism similar to that previously observed for AGEs or advanced lipoxidation end products and (ii) a stabilizing role for subdomain IA. Albeit a moderate affinity for complex formation, the high plasma levels of early-glycated albumin and high percentage of glycation at Lys525 in diabetic patients make this interaction of possible pathological relevance. Graphical abstract.

Molecular Epitope Determination of Aptamer Complexes of the Multidomain Protein C-Met by Proteolytic Affinity-Mass Spectrometry.

C-Met protein is a glycosylated receptor tyrosine kinase of the hepatocyte growth factor (HGF), composed of an α and a ß chain. Upon ligand binding, C-Met transmits intracellular signals by a unique multi-substrate docking site. C-Met can be aberrantly activated leading to tumorigenesis and other diseases, and has been recognized as a biomarker in cancer diagnosis. C-Met aptamers have been recently considered a useful tool for detection of cancer biomarkers. Herein we report a molecular interaction study of human C-Met expressed in kidney cells with two DNA aptamers of 60 and 64 bases (CLN0003 and CLN0004), obtained using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) procedure. Epitope peptides of aptamer-C-Met complexes were identified by proteolytic affinity-mass spectrometry in combination with SPR biosensor analysis (PROTEX-SPR-MS), using high-pressure proteolysis for efficient digestion. High affinities (KD , 80-510 nM) were determined for aptamer-C-Met complexes, with two-step binding suggested by kinetic analysis. A linear epitope, C-Met (381-393) was identified for CLN0004, while the CLN0003 aptamer revealed an assembled epitope comprised of two peptide sequences, C-Met (524-543) and C-Met (557-568). Structure modeling of C-Met-aptamers were consistent with the identified epitopes. Specificities and affinities were ascertained by SPR analysis of the synthetic epitope peptides. The high affinities of aptamers to C-Met, and the specific epitopes revealed render them of high interest for cellular diagnostic studies.

Epitope and affinity determination of recombinant Mycobacterium tuberculosis Ag85B antigen towards anti-Ag85 antibodies using proteolytic affinity-mass spectrometry and biosensor analysis.

Tuberculosis (TB) is the first cause of death from infectious diseases worldwide. Only a single anti-TB vaccine is currently available for clinical use, but its efficacy is not achieved with certainty. The aim of this work is to provide a basis for the rational design of a neo-glycoconjugate vaccine against TB. Structural characterization of recombinant antigenic proteins from Mycobacterium tuberculosis (MTB) Ag85B (rAg85B, variants, and semi-synthetic glycoconjugates) was initially carried out. Identification of antibody epitope analyses by proteolytic affinity-mass spectrometry and surface plasmon resonance (SPR) biosensor analyses were performed in order to qualitatively identify and quantitatively characterize interaction structures of the antigens with antibodies from different sources. A commercial monoclonal antibody and polyclonal antibodies from different sources (patients with active TB, vaccinated individuals, and a healthy control) were employed to analyze antigen-antibody interactions. These combined approaches provided the identification of different assembled epitope regions on the recombinant MTB antigens, their affinity binding constants in the interactions with specific antibodies, and revealed the importance of protection from excessive glycosylation. The identified epitope peptides should constitute a suitable basis for the design of new specific target vaccines. Graphical abstract ᅟ.

Epitope Ligand Binding Sites of Blood Group Oligosaccharides in Lectins Revealed by Pressure-Assisted Proteolytic Excision Affinity Mass Spectrometry.

Affinity mass spectrometry using selective proteolytic excision and extraction combined with MALDI and ESI mass spectrometry has been applied to the identification of epitope binding sites of lactose, GalNac, and blood group oligosaccharides in two blood group-specific lectins, human galectin-3 and glycine max lectin. The epitope peptides identified comprise all essential amino acids involved in carbohydrate recognition, in complete agreement with available X-ray structures. Tryptic and chymotryptic digestion of lectins for proteolytic extraction/excision-MS was substantially improved by pressure-enhanced digestion using an automated Barocycler procedure (40 kpsi). Both previously established immobilization on affinity microcolumns using divinyl sulfone and coupling of a specific peptide glycoprobe to the gold surface of a biosensor chip were successfully employed for proteolytic excision and extraction of carbohydrate epitopes and affinity measurements. The identified epitope peptides could be differentiated according to the carbohydrate employed, thus demonstrating the specificity of the mass spectrometric approach. The specificities of the epitope ligands for individual carbohydrates were further ascertained by affinity studies using synthetic peptide ligands with immobilized carbohydrates. Binding affinities of the synthetic ligand peptides to lactose, in comparison to the intact full-length lectins, were determined by surface acoustic wave (SAW) biosensor analysis and provided micromolar KD values for the intact lectins, in agreement with results of previous ITC and SPR studies. Binding affinities of the epitope peptides were approximately two orders of magnitude lower, consistent with their smaller size and assembled arrangement in the carbohydrate recognition domains. Graphical Abstract ᅟ.

Antibody Epitope of Human α-Galactosidase†A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease.

α-Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α-galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α-galactosidase A is known as Fabry disease or Fabry-Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy-limiting, and eventually life-threatening complications of ERT. The present study focused on the epitope determination of human α-galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309-332) recognized by a human monoclonal anti-αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309-332), was synthesized by solid-phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (KD =39×10-9 m), which is nearly identical to that of the full-length enzyme (KD =16×10-9 m). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full-length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.

Molecular characterization of the ß-amyloid(4-10) epitope of plaque specific Aß antibodies by affinity-mass spectrometry using alanine site mutation.

Alzheimer disease is a neurodegenerative disease affecting an increasing number of patients worldwide. Current therapeutic strategies are directed to molecules capable to block the aggregation of the ß-amyloid(1-42) (Aß) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aß-specific antibodies have been recently developed as powerful antiaggregation tools. The identification and functional characterization of the epitope structures of Aß antibodies contributes to the elucidation of their mechanism of action in the human organism. In previous studies, the Aß(4-10) peptide has been identified as an epitope for the polyclonal anti-Aß(1-42) antibody that has been shown capable to reduce amyloid deposition in a transgenic Alzheimer disease mouse model. To determine the functional significance of the amino acid residues involved in binding to the antibody, we report here the effects of alanine single-site mutations within the Aß-epitope sequence on the antigen-antibody interaction. Specific identification of the essential affinity preserving mutant peptides was obtained by exposing a Sepharose-immobilized antibody column to an equimolar mixture of mutant peptides, followed by analysis of bound peptides using high-resolution MALDI-Fourier transform-Ion Cyclotron Resonance mass spectrometry. For the polyclonal antibody, affinity was preserved in the H6A, D7A, S8A, and G9A mutants but was lost in the F4, R5, and Y10 mutants, indicating these residues as essential amino acids for binding. Enzyme-linked immunosorbent assays confirmed the binding differences of the mutant peptides to the polyclonal antibody. In contrast, the mass spectrometric analysis of the mutant Aß(4-10) peptides upon affinity binding to a monoclonal anti-Aß(1-17) antibody showed complete loss of binding by Ala-site mutation of any residue of the Aß(4-10) epitope. Surface plasmon resonance affinity determination of wild-type Aß(1-17) to the monoclonal Aß antibody provided a binding constant KD in the low nanomolar range. These results provide valuable information in the elucidation of the binding mechanism and the development of Aß-specific antibodies with improved therapeutic efficacy.