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 (K) of a protein-antibody complex is first determined by immobilizing an antibody or DNA-aptamer on an SPR chip.

Enzymatic diagnosis of neuronal lipofuscinoses in dried blood spots using substrates for concomitant tandem mass spectrometry and fluorimetry.

Neuronal ceroid lipofuscinoses (NCLs) are a group of neurodegenerative diseases predominantly in childhood that are characterized by psychomotor deterioration, epilepsy, and early death of patients. The NCLs analyzed in the present study are caused by defects of the specific enzymes, CLN1 (palmitoyl protein thioesterase 1; PPT1), CLN2 (tripeptidyl peptidase 1; TPP1), and CLN10 (cathepsin D). Specific and sensitive diagnostic assays of NCLs were the main goal of this study.

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.

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.

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.

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 β 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.

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.

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).

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.

Molecular characterization of the β-amyloid(4-10) epitope of plaque specific Aβ 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 β-amyloid(1-42) (Aβ) peptide and its shorter naturally occurring peptide fragments into toxic oligomers and amyloid fibrils. Aβ-specific antibodies have been recently developed as powerful antiaggregation tools.

Erratum: Growth of organic crystals via attachment and transformation of nanoscopic precursors.

This corrects the article DOI: 10.1038/ncomms15933.

Growth of organic crystals via attachment and transformation of nanoscopic precursors.

A key requirement for the understanding of crystal growth is to detect how new layers form and grow at the nanoscale. Multistage crystallization pathways involving liquid-like, amorphous or metastable crystalline precursors have been predicted by theoretical work and have been observed experimentally. Nevertheless, there is no clear evidence that any of these precursors can also be relevant for the growth of crystals of organic compounds.

Substrate and Substrate-Mimetic Chaperone Binding Sites in Human α-Galactosidase A Revealed by Affinity-Mass Spectrometry.

Fabry disease (FD) is a rare metabolic disorder of a group of lysosomal storage diseases, caused by deficiency or reduced activity of the enzyme α-galactosidase. Human α-galactosidase A (hαGAL) hydrolyses the terminal α-galactosyl moiety from glycosphingolipids, predominantly globotriaosylceramide (Gb3). Enzyme deficiency leads to incomplete or blocked breakdown and progressive accumulation of Gb3, with detrimental effects on normal organ functions.

An HLA-B27 Homodimer Specific Antibody Recognizes a Discontinuous Mixed-Disulfide Epitope as Identified by Affinity-Mass Spectrometry.

HLA-B27 homodimer formation is believed to be a hallmark of HLA-B27 associated spondyloarthritides. Recently, we have generated a homodimer-specific monoclonal antibody (HD6) and have demonstrated that HLA-B27 homodimer complexes are present on monocytes of healthy HLA-B27 gene carriers at low levels, with significantly increased levels at active disease. The capability of the HD6 antibody to discriminate between correctly formed HLA-B27 heterotrimers and pathology-associated homodimers is striking and cannot be explained by the primary structure of HLA-B27.

β-Lactoglobulin detected in human milk forms noncovalent complexes with maltooligosaccharides as revealed by chip-nanoelectrospray high-resolution tandem mass spectrometry.

Cow's milk protein allergy in exclusively breastfed infants, the main cause of food intolerance during the first 6 months of life, is triggered by the mother's diet. β-Lactoglobulin (BLG) present in cow's milk is one of the most potent allergens for newborns. Since no prophylactic treatment is available, finding ligands capable of binding BLG and reducing its allergenicity is currently the focus of research.

Identification and affinity-quantification of ß-amyloid and α-synuclein polypeptides using on-line SAW-biosensor-mass spectrometry.

Bioaffinity analysis using a variety of biosensors has become an established tool for detection and quantification of biomolecular interactions. Biosensors, however, are generally limited by the lack of chemical structure information of affinity-bound ligands. On-line bioaffinity-mass spectrometry using a surface-acoustic wave biosensor (SAW-MS) is a new combination providing the simultaneous affinity detection, quantification, and mass spectrometric structural characterization of ligands.

Increased levels of antigen-bound β-amyloid autoantibodies in serum and cerebrospinal fluid of Alzheimer's disease patients.

Recent studies have suggested a protective role of physiological β-amyloid autoantibodies (Aβ-autoantibodies) in Alzheimer's disease (AD). However, the determination of both free and dissociated Aβ-autoantibodies in serum hitherto has yielded inconsistent results regarding their function and possible biomarker value. Here we report the application of a new sandwich enzyme-linked immunosorbent assay (ELISA) for the determination of antigen-bound Aβ-autoantibodies (intact Aβ-IgG immune complexes) in serum and cerebrospinal fluid (CSF) of a total number of 112 AD patients and age- and gender-matched control subjects.

Testing the feasibility of fully automated chip-based nanoelectrospray ionization mass spectrometry as a novel tool for rapid diagnosis of Fabry disease.

Fabry condition, a lysosomal storage disease (LSD) is characterized by the absence or reduction of the α-galactosidase A activity. Recently, a new diagnostic method for detection of α-galactosidase activity from dried blood spots (DBS) using a chemical substrate and quantification of reaction mixture was developed. To improve this method in the terms of automation, reproducibility, sensitivity, and data reliability, we introduce here an innovative analytical approach based on chip-nanoESI MS.

Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.

ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants.

Antigen-bound and free β-amyloid autoantibodies in serum of healthy adults.

Physiological β-amyloid autoantibodies (Aβ-autoantibodies) are currently investigated as potential diagnostic and therapeutic tools for Alzheimer's disease (AD). In previous studies, their determination in serum and cerebrospinal fluid (CSF) using indirect ELISA has provided controversial results, which may be due to the presence of preformed Aβ antigen-antibody immune complexes. Based on the epitope specificity of the Aβ-autoantibodies, recently elucidated in our laboratory, we developed (a) a sandwich ELISA for the determination of circulating Aβ-IgG immune complexes and (b) an indirect ELISA for the determination of free Aβ-autoantibodies.

When is mass spectrometry combined with affinity approaches essential? A case study of tyrosine nitration in proteins.

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration.

Characterization of oligomerization-aggregation products of neurodegenerative target proteins by ion mobility mass spectrometry.

Protein amyloidogenesis is generally considered to be a major cause of two most severe neurodegenerative disorders, Parkinson's disease (PD) and Alzheimer's disease (AD). Formation and accumulation of fibrillar aggregates and plaques derived from α-synuclein (α-Syn) and ß-amyloid (Aß) polypeptide in brain have been recognized as characteristics of Parkinson's disease and Alzheimer's disease. Oligomeric aggregates of α-Syn and Aß are considered as neurotoxic intermediate products leading to progressive neurodegeneration.

Why the structure but not the activity of the immunoproteasome subunit low molecular mass polypeptide 2 rescues antigen presentation.

The proteasome is responsible for the generation of most epitopes presented on MHC class I molecules. Treatment of cells with IFN-γ leads to the replacement of the constitutive catalytic subunits β1, β2, and β5 by the inducible subunits low molecular mass polypeptide (LMP) 2 (β1i), multicatalytic endopeptidase complex-like-1 (β2i), and LMP7 (β5i), respectively. The incorporation of these subunits is required for the production of numerous MHC class I-restricted T cell epitopes.

Application of MALDI-TOF-mass spectrometry to proteome analysis using stain-free gel electrophoresis.

The combination of MALDI-TOF-mass spectrometry with gel electrophoretic separation using protein visualization by staining procedures involving such as Coomassie Brilliant Blue has been established as a widely used approach in proteomics. Although this approach has been shown to present high detection sensitivity, drawbacks and limitations frequently arise from the significant background in the mass spectrometric analysis. In this chapter we describe an approach for the application of MALDI-MS to the mass spectrometric identification of proteins from one-dimensional (1D) and two-dimensional (2D) gel electrophoretic separation, using stain-free detection and visualization based on native protein fluorescence.