Native and compactly folded in-solution conformers of pepsin are revealed and distinguished by mass spectrometric ITEM-TWO analyses of gas-phase pepstatin A - pepsin complex binding strength differences.

Pepsin, because of its optimal activity at low acidic pH, has gained importance in mass spectrometric proteome research as a readily available and easy-to-handle protease. Pepsin has also been study object of protein higher-order structure analyses, but questions about how to best investigate pepsin in-solution conformers still remain. We first determined dependencies of pepsin ion charge structures on solvent pH which indicated the in-solution existence of (a) natively folded pepsin (N) which by nanoESI-MS analysis gave rise to a narrow charge state distribution with an 11-fold protonated most intense ion signal, (b) unfolded pepsin (U) with a rather broad ion charge state distribution whose highest ion signal carried 25 protons, and (c) a compactly folded pepsin conformer (C) with a narrow charge structure and a 12-fold protonated ion signal in the center of its charge state envelope. Because pepsin is a protease, unfolded pepsin became its own substrate in solution at pH 6.6 since at this pH some portion of pepsin maintained a compact/native fold which displayed enzymatic activity. Subsequent mass spectrometric ITEM-TWO analyses of pepstatin A - pepsin complex dissociation reactions in the gas phase confirmed a very strong binding of pepstatin A by natively folded pepsin (N). ITEM-TWO further revealed the existence of two compactly folded in-solution pepsin conformers (Ca and Cb) which also were able to bind pepstatin A. Binding strengths of the respective compactly folded pepsin conformer-containing complexes could be determined and apparent gas phase complex dissociation constants and reaction enthalpies differentiated these from each other and from the pepstatin A - pepsin complex which had been formed from natively folded pepsin. Thus, ITEM-TWO turned out to be well suited to pinpoint in-solution pepsin conformers by interrogating quantitative traits of pepstatin A - pepsin complexes in the gas phase.

Epitope Fine Mapping by Mass Spectrometry: Investigations of Immune Complexes Consisting of Monoclonal Anti-HpTGEKP Antibody and Zinc Finger Protein Linker Phospho-Hexapeptides.

Accurate formation of antibody-antigen complexes has been relied on in both, multitudes of scientific projects and ample therapeutic and diagnostic applications. Mass spectrometrically determined dissociation behavior of immune complexes with the anti-HpTGEKP antibody revealed that the ten most frequently occurring phospho-hexapeptide linker sequences from C2H2 zinc finger proteins could be divided into two classes: orthodox binders, where strong noncovalent interactions developed as anticipated, and unorthodox binders with deviating structures and weaker binding. Phosphorylation of threonine was compulsory for antibody binding in an orthodox manner. Gas phase dissociation energy determinations of seven C2H2 zinc finger protein linker phospho-hexapeptides with orthodox binding properties revealed a bipolar binding motif of the antibody paratope. Epitope peptides, which in addition to the negatively charged phospho-threonine residue were C-terminally flanked by positively charged residues provided stronger binding, i. e. dissociation was endothermic, than peptides with acidic amino acid residues at these positions, for which dissociation was exothermic.

Clinical and genetic characterization of a cohort of 97 CLN6 patients tested at a single center.

BACKGROUND: Ceroid lipofuscinoses neuronal 6 (CLN6) disease belongs to the neuronal ceroid lipofuscinoses (NCLs), complex and genetically heterogeneous disorders with wide geographical and phenotypic variation. The first clinical signs usually appear between 18 months and 8 years, but examples of later-onset have also been reported. Common manifestations include ataxia, seizures, vision impairment, and developmental regression. Because these are shared by other neurological diseases, identification of CLN6 genetic variants is imperative for early diagnosis. RESULTS: We present one of the largest cohorts to date of genetically diagnosed CLN6 patients screened at a single center. In total 97 subjects, originating from 20 countries were screened between 2010 and 2020. They comprised 86 late-infantile, eight juvenile, and three adult-onset cases (two patients with Kufs disease type A, and one with teenage progressive myoclonic epilepsy). The male to female ratio was 1.06: 1.00. The age at referral was between six months and 33 years. The time from disease onset to referral ranged from less than 1 month to 8.3 years. The clinical phenotype consisted of a combination of symptoms, as reported before. We characterized a total of 45 distinct variants defining 45 distinct genotypes. Twenty-four were novel variants, some with distinct geographic associations. Remarkably, c.257A > G (p.H86R) was present in five out of 23 unrelated Egyptian individuals but in no patients from other countries. The most common genotype was homozygosity for the c.794_796del in-frame deletion. It was present in about one-third of CLN6 patients (28 unrelated cases, and 2 familial cases), all with late-infantile onset. Variants with a high likelihood of causing loss of CLN6 function were found in 21% of cases and made up 33% of all distinct variants. Forty-four percent of variants were classified as pathogenic or likely pathogenic. CONCLUSIONS: Our study significantly expands the number of published clinical cases and the mutational spectrum of disease-associated CLN6 variants, especially for the Middle Eastern and North African regions. We confirm previous observations regarding the most prevalent symptoms and recommend including CLN6 in the genetic diagnosis of patients presenting with early-onset abnormalities of the nervous system, musculoskeletal system, and eye.

Mass Spectrometric and Bio-Computational Binding Strength Analysis of Multiply Charged RNAse S Gas-Phase Complexes Obtained by Electrospray Ionization from Varying In-Solution Equilibrium Conditions.

We investigated the influence of a solvent's composition on the stability of desorbed and multiply charged RNAse S ions by analyzing the non-covalent complex's gas-phase dissociation processes. RNAse S was dissolved in electrospray ionization-compatible buffers with either increasing organic co-solvent content or different pHs. The direct transition of all the ions and the evaporation of the solvent from all the in-solution components of RNAse S under the respective in-solution conditions by electrospray ionization was followed by a collision-induced dissociation of the surviving non-covalent RNAse S complex ions. Both types of changes of solvent conditions yielded in mass spectrometrically observable differences of the in-solution complexation equilibria. Through quantitative analysis of the dissociation products, i.e., from normalized ion abundances of RNAse S, S-protein, and S-peptide, the apparent kinetic and apparent thermodynamic gas-phase complex properties were deduced. From the experimental data, it is concluded that the stability of RNAse S in the gas phase is independent of its in-solution equilibrium but is sensitive to the complexes' gas-phase charge states. Bio-computational in-silico studies showed that after desolvation and ionization by electrospray, the remaining binding forces kept the S-peptide and S-protein together in the gas phase predominantly by polar interactions, which indirectly stabilized the in-bulk solution predominating non-polar intermolecular interactions. As polar interactions are sensitive to in-solution protonation, bio-computational results provide an explanation of quantitative experimental data with single amino acid residue resolution.

Mass Spectrometric Analysis of Antibody-Epitope Peptide Complex Dissociation: Theoretical Concept and Practical Procedure of Binding Strength Characterization.

Electrospray mass spectrometry is applied to determine apparent binding energies and quasi equilibrium dissociation constants of immune complex dissociation reactions in the gas phase. Myoglobin, a natural protein-ligand complex, has been used to develop the procedure which starts from determining mean charge states and normalized and averaged ion intensities. The apparent dissociation constant KD m0g#= 3.60 × 10-12 for the gas phase heme dissociation process was calculated from the mass spectrometry data and by subsequent extrapolation to room temperature to mimic collision conditions for neutral and resting myoglobin. Similarly, for RNAse S dissociation at room temperature a KD m0g#= 4.03 × 10-12 was determined. The protocol was tested with two immune complexes consisting of epitope peptides and monoclonal antibodies. For the epitope peptide dissociation reaction of the FLAG peptide from the antiFLAG antibody complex an apparent gas phase dissociation constant KD m0g#= 4.04 × 10-12 was calculated. Likewise, an apparent KD m0g#= 4.58 × 10-12 was calculated for the troponin I epitope peptide-antiTroponin I antibody immune complex dissociation. Electrospray mass spectrometry is a rapid method, which requires small sample amounts for either identification of protein-bound ligands or for determination of the apparent gas phase protein-ligand complex binding strengths.

ITEM-THREE analysis of a monoclonal anti-malaria antibody reveals its assembled epitope on the pfMSP119 antigen.

Rapid diagnostic tests are first-line assays for diagnosing infectious diseases, such as malaria. To minimize false positive and false negative test results in population-screening assays, high-quality reagents and well-characterized antigens and antibodies are needed. An important property of antigen-antibody binding is recognition specificity, which best can be estimated by mapping an antibody's epitope on the respective antigen. We have cloned a malarial antigen-containing fusion protein, MBP-pfMSP119, in Escherichia coli, which then was structurally and functionally characterized before and after high pressure-assisted enzymatic digestion. We then used our previously developed method, intact transition epitope mapping-targeted high-energy rupture of extracted epitopes (ITEM-THREE), to map the area on the MBP-pfMSP119 antigen surface that is recognized by the anti-pfMSP119 antibody G17.12. We identified three epitope-carrying peptides, 386GRNISQHQCVKKQCPQNSGCFRHLDE411, 386GRNISQHQCVKKQCPQNSGCFRHLDEREE414, and 415CKCLLNYKQE424, from the GluC-derived peptide mixture. These peptides belong to an assembled (conformational) epitope on the MBP-pfMSP119 antigen whose identification was substantiated by positive and negative control experiments. In conclusion, our data help to establish a workflow to obtain high-quality control data for diagnostic assays, including the use of ITEM-THREE as a powerful analytical tool. Data are available via ProteomeXchange: PXD019717.

Intact Transition Epitope Mapping - Thermodynamic Weak-force Order (ITEM - TWO).

We have developed an electrospray mass spectrometry method which is capable to determine antibody affinity in a gas phase experiment. A solution with the immune complex is electrosprayed and multiply charged ions are translated into the gas phase. Then, the intact immune-complex ions are separated from unbound peptide ions. Increasing the voltage difference in a collision cell results in collision induced dissociation of the immune-complex by which bound peptide ions are released. When analyzing a peptide mixture, measuring the mass of the complex-released peptide ions identifies which of the peptides contains the epitope. A step-wise increase in the collision cell voltage difference changes the intensity ratios of the surviving immune complex ions, the released peptide ions, and the antibody ions. From all the ions´ normalized intensity ratios are deduced the thermodynamic quasi equilibrium dissociation constants (KDm0g#) from which are calculated the apparent gas phase Gibbs energies of activation over temperature (ΔGm0g#T). The order of the apparent gas phase dissociation constants of four antibody - epitope peptide pairs matched well with those obtained from in-solution measurements. The determined gas phase values for antibody affinities are independent from the source of the investigated peptides and from the applied instrument. Data are available via ProteomeXchange with identifier PXD016024. SIGNIFICANCE: ITEM - TWO enables rapid epitope mapping and determination of apparent dissociation energies of immune complexes with minimal in-solution handling. Mixing of antibody and antigen peptide solutions initiates immune complex formation in solution. Epitope binding strengths are determined in the gas phase after electrospraying the antibody / antigen peptide mixtures and mass spectrometric analysis of immune complexes under different collision induced dissociation conditions. Since the order of binding strengths in the gas phase is the same as that in solution, ITEM - TWO characterizes two most important antibody properties, specificity and affinity.

Intact Transition Epitope Mapping - Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE).

Epitope mapping, which is the identification of antigenic determinants, is essential for the design of novel antibody-based therapeutics and diagnostic tools. ITEM-THREE is a mass spectrometry-based epitope mapping method that can identify epitopes on antigens upon generating an immune complex in electrospray-compatible solutions by adding an antibody of interest to a mixture of peptides from which at least one holds the antibody's epitope. This mixture is nano-electrosprayed without purification. Identification of the epitope peptide is performed within a mass spectrometer that provides an ion mobility cell sandwiched in-between two collision cells and where this ion manipulation setup is flanked by a quadrupole mass analyzer on one side and a time-of-flight mass analyzer on the other side. In a stepwise fashion, immune-complex ions are separated from unbound peptide ions and dissociated to release epitope peptide ions. Immune complex-released peptide ions are separated from antibody ions and fragmented by collision induced dissociation. Epitope-containing peptide fragment ions are recorded, and mass lists are submitted to unsupervised data base search thereby retrieving both, the amino acid sequence of the epitope peptide and the originating antigen. ITEM-THREE was developed with antiTRIM21 and antiRA33 antibodies for which the epitopes were known, subjecting them to mixtures of synthetic peptides of which one contained the respective epitope. ITEM-THREE was then successfully tested with an enzymatic digest of His-tagged recombinant human ß-actin and an antiHis-tag antibody, as well as with an enzymatic digest of recombinant human TNFα and an antiTNFα antibody whose epitope was previously unknown.

Mass spectrometric characterization of protein structures and protein complexes in condensed and gas phase.

Proteins are essential for almost all physiological processes of life. They serve a myriad of functions which are as varied as their unique amino acid sequences and their corresponding three-dimensional structures. To fulfill their tasks, most proteins depend on stable physical associations, in the form of protein complexes that evolved between themselves and other proteins. In solution (condensed phase), proteins and/or protein complexes are in constant energy exchange with the surrounding solvent. Albeit methods to describe in-solution thermodynamic properties of proteins and of protein complexes are well established and broadly applied, they do not provide a broad enough access to life-science experimentalists to study all their proteins' properties at leisure. This leaves great desire to add novel methods to the analytical biochemist's toolbox. The development of electrospray ionization created the opportunity to characterize protein higher order structures and protein complexes rather elegantly by simultaneously lessening the need of sophisticated sample preparation steps. Electrospray mass spectrometry enabled us to translate proteins and protein complexes very efficiently into the gas phase under mild conditions, retaining both, intact protein complexes, and gross protein structures upon phase transition. Moreover, in the environment of the mass spectrometer (gas phase, in vacuo), analyte molecules are free of interactions with surrounding solvent molecules and, therefore, the energy of inter- and intramolecular forces can be studied independently from interference of the solvating environment. Provided that gas phase methods can give information which is relevant for understanding in-solution processes, gas phase protein structure studies and/or investigations on the characterization of protein complexes has rapidly gained more and more attention from the bioanalytical scientific community. Recent reports have shown that electrospray mass spectrometry provides direct access to six prime protein complex properties: stabilities, compositions, binding surfaces (epitopes), disassembly processes, stoichiometries, and thermodynamic parameters.

Apparent activation energies of protein-protein complex dissociation in the gas-phase determined by electrospray mass spectrometry.

We have developed a method to determine apparent activation energies of dissociation for ionized protein-protein complexes in the gas phase using electrospray ionization mass spectrometry following the Rice-Ramsperger-Kassel-Marcus quasi-equilibrium theory. Protein-protein complexes were formed in solution, transferred into the gas phase, and separated from excess free protein by ion mobility filtering. Afterwards, complex disassembly was initiated by collision-induced dissociation with step-wise increasing energies. Relative intensities of ion signals were used to calculate apparent activation energies of dissociation in the gas phase by applying linear free energy relations. The method was developed using streptavidin tetramers. Experimentally determined apparent gas-phase activation energies for dissociation ([Formula: see text]) of complexes consisting of Fc parts from immunoglobulins (IgG-Fc) and three closely related protein G' variants (IgG-Fc•protein G'e, IgG-Fc•protein G'f, and IgG-Fc•protein G'g) show the same order of stabilities as can be inferred from their in-solution binding constants. Differences in stabilities between the protein-protein complexes correspond to single amino acid residue exchanges in the IgG-binding regions of the protein G' variants. Graphical abstract Electrospray mass spectrometry and collision-induced dissociation delivers apparent activation energies and supramolecular bond force constants of protein-protein complexes in the gas phase.

Intact Transition Epitope Mapping (ITEM).

Intact transition epitope mapping (ITEM) enables rapid and accurate determination of protein antigen-derived epitopes by either epitope extraction or epitope excision. Upon formation of the antigen peptide-containing immune complex in solution, the entire mixture is electrosprayed to translate all constituents as protonated ions into the gas phase. There, ions from antibody-peptide complexes are separated from unbound peptide ions according to their masses, charges, and shapes either by ion mobility drift or by quadrupole ion filtering. Subsequently, immune complexes are dissociated by collision induced fragmentation and the ion signals of the "complex-released peptides," which in effect are the epitope peptides, are recorded in the time-of-flight analyzer of the mass spectrometer. Mixing of an antibody solution with a solution in which antigens or antigen-derived peptides are dissolved is, together with antigen proteolysis, the only required in-solution handling step. Simplicity of sample handling and speed of analysis together with very low sample consumption makes ITEM faster and easier to perform than other experimental epitope mapping methods. Graphical Abstract ᅟ.