From Free Binding Energy Calculations of SARS-CoV-2-Receptor Interactions to Cellular Immune Responses.

Our study focuses on free energy calculations of SARS-CoV-2 spike protein receptor binding motives (RBMs) from wild type and variants of concern (VOCs), with emphasis on SARS-CoV-2 Omicron. Our computational analysis underlines the occurrence of positive selection processes that specify Omicron host adaption and bring changes on the molecular level into context with clinically relevant observations. Our free energy calculation studies regarding the interaction of Omicron's RBM with human angiotensin converting enzyme 2 (hACE2) indicate weaker binding to the receptor than Alpha's or Delta's RBMs. Upon weaker binding, fewer viruses are predicted to be generated in time per infected cell, resulting in a delayed induction of danger signals as a trade-off. Along with delayed immunogenicity and pathogenicity, more viruses may be produced in the upper respiratory tract, explaining enhanced transmissibility. Since in interdependence on the human leukocyte antigen type (HLA type), more SARS-CoV-2 Omicron viruses are assumed to be required to initiate inflammatory immune responses, and because of pre-existing partial immunity through previous infections and/or vaccinations, which mostly guard the lower respiratory tract, overall disease severity is expected to be reduced.

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.

Prevalence, patterns and beliefs about the use of herbal medicinal products in Ghana: a multi-centre community-based cross-sectional study.

OBJECTIVE: Worldwide, herbal medicinal products (HMPs) play key roles in healthcare systems, especially in developing countries, yet there is inconsistent evidence about their prevalence and patterns of use in Ghana. This study therefore sought to determine the prevalence, patterns and beliefs about the use of HMPs in Ghana. METHODS: A descriptive community-based cross-sectional survey was conducted using a researcher-administered questionnaire on 1364 adults, selected from five communities for each of the ecological zones in Ghana using a multi-stage sampling procedure. The study was conducted between December 2019 and March 2020. RESULTS: The prevalence of ever use of HMPs was 76.5% with 73.0% of respondents using these products within the past year. Almost 60% of respondents reported using HMPs that were registered by the Ghana Food and Drugs Authority. About 56.7% used these products to cure diseases. All the sociodemographic characteristics (age, religion, marital status, educational level and employment status) except for sex were significantly associated with the use of HMPs (P < 0.001). For beliefs about HMPs, the proportion of respondents classified to be accepting, ambivalent, indifferent and sceptical was 14.3%, 25.2%, 47.5% and 13.07%, respectively. About 62.2% of study participants had plans to use HMPs in the future, and 69.1% were willing to encourage others to use such products. However, 51.6% of the participants did not perceive HMPs as more effective and safe compared with orthodox products. CONCLUSION: The prevalence of use of HMPs in Ghana is high, thus suggesting an appropriate public health policy to improve the regulation of these products and also provide basis for the integration of HMPs into the healthcare system in Ghana.

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.

Monitoring and risk assessment of pesticide residues in selected herbal medicinal products in Ghana.

The high patronage of herbal medicinal products in Ghana for the treatment of diverse disease conditions raises concerns about patient safety, given that much of the raw materials for production are obtained from the wild or farmlands potentially exposed to varied agrochemical residues. Therefore, the work sought to investigate the contamination of herbal medicinal products with pesticide residues and assess the potential risk posed to patients. As a result, validated gas chromatography with mass spectrometry as a detector was used to determine forty-two pesticides in thirty herbal medicinal products. The performance parameters of the method such as linearity, accuracy, and precision were found as acceptable. Pesticide residues such as chlorpyrifos and/or bifenthrin were found in 4/30 herbal medicinal products. Specifically, 3/30 herbal medicinal products contained only one pesticide, while 1/30 was contaminated with both pesticide residues. The levels of pesticide residue contamination ranged between 2.5 and 5.0 µg/kg. The acute hazard quotient and chronic hazard quotient for the two pesticide residues were evaluated and ranged between 0.21 and 0.92% and between 8.21 × 10-4 and 5.88 × 10-3%. The detected pesticide residue levels are below the maximum residue limit values, which may not cause acute and chronic health risks due to intake of the selected herbal medicinal product. Nevertheless, patient safety and potential public health risk can be reduced by regular monitoring, and regulation of pesticide residue levels in herbal medicinal products.

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.

Mass spectrometric epitope mapping.

Mass spectrometric epitope mapping has become a versatile method to precisely determine a soluble antigen's partial structure that directly interacts with an antibody in solution. Typical lengths of investigated antigens have increased up to several 100 amino acids while experimentally determined epitope peptides have decreased in length to on average 10-15 amino acids. Since the early 1990s more and more sophisticated methods have been developed and have forwarded a bouquet of suitable approaches for epitope mapping with immobilized, temporarily immobilized, and free-floating antibodies. While up to now monoclonal antibodies have been mostly used in epitope mapping experiments, the applicability of polyclonal antibodies has been proven. The antibody's resistance towards enzymatic proteolysis has been of key importance for the two mostly applied methods: epitope excision and epitope extraction. Sample consumption has dropped to low pmol amounts on both, the antigen and the antibody. While adequate in-solution sample handling has been most important for successful epitope mapping, mass spectrometric analysis has been found the most suitable read-out method from early on. The rapidity by which mass spectrometric epitope mapping nowadays is executed outperforms all alternative methods. Thus, it can be asserted that mass spectrometric epitope mapping has reached a state of maturity, which allows it to be used in any mass spectrometry laboratory. After 25 years of constant and steady improvements, its application to clinical samples, for example, for patient characterization and stratification, is anticipated in the near future. © 2016 Wiley Periodicals, Inc. Mass Spec Rev 37:229-241, 2018.

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.

A Dynamic Model of pH-Induced Protein G'e Higher Order Structure Changes derived from Mass Spectrometric Analyses.

To obtain insight into pH change-driven molecular dynamics, we studied the higher order structure changes of protein G'e at the molecular and amino acid residue levels in solution by using nanoESI- and IM-mass spectrometry, CD spectroscopy, and protein chemical modification reactions (protein footprinting). We found a dramatic change of the overall tertiary structure of protein G'e when the pH was changed from neutral to acidic, whereas its secondary structure features remained nearly invariable. Limited proteolysis and surface-topology mapping of protein G'e by fast photochemical oxidation of proteins (FPOP) under neutral and acidic conditions reveal areas where higher order conformational changes occur on the amino-acid residue level. Under neutral solution conditions, lower oxidation occurs for residues of the first linker region, whereas greater oxidative modifications occur for amino-acid residues of the IgG-binding domains I and II. We propose a dynamic model of pH-induced structural changes in which protein G'e at neutral pH adopts an overall tight conformation with all four domains packed in a firm assembly, whereas at acidic pH, the three IgG-binding domains form an elongated alignment, and the N-terminal, His-tag-carrying domain unfolds. At the same time the individual IgG-binding domains themselves seem to adopt a more compacted fold. As the secondary structure features are nearly unchanged at either pH, interchange between both conformations is highly reversible, explaining the high reconditioning power of protein G'e-based affinity chromatography columns.

A novel strategy for the rapid preparation and isolation of intact immune complexes from peptide mixtures.

The development and application of a miniaturized affinity system for the preparation and release of intact immune complexes are demonstrated. Antibodies were reversibly affinity-adsorbed on pipette tips containing protein G´ and protein A, respectively. Antigen proteins were digested with proteases and peptide mixtures were exposed to attached antibodies; forming antibody-epitope complexes, that is, immune complexes. Elution with millimolar indole propionic acid (IPA)-containing buffers under neutral pH conditions allowed to effectively isolate the intact immune complexes in purified form. Size exclusion chromatography was performed to determine the integrity of the antibody-epitope complexes. Mass spectrometric analysis identified the epitope peptides in the respective SEC fractions. His-tag-containing recombinant human glucose-6-phosphate isomerase in combination with an anti-His-tag monoclonal antibody was instrumental to develop the method. Application was extended to the isolation of the intact antibody-epitope complex of a recombinant human tripartite motif 21 (rhTRIM21) auto-antigen in combination with a rabbit polyclonal anti-TRIM21 antibody. Peptide chip analysis showed that antibody-epitope binding of rhTRIM21 peptide antibody complexes was not affected by the presence of IPA in the elution buffer. By contrast, protein G´ showed an ion charge structure by electrospray mass spectrometry that resembled a denatured conformation when exposed to IPA-containing buffers. The advantages of this novel isolation strategy are low sample consumption and short experimental duration in addition to the direct and robust methodology that provides easy access to intact antibody-antigen complexes under neutral pH and low salt conditions for subsequent investigations.

Dried serum spots on pre-punched filter paper discs are ready-to-use storage and shipping devices for blood-borne antigens and antibodies.

Dried serum spots that are well prepared can be attractive alternatives to frozen serum samples for shelving specimens in a medical or research center's biobank and mailing freshly prepared serum to specialized laboratories. During the pre-analytical phase, complications can arise which are often challenging to identify or are entirely overlooked. These complications can lead to reproducibility issues, which can be avoided in serum protein analysis by implementing optimized storage and transfer procedures. With a method that ensures accurate loading of filter paper discs with donor or patient serum, a gap in dried serum spot preparation and subsequent serum analysis shall be filled. Pre-punched filter paper discs with a 3 mm diameter are loaded within seconds in a highly reproducible fashion (approximately 10% standard deviation) when fully submerged in 10 µl of serum, named the "Submerge and Dry" protocol. Such prepared dried serum spots can store several hundred micrograms of proteins and other serum components. Serum-borne antigens and antibodies are reproducibly released in 20 µl elution buffer in high yields (approximately 90%). Dried serum spot-stored and eluted antigens kept their epitopes and antibodies their antigen binding abilities as was assessed by SDS-PAGE, 2D gel electrophoresis-based proteomics, and Western blot analysis, suggesting pre-punched filter paper discs as handy solution for serological tests.

Contamination of herbal medicinal products in low-and-middle-income countries: A systematic review.

The use of herbal medicinal products (HMPs) has grown significantly across low-and-middle-income countries (LMICs). Consequently, the safety of these products due to contamination is a significant public health concern. This systematic review aimed to determine the prevalence, types, and levels of contaminants in HMPs from LMICs. A search was performed in seven online databases, i.e., Africa journal online (AJOL), Cumulative Index to Nursing and Allied Health Literature (CINAHL), Directory of Open Access Journals (DOAJ), Health Inter-Network Access to Research Initiative (HINARI), World Health Organization Global Index Medicus (WHO GIM), Scopus, and PubMed using appropriate search queries and reported as per the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) guidelines. Ninety-one peer-reviewed articles published from 1982 to 2021 from 28 different countries across four continents were included in the study. Although metals, microbial, mycotoxins, pesticides, and residual solvents were the reported contaminants in the 91 articles, metals (56.0%, 51/91), microbial (27.5%, 25/91), and mycotoxins (18.7%, 17/91) were the most predominant. About 16.4% (1236/7518) of the samples had their contaminant levels above the regulatory limits. Samples tested for microbial contaminants had the highest proportion (46.4%, 482/1039) of contaminants exceeding the regulatory limit, followed by mycotoxins (25.8%, 109/423) and metals (14.3%, 591/4128). The proportion of samples that had their average non-essential metal contaminant levels above the regulatory limit was (57.6%, 377/655), 18.3% (88/480), 10.7% (24/225), and 11.3% (29/257) for Pb, Cd, Hg, and As, respectively. The commonest bacteria species found were Escherichia coli (52.3%, 10/19) and Salmonella species (42.1%, 8/19). This review reported that almost 90% of Candida albicans and more than 80% of moulds exceeded the required regulatory limits. HMP consumption poses profound health implications to consumers and patients. Therefore, designing and/or implementing policies that effectively regulate HMPs to minimize the health hazards related to their consumption while improving the quality of life of persons living in LMICs are urgently needed.

Bioanalysis of aminoglycosides using high-performance liquid chromatography.

Aminoglycosides are broad-spectrum antibiotics used in the treatment of gram-negative bacterial infections. Due to their nephrotoxic and ototoxic potential (narrow therapeutic index), the use of aminoglycoside for clinical indications requires monitoring. The objective of this review was to identify relevant literature reporting liquid chromatographic methods for the bioanalysis of aminoglycosides in both preclinical and clinical settings/experiments. Data on liquid chromatographic methods were collected from articles in an online academic database (PubMed, Science Direct, Scopus, and Google Scholar). All 71 articles published from 1977 to 2020 were included in the review. Reversed-phase liquid chromatography was the most used method for the bioanalysis of aminoglycosides. Fluorescence or ultraviolet detection methods were mostly used from 1977 to 2002 (51 articles), while mass spectrometry was predominantly used as a detector from 2003 to 2020 (15 articles). Sixty-seven articles reported calibration ranges, which varied significantly for the various drugs assayed: some in the range of 0.1-0.5 ng/mL and others 1250-200000 ng/mL. Also, 61 articles reported R2 values (0.964-1.0) for almost all analytes under consideration. Sixty-three articles reported percent recoveries mostly between 61.0 % to 114.0 %, with only two articles reporting recoveries of 4.9 % and 36 %. Out of the 71 reviewed articles, 56 reported intermediate precision values ranging between 0.331 % to 19.76 %, which is within the acceptable limit of 20 %. This review will serve as a guide for research and/or routine clinical monitoring of aminoglycosides in biological matrices.

The Unethical Use of Paracetamol As a Food Tenderizer in Four Selected African Countries: A Major Public Health Concern?

Paracetamol poisoning is the commonest cause of acute liver injury. Therefore, the unethical use of paracetamol as a food tenderizer poses a threat to human health. Although this is a common practice in Ghana, Uganda, Nigeria, and Kenya, there are few or no scientific records on the use of paracetamol as a food tenderizer and its deleterious effects, thus making it difficult to regulate this practice. This review aims to fully collate and present a systematic overview of the literature on the use of paracetamol as a food tenderizer in these countries, the potentially harmful effects posed by the practice, and measures in place to curb the situation. Additionally, this review aims to reveal the scientific gaps and areas requiring more research, thus providing a reference for further research to regulate this unscrupulous practice. From our extensive review of the literature, the high cost of fuel used in cooking and longer cooking times are the main reasons for the inappropriate use of paracetamol as a food tenderizer. Also, this review concludes that little has been done to create public awareness of this unethical practice. Furthermore, few ways to monitor, control and regulate this practice have been proposed.

High performance liquid chromatography: A versatile tool for assaying antiepileptic drugs in biological matrices.

Epilepsy is a recurrent long-term illness occurring in approximately 1.0% of the world's population. There are currently about 29 approved antiepileptic drugs for the management of epilepsy. Due to narrow therapeutic indices of most antiepileptic drugs, clinical pharmacokinetic characteristics and therapeutic drug monitoring of these drugs are imperative. The objectives of this review were to identify common chromatographic principles, requirements and/or conditions for high-performance liquid chromatography as applied to assay of antiepileptic drugs in biological matrices. The review was conducted using 66 peer reviewed articles (1990 to 2020) from 29 journals that were sought via PubMed, Science Direct and Google Scholar. In all, 29 antiepileptic drugs were assayed from 6 different biological matrices. Forty-three of the reviewed articles estimated the concentration of only one antiepileptic drug, whilst 23 articles focused on simultaneous determination of two or more antiepileptic drugs. Thirty-four, 20, and 14 articles reported using liquid-liquid extraction, protein precipitation, or solid phase extraction for sample clean up, respectively. The ratio of reversed-phase to normal phase, LC-UV to LC-MS and isocratic elution to gradient elution were 61:3, 43:7 and 55:11, respectively. With the exception of one article the reported recoveries ranged from 60.3% to 109.6%. It is noteworthy, that, the performance metrics of high-performance liquid chromatography are better compared to other assays of antiepileptic drugs in biological matrices. This review describes the relevant liquid chromatographic method conditions over the past 30 years for the analysis of this class of drugs, which provides a basis for further method development and optimization.

Utilization of complementary and alternative medicine for the prevention of COVID-19 infection in Ghana: A national cross-sectional online survey.

Complementary and alternative medicine (CAM) use is widespread and has played critical roles in preventing infections, including previous coronaviruses. This study sought to document current practices in the use of CAM for the prevention of COVID-19 disease in Ghana. An anonymous electronic survey was conducted from February 1, 2021 to April 30, 2021. Data on demographic characteristics, basic clinical information, illness perceptions about COVID-19, and CAM use during the pandemic period were generated. While about 82.5% (986/1195) of the participants used CAM during the COVID-19 period, 69.1% (681/986) of CAM users intented it for COVID-19 infection prevention. Vitamin supplements (88.1%, 869/986), spiritual healing/prayer (23.3%, 230/986), mineral supplements (22.3%, 220/986), botanical/herbal medicines (22.2%, 219/986), and diet therapy (19.4%, 191/986) were the main types of CAM used. From the adjusted binary logistic regression model, current age (aOR: 1.03, 95%CI: 1.01-1.05), sex (aOR: 1.41, 95%CI: 1.02-1.95), participants' perceptions of consequences (aOR: 1.10, 95%CI: 1.04-1.17), identity (aOR: 1.15, 95%CI: 1.06-1.25) and concerns about COVID-19 (aOR: 0.91, 95%CI: 0.85-0.97) were statistically significant predictors of CAM use. These results suggest the need for appropriate public health policy on COVID-19 and CAM use in addition to directing further research initiatives toward an optimized COVID-19 prevention scheme using clinically validated CAM treatments. Research to validate the clinical efficacy of these products, especially the herbs, for COVID-19 prevention while isolating lead compounds that could be optimized and used for the treatment and prevention of COVID-19 is also recommended.

Ultraviolet-Visible Spectroscopy and Chemometric Strategy Enable the Classification and Detection of Expired Antimalarial Herbal Medicinal Product in Ghana.

To meet the growing demand for complementary and alternative treatment for malaria, manufacturers produce several antimalarial herbal medicinal products. Herbal medicinal products regulation is difficult due to their complex chemical nature, requiring cumbersome, expensive, and time-consuming methods of analysis. The aim of this study was to develop a simple spectroscopic method together with a chemometric model for the classification and the identification of expired liquid antimalarial herbal medicinal products. Principal component analysis model was successfully used to distinguish between different herbal medicinal products and identify expired products. Principal component analysis showed a clear class separation between all five herbal medicinal products (HMP) studied, with explained variance for first and second principal components as 37.51% and 26.38%, respectively, while the third principal component had 18.74%. Support vector machine classification gave specificity and accuracy of 1.00 (100%) for training set data for all the products. The validation set HMP1, HMP2, and HMP3 had sensitivity, specificity, and accuracy of 1.00. HMP4 and HMP5 had sensitivity and specificity of 0.90 and 1.00, respectively, and an accuracy of 0.98. The support vector machine classification and principal component analysis models were successfully used to identify expired herbal medicinal products. This strategy can be used for rapid field detection of expired liquid antimalarial herbal medicinal products.

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 (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.

"De-novo" amino acid sequence elucidation of protein G'e by combined "top-down" and "bottom-up" mass spectrometry.

Mass spectrometric de-novo sequencing was applied to review the amino acid sequence of a commercially available recombinant protein G´ with great scientific and economic importance. Substantial deviations to the published amino acid sequence (Uniprot Q54181) were found by the presence of 46 additional amino acids at the N-terminus, including a so-called "His-tag" as well as an N-terminal partial α-N-gluconoylation and α-N-phosphogluconoylation, respectively. The unexpected amino acid sequence of the commercial protein G' comprised 241 amino acids and resulted in a molecular mass of 25,998.9 ± 0.2 Da for the unmodified protein. Due to the higher mass that is caused by its extended amino acid sequence compared with the original protein G' (185 amino acids), we named this protein "protein G'e." By means of mass spectrometric peptide mapping, the suggested amino acid sequence, as well as the N-terminal partial α-N-gluconoylations, was confirmed with 100% sequence coverage. After the protein G'e sequence was determined, we were able to determine the expression vector pET-28b from Novagen with the Xho I restriction enzyme cleavage site as the best option that was used for cloning and expressing the recombinant protein G'e in E. coli. A dissociation constant (K(d)) value of 9.4 nM for protein G'e was determined thermophoretically, showing that the N-terminal flanking sequence extension did not cause significant changes in the binding affinity to immunoglobulins.