Biochimica et biophysica acta. Proteins and proteomics最新文献

{"title":"Mapping the Periostin splice isoforms in atopic dermatitis and an in vitro asthma model – A multi-platform analysis using mass spectrometry and RT-qPCR","authors":"Christian E. Rusbjerg-Weberskov ,&nbsp;Anne Kruse Hollensen ,&nbsp;Christian Kroun Damgaard ,&nbsp;Marianne Bengtson Løvendorf ,&nbsp;Lone Skov ,&nbsp;Jan J. Enghild ,&nbsp;Nadia Sukusu Nielsen","doi":"10.1016/j.bbapap.2024.141031","DOIUrl":"10.1016/j.bbapap.2024.141031","url":null,"abstract":"<div><p>Periostin is a matricellular protein known to be alternatively spliced to produce ten isoforms with a molecular weight of 78–91 kDa. Within the extracellular matrix, periostin attaches to cell surfaces to induce signaling <em>via</em> integrin-binding and actively participates in fibrillogenesis, orchestrating the arrangement of collagen in the extracellular environment. In atopic diseases such as atopic dermatitis (AD) and asthma, periostin is known to participate in driving the disease-causing type 2 inflammation. The periostin isoforms expressed in these diseases and the implication of the alternative splicing events are unknown. Here, we present two universal assays to map the expression of periostin isoforms at the mRNA (RT-qPCR) and protein (PRM-based mass spectrometry) levels. We use these assays to study the splicing profile of periostin in AD lesions as well as in <em>in vitro</em> models of AD and asthma. In these conditions, periostin displayed overexpression with isoforms 3 and 5 standing out as highly overexpressed. Notably, isoforms 9 and 10 exhibited a divergent pattern relative to the remaining isoforms. Isoforms 9 and 10 are often overlooked in periostin research and this paper presents the first evidence of their expression at the protein level. This underlines the necessity to include isoforms 9 and 10 in future research addressing periostin splice isoforms. The assays presented in this paper hold the potential to improve our insight into the splicing profile of periostin in tissues and diseases of interest. The application of these assays to AD lesions and <em>in vitro</em> models demonstrated their potential for identifying isoforms of particular significance, warranting a further in-depth investigation.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 5","pages":"Article 141031"},"PeriodicalIF":2.5,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570963924000384/pdfft?md5=c12be11dda803cdf95887f728e83f8b0&pid=1-s2.0-S1570963924000384-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141557898","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Robust assessment of sample preparation protocols for proteomics of cells and tissues","authors":"Francielle Aguiar Gomes,&nbsp;Douglas Ricardo Souza Junior,&nbsp;Mariana Pereira Massafera,&nbsp;Graziella Eliza Ronsein","doi":"10.1016/j.bbapap.2024.141030","DOIUrl":"10.1016/j.bbapap.2024.141030","url":null,"abstract":"<div><p>In proteomic studies, the reliability and reproducibility of results hinge on well-executed protein extraction and digestion protocols. Here, we systematically compared three established digestion methods for macrophages, namely filter-assisted sample preparation (FASP), in-solution, and in-gel digestion protocols. We also compared lyophilization and manual lysis for liver tissue protein extraction, each of them tested using either sodium deoxycholate (SDC)- or RIPA-based lysis buffer. For the macrophage cell line, FASP using passivated filter units outperformed the other tested methods regarding the number of identified peptides and proteins. However, a careful standardization has shown that all three methods can yield robust results across a wide range of starting material (even starting with 1 μg of proteins). Importantly, inter and intra-day coefficients of variance (CVs) were determined for all sample preparation protocols. Thus, the median inter-day CVs for in-solution, in-gel and FASP protocols were respectively 10, 8 and 9%, very similar to the median CVs obtained for the intra-day analysis (9, 8 and 8%, respectively). Moreover, FASP digestion presented 80% of proteins with a CV lower than 25%, followed closely by in-gel digestion (78%) and in-solution sample preparation (72%) protocols. For tissue proteomics, both manual lysis and lyophilization presented similar proteome coverage and reproducibility, but the efficiency of protein extraction depended on the lysis buffer used, with RIPA buffer showing better results. In conclusion, although each sample preparation method has its own particularity, they are all suited for successful proteomic experiments if a careful standardization of the sample preparation workflow is carried out.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 5","pages":"Article 141030"},"PeriodicalIF":2.5,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141465889","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Co-chaperonin GroES subunit exchange as dependent on time, pH, protein concentration, and urea","authors":"Victor Marchenkov ,&nbsp;Alexey Surin ,&nbsp;Victor Ugarov ,&nbsp;Nina Kotova ,&nbsp;Natalia Marchenko ,&nbsp;Alexey Fedorov ,&nbsp;Alexei Finkelstein ,&nbsp;Vladimir Filimonov ,&nbsp;Gennady Semisotnov","doi":"10.1016/j.bbapap.2024.141032","DOIUrl":"10.1016/j.bbapap.2024.141032","url":null,"abstract":"<div><p>The discovery of a subunit exchange in some oligomeric proteins, implying short-term dissociation of their oligomeric structure, requires new insights into the role of the quaternary structure in oligomeric protein stability and function. Here we demonstrate the effect of pH, protein concentration, and urea on the efficiency of GroES heptamer (GroES₇) subunit exchange. A mixture of equimolar amounts of wild-type (WT) GroES₇ and its Ala97Cys mutant modified with iodoacetic acid (97-carboxymethyl cysteine or CMC-GroES₇) was incubated in various conditions and subjected to isoelectric focusing (IEF) in polyacrylamide gel. For each sample, there are eight Coomassie-stained electrophoretic bands showing different charges that result from a different number of included mutant subunits, each carrying an additional negative charge. The intensities of these bands serve to analyze the protein subunit exchange. The protein stability is evaluated using the transverse urea gradient gel electrophoresis (TUGGE). At pH 8.0, the intensities of the initial bands corresponding to WT-GroES₇ and CMC-GroES₇ are decreased with a half-time of (23 ± 2) min. The exchange decreases with decreasing pH and seems to be strongly hindered at pH 5.2 due to the protonation of groups with pK ∼ 6.3, which stabilizes the protein quaternary structure. The destabilization of the protein quaternary structure caused by increased pH, decreased protein concentration, or urea accelerates the GroES subunit exchange. This study allows visualizing the subunit exchange in oligomeric proteins and confirms its direct connection with the stability of the protein quaternary structure.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 5","pages":"Article 141032"},"PeriodicalIF":2.5,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141615872","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Plumbagin accelerates serum albumin's amyloid aggregation kinetics and generates fibril polymorphism by inducing non-native β-sheet structures","authors":"Chanchal Chauhan ,&nbsp;Poonam Singh ,&nbsp;Shivani A. Muthu ,&nbsp;Suhel Parvez ,&nbsp;Angamuthu Selvapandiyan ,&nbsp;Basir Ahmad","doi":"10.1016/j.bbapap.2024.141028","DOIUrl":"10.1016/j.bbapap.2024.141028","url":null,"abstract":"<div><p>The ligand-induced conformational switch of proteins has great significance in understanding the biophysics and biochemistry of their self-assembly. In this work, we have investigated the ability of plumbagin (PL), a hydroxynaphthoquinone compound found in the root of the medicinal plant <em>Plumbago zeylanica</em>, to modulate aggregation precursor state, aggregation kinetics and generate distinct fibril of human serum albumin (HSA). PL was found to moderately bind (binding constant K_a ∼ 10⁻⁴ M⁻¹)) to domain-II of HSA in the stoichiometric ratio of 1:1. We found that PL-HSA complex aggregation was accelerated as compared to that of HSA aggregation and it may be through an independent pathway. We also detected that fibril produced in the presence of PL is wider in diameter, contains a higher amount of β-sheet (∼18%) and disordered (∼46%) structures, and is less stable. We concluded that the acceleration of aggregation reaction and generation of fibril polymorphism was mainly because of the higher extent of unfolding and high content of non-native β-sheet structure in the aggregation precursor state of PL-HSA complex. This study offers opportunities to explore the ability of ligand binding to modulate aggregation reactions and generate polymorphic protein fibrils.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 5","pages":"Article 141028"},"PeriodicalIF":3.2,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141287674","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The regulation of the thermal stability and affinity of the HSPA5 (Grp78/BiP) by clients and nucleotides is modulated by domains coupling","authors":"Noeli S.M. Silva ,&nbsp;Bruna Siebeneichler ,&nbsp;Carlos S. Oliveira ,&nbsp;Paulo R. Dores-Silva ,&nbsp;Júlio C. Borges","doi":"10.1016/j.bbapap.2024.141034","DOIUrl":"10.1016/j.bbapap.2024.141034","url":null,"abstract":"<div><p>The HSPA5 protein (BiP/Grp78) serves as a pivotal chaperone in maintaining cellular protein quality control. As a member of the human HSP70 family, HSPA5 comprises two distinct domains: a nucleotide-binding domain (NBD) and a peptide-binding domain (PBD). In this study, we investigated the interdomain interactions of HSPA5, aiming to elucidate how these domains regulate its function as a chaperone. Our findings revealed that HSPA5-FL, HSPA5-T, and HSPA5-N exhibit varying affinities for ATP and ADP, with a noticeable dependency on Mg²⁺ for optimal interactions. Interestingly, in ADP assays, the presence of the metal ion seems to enhance NBD binding only for HSPA5-FL and HSPA5-T. Moreover, while the truncation of the C-terminus does not significantly impact the thermal stability of HSPA5, experiments involving MgATP underscore its essential role in mediating interactions and nucleotide hydrolysis. Thermal stability assays further suggested that the NBD-PBD interface enhances the stability of the NBD, more pronounced for HSPA5 than for the orthologous HSPA1A, and prevents self-aggregation through interdomain coupling. Enzymatic analyses indicated that the presence of PBD enhances NBD ATPase activity and augments its nucleotide affinity. Notably, the intrinsic chaperone activity of the PBD is dependent on the presence of the NBD, potentially due to the propensity of the PBD for self-oligomerization. Collectively, our data highlight the pivotal role of allosteric mechanisms in modulating thermal stability, nucleotide interaction, and ATPase activity of HSPA5, underscoring its significance in protein quality control within cellular environments.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 5","pages":"Article 141034"},"PeriodicalIF":2.5,"publicationDate":"2024-09-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141619204","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Human apo-metallothionein 1a is not a random coil: Evidence from guanidinium chloride, high temperature, and acidic pH unfolding studies","authors":"Natalie C. Korkola,&nbsp;Martin J. Stillman","doi":"10.1016/j.bbapap.2024.141010","DOIUrl":"10.1016/j.bbapap.2024.141010","url":null,"abstract":"<div><p>The structures of apo-metallothioneins (apo-MTs) have been relatively elusive due to their fluxional, disordered state which has been difficult to characterize. However, intrinsically disordered protein (IDP) structures are rather diverse, which raises questions about where the structure of apo-MTs fit into the protein structural spectrum. In this paper, the unfolding transitions of apo-MT1a are discussed with respect to the effect of the chemical denaturant GdmCl, temperature conditions, and pH environment. Cysteine modification in combination with electrospray ionization mass spectrometry was used to probe the unfolding transition of apo-MT1a in terms of cysteine exposure. Circular dichroism spectroscopy was also used to monitor the change in secondary structure as a function of GdmCl concentration. For both of these techniques, cooperative unfolding was observed, suggesting that apo-MT1a is not a random coil. More GdmCl was required to unfold the protein backbone than to expose the cysteines, indicating that cysteine exposure is likely an early step in the unfolding of apo-MT1a. MD simulations complement the experimental results, suggesting that apo-MT1a adopts a more compact structure than expected for a random coil. Overall, these results provide further insight into the intrinsically disordered structure of apo-MT.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141010"},"PeriodicalIF":3.2,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570963924000177/pdfft?md5=1e2b99a81d2977a21cf2abac24f96759&pid=1-s2.0-S1570963924000177-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140136421","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Conformational and dynamic properties of the KH1 domain of FMRP and its fragile X syndrome linked G266E variant","authors":"Flavia Catalano ,&nbsp;Daniele Santorelli ,&nbsp;Alessandra Astegno ,&nbsp;Filippo Favretto ,&nbsp;Marco D'Abramo ,&nbsp;Alessandra Del Giudice ,&nbsp;Maria Laura De Sciscio ,&nbsp;Francesca Troilo ,&nbsp;Giorgio Giardina ,&nbsp;Adele Di Matteo ,&nbsp;Carlo Travaglini-Allocatelli","doi":"10.1016/j.bbapap.2024.141019","DOIUrl":"https://doi.org/10.1016/j.bbapap.2024.141019","url":null,"abstract":"<div><p>The Fragile X messenger ribonucleoprotein (FMRP) is a multi-domain protein involved in interactions with various macromolecules, including proteins and coding/non-coding RNAs. The three KH domains (KH0, KH1 and KH2) within FMRP are recognized for their roles in mRNA binding. In the context of Fragile X syndrome (FXS), over-and-above CGG triplet repeats expansion, three specific point mutations have been identified, each affecting one of the three KH domains (^R138QKH0, ^G266EKH1, and ^I304NKH2) resulting in the expression of non-functional FMRP. This study aims to elucidate the molecular mechanism underlying the loss of function associated with the ^G266EKH1 pathological variant. We investigate the conformational and dynamic properties of the isolated KH1 domain and the two KH1 site-directed mutants ^G266EKH1 and ^G266AKH1. Employing a combined <em>in vitro</em> and <em>in silico</em> approach, we reveal that the ^G266EKH1 variant lacks the characteristic features of a folded domain. This observation provides an explanation for functional impairment observed in FMRP carrying the G266E mutation within the KH1 domain, as it renders the domain unable to fold properly. Molecular Dynamics simulations suggest a pivotal role for residue 266 in regulating the structural stability of the KH domains, primarily through stabilizing the α-helices of the domain. Overall, these findings enhance our comprehension of the molecular basis for the dysfunction associated with the ^G266EKH1 variant in FMRP.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141019"},"PeriodicalIF":3.2,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140645123","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A role of methionines in the functioning of oxidatively modified fibrinogen","authors":"L.V. Yurina ,&nbsp;A.D. Vasilyeva ,&nbsp;E.S. Gavrilina ,&nbsp;V.S. Ivanov ,&nbsp;S.I. Obydennyi ,&nbsp;I.A. Chabin ,&nbsp;M.I. Indeykina ,&nbsp;A.S. Kononikhin ,&nbsp;E.N. Nikolaev ,&nbsp;M.A. Rosenfeld","doi":"10.1016/j.bbapap.2024.141013","DOIUrl":"https://doi.org/10.1016/j.bbapap.2024.141013","url":null,"abstract":"<div><p>Posttranslational modifications in fibrinogen resulting from induced oxidation or oxidative stress in the organism can have deleterious influence on optimal functioning of fibrinogen, causing a disturbance in assembly and properties of fibrin. The protective mechanism supporting the ability of fibrinogen to function in ROS-generating environment remains completely unexplored. The effects of very low and moderately low HOCl/⁻OCl concentrations on fibrinogen oxidative modifications, the fibrin network structure as well as the kinetics of both fibrinogen-to-fibrin conversion and fibrin hydrolysis have been explored in the current study. As opposed to 25 Μm, HOCl/⁻OCl, 10 μM HOCl/⁻OCl did not affect the functional activity of fibrinogen. It is shown for the first time that a number of Met residues, AαMet476, AαMet517, AαMet584, BβMet367, γMet264, and γMet94, identified in 10 μM HOCl/–OCl fibrinogen by the HPLC-MS/MS method, operate as ROS scavengers, performing an important antioxidant function. In turn, this indicates that the fibrinogen structure is adapted to the detrimental action of ROS. The results obtained in our study provide evidence for a protective mechanism responsible for maintaining the structure and functioning of fibrinogen molecules in the bloodstream under conditions of mild and moderate oxidative stress.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141013"},"PeriodicalIF":3.2,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140539713","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Active roles of lytic polysaccharide monooxygenases in human pathogenicity","authors":"Daniel Kracher ,&nbsp;Tina Lanzmaier ,&nbsp;Leonor Vieira Carneiro","doi":"10.1016/j.bbapap.2024.141012","DOIUrl":"10.1016/j.bbapap.2024.141012","url":null,"abstract":"<div><p>Lytic polysaccharide monooxygenases (LPMOs) are redox enzymes widely studied for their involvement in microbial and fungal biomass degradation. The catalytic versatility of these enzymes is demonstrated by the recent discovery of LPMOs in arthropods, viruses, insects and ferns, where they fulfill diverse functions beyond biomass conversion. This mini-review puts a spotlight on a recently recognized aspect of LPMOs: their role in infectious processes in human pathogens. It discusses the occurrence and potential biological mechanisms of LPMOs associated with human pathogens and provides an outlook on future avenues in this emerging and exciting research field.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141012"},"PeriodicalIF":3.2,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570963924000190/pdfft?md5=2314ad039f36a086c7f7968919f837c0&pid=1-s2.0-S1570963924000190-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140139795","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls","authors":"Daniel Nyberg Larsen ,&nbsp;Jakub Zbigniew Kaczmarek ,&nbsp;Yaseelan Palarasah ,&nbsp;Jonas Heilskov Graversen ,&nbsp;Peter Højrup","doi":"10.1016/j.bbapap.2024.141011","DOIUrl":"10.1016/j.bbapap.2024.141011","url":null,"abstract":"<div><p>Understanding protein-protein interactions is crucial for drug design and investigating biological processes. Various techniques, such as CryoEM, X-ray spectroscopy, linear epitope mapping, and mass spectrometry-based methods, can be employed to map binding regions on proteins. Commonly used mass spectrometry-based techniques are cross-linking and hydrogen‑deuterium exchange (HDX). Another approach, hydroxyl radical protein footprinting (HRPF), identifies binding residues on proteins but faces challenges due to high initial costs and complex setups.</p><p>This study introduces a generally applicable method using Fenton chemistry for epitope mapping in a standard mass spectrometry laboratory. It emphasizes the importance of controls, particularly the inclusion of a negative antibody control, not widely utilized in HRPF epitope mapping. Quantification by TMT labelling is introduced to reduce false positives, enabling direct comparison between sample conditions and biological triplicates. Additionally, six technical replicates were incorporated to enhance the depth of analysis.</p><p>Observations on the receptor-binding domain (RBD) of SARS-CoV-2 Spike Protein, Alpha and Delta variants, revealed both binding and opening regions. Significantly changed peptides upon mixing with a negative control antibody suggested structural alterations or nonspecific binding induced by the antibody alone. Integration of negative control antibody experiments and high overlap between biological triplicates led to the exclusion of 40% of significantly changed regions. The final identified binding region correlated with existing literature on neutralizing antibodies against RBD.</p><p>The presented method offers a straightforward implementation for HRPF analysis in a generic mass spectrometry-based laboratory. Enhanced data reliability was achieved through increased technical and biological replicates alongside negative antibody controls.</p></div>","PeriodicalId":8760,"journal":{"name":"Biochimica et biophysica acta. Proteins and proteomics","volume":"1872 4","pages":"Article 141011"},"PeriodicalIF":3.2,"publicationDate":"2024-07-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1570963924000189/pdfft?md5=e1464e1e200d56909b302bd9a8c71d90&pid=1-s2.0-S1570963924000189-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140157531","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}