Analysis of Polypeptides by Amino Acid Analysis.

Amino acid analysis is an accurate method for the composition and quantitation of polypeptides and among these synthetic peptides. Combined with mass spectrometry, it yields a reliable control of peptide quality and quantity prior to conjugation and immunization.Initially peptides are hydrolyzed, preferably in the gas phase, with 6-M HCl at 110 °C for 20-24 h and the resulting amino acids analyzed by chromatography, where the most reliable form is ion exchange chromatography with post-column ninhydrin derivatization. Depending on the hydrolysis conditions, tryptophan is destroyed, and likewise cysteine, unless derivatized, and the amides, glutamine, and asparagine are deamidated to glutamic acid and aspartic acid, respectively. Three different ways of calculating results are suggested, and taking the above limitations into account, a quantitation better than 5% can usually be obtained.

Characterization of Synthetic Peptides by Mass Spectrometry.

In the quality control of synthetic peptides, mass spectroscopy (MS) serves as an optimal method for evaluating authenticity and integrity. Typically, the sequence of a synthetic peptide is already established, thereby directing the focus of analysis towards validating its identity and purity. This chapter outlines straightforward methodologies for conducting MS analyses specifically tailored for synthetic peptides.

Interpretation of Tandem Mass Spectrometry (MS-MS) Spectra for Peptide Analysis.

The aim of this chapter is to give a short introduction to peptide analysis by mass spectrometry (MS) and interpretation of fragment mass spectra. Through examples and guidelines, we will demonstrate how to understand and validate search results and how to perform de novo sequencing based on the often very complex fragmentation pattern obtained by tandem mass spectrometry (also referred to as MSMS). The focus will be on simple rules for interpretation of MSMS spectra of tryptic as well as non-tryptic peptides.

Increased Protein Kinase A Activity Induces Fibrolamellar Hepatocellular Carcinoma Features Independent of DNAJB1.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare liver cancer that is driven by a fusion of DNAJB1 and PRKACA, the catalytic subunit of protein kinase A (PKA). PKA activity is controlled through regulatory proteins that both inhibit catalytic activity and control localization, and an excess of regulatory subunits ensures PRKACA activity is inhibited. Here, we found an increase in the ratio of catalytic to regulatory units in FLC patient tumors driven by DNAJB1::PRKACA using mass spectrometry, biochemistry, and immunofluorescence, with increased nuclear localization of the kinase. Overexpression of DNAJB1::PRKACA, ATP1B1::PRKACA, or PRKACA, but not catalytically inactive kinase, caused similar transcriptomic changes in primary human hepatocytes, recapitulating the changes observed in FLC. Consistently, tumors in patients missing a regulatory subunit or harboring an ATP1B1::PRKACA fusion were indistinguishable from FLC based on the histopathological, transcriptomic, and drug-response profiles. Together, these findings indicate that the DNAJB1 domain of DNAJB1::PRKACA is not required for FLC. Instead, changes in PKA activity and localization determine the FLC phenotype.

Epitope mapping of SARS-CoV-2 RBDs by hydroxyl radical protein footprinting reveals the importance of including negative antibody controls.

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 hydrogen­deuterium 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. 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. 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. 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.

Citrullination of myelin basic protein induces a Th17-cell response in healthy individuals and enhances the presentation of MBP85-99 in patients with multiple sclerosis.

The post-translational modification citrullination has been proposed to play a role in the pathogenesis of multiple sclerosis (MS). Myelin basic protein (MBP) is a candidate autoantigen which is citrullinated to a minor extent under physiological conditions and hypercitrullinated in MS. We examined immune cell responses elicited by hypercitrullinated MBP (citMBP) in cultures of mononuclear cells from 18 patients with MS and 42 healthy donors (HDs). The immunodominant peptide of MBP, MBP85-99, containing citrulline in position 99, outcompeted the binding of native MBP85-99 to HLA-DR15, which is strongly linked to MS. Moreover, using the monoclonal antibody MK16 as probe, we observed that B cells and monocytes from HLA-DR15+ patients with MS presented MBP85-99 more efficiently after challenge with citMBP than with native MBP. Both citMBP and native MBP induced proliferation of CD4+ T cells from patients with MS as well as TNF-α production by their B cells and CD4+ T cells, and citrullination of MBP tended to enhance TNF-α secretion by CD4+ T cells from HLA-DR15+ patients. Unlike native MBP, citMBP induced differentiation into Th17 cells in cultures from HDs, while neither form of MBP induced Th17-cell differentiation in cultures from patients with MS. These data suggest a role for citrullination in the breach of tolerance to MBP in healthy individuals and in maintenance of the autoimmune response to MBP in patients with MS.

Elevated Antibody Titers to Epstein-Barr Virus and Cytomegalovirus in Patients with Drug-Induced Lupus.

Systemic lupus erythematosus (SLE) is an autoimmune disease, which has been associated with Epstein-Barr virus (EBV) and Cytomegalovirus (CMV) infection. Drug-induced lupus (DIL) is a lupus-like disease caused by the intake of therapeutic drugs, which has been estimated to cause approximately 10-15% of lupus-like cases. Although SLE and DIL share common clinical symptoms, there are some fundamental differences between DIL and SLE onset. Moreover, it remains to be examined whether environmental factors, such as EBV and CMV infections, may contribute to the development of DIL. This study focused on examining the possible association between DIL and EBV and CMV infections, by examining IgG titers to EBV and CMV antigens in serum samples by enzyme-linked immunosorbent assays. Antibody titers to EBV early antigen-diffuse and CMV pp52 were found to be significantly elevated in both SLE and DIL patients compared to healthy controls, although no correlation was found for antibodies to the two virus antigens in the respective disease groups. Moreover, total IgG titers were reduced in SLE and DIL serum samples, which may reflect a general lymphocytopenia, which commonly is associated with SLE. The current findings support that EBV and CMV infections may contribute to the development of DIL and that onset of both diseases are related.

Defensin Interactions in Relation to Monoclonal and Disease-Related Proteinase 3 Antibodies Binding at the Catalytic Site.

Proteinase 3 (PR3) is a neutrophil granulocyte enzyme and an autoantigen found in several forms of vasculitis. Due to the diagnostic and clinical importance of antibodies (Abs) to PR3, it is important to characterize the protein and the nature of its epitopes. Here, we have characterized PR3 monoclonal antibodies (MAbs) and disease-associated Abs and their dependency on the PR3 structure and modifications, especially interactions with α-defensins. Three MAbs (HYB 172-01, 172-04, 172-05), which bind to PR3 in its native and denatured forms and provide the disulphide bridges, were intact. α-1-antitrypsin (AT) binds to purified human neutrophil granulocyte PR3 and inhibits its proteolytic activity, towards a small synthetic peptide substrate and a large protein substrate (casein). AT also inhibited the binding of the three MAbs to PR3, indicating that they bind in a region affected by AT binding. However, the MAbs did not inhibit PR3 proteolytic activity with a small substrate, showing that they bound at the active site without restricting access to the substrate cleft. Patient-derived Abs showed essentially the same characteristics as the MAbs, with important implications for vasculitis diagnostics and pathophysiology. Current findings illustrate that PR3 epitopes depend on the three-dimensional structure of the PR3/defensin complex, and that the epitopes depend to a smaller or larger degree on PR3/defensin associations.

Haptoglobin-related protein in human plasma correlates to haptoglobin concentrations and phenotypes.

Haptoglobin-related protein (Hpr) is a plasma protein with high sequence similarity to haptoglobin (Hp). Like Hp, Hpr also binds hemoglobin (Hb) with high affinity, but it does not bind to the Hb-Hp receptor CD163 on macrophages. The Hpr concentration is markedly lower than Hp in plasma and its regulation is not understood. In the present study, we have developed non-crossreactive antibodies to Hpr to analyze the Hpr concentration in 112 plasma samples from anonymized individuals and compared it to Hp. The results show that plasma Hpr correlated with Hp concentrations (rho = 0.46, p = .0001). Hpr accounts for on average 0.35% of the Hp/Hpr pool but up to 29% at low Hp levels. Furthermore, the Hpr concentrations were significantly lower in individuals with the Hp2-2 phenotype compared to those with the Hp2-1 or Hp1-1 phenotypes. Experimental binding analysis did not provide evidence that Hpr associates with Hp and in this way is removed via CD163. In conclusion, the Hpr concentration correlates to Hp concentrations and Hp-phenotypes by yet unknown mechanisms independent of CD163-mediated removal of Hb-Hp complexes.

Production and Characterization of Peptide Antibodies to the C-Terminal of Frameshifted Calreticulin Associated with Myeloproliferative Diseases.

Myeloproliferative Neoplasms (MPNs) constitute a group of rare blood cancers that are characterized by mutations in bone marrow stem cells leading to the overproduction of erythrocytes, leukocytes, and thrombocytes. Mutations in calreticulin (CRT) genes may initiate MPNs, causing a novel variable polybasic stretch terminating in a common C-terminal sequence in the frameshifted CRT (CRTfs) proteins. Peptide antibodies to the mutated C-terminal are important reagents for research in the molecular mechanisms of MPNs and for the development of new diagnostic assays and therapies. In this study, eight peptide antibodies targeting the C-terminal of CRTfs were produced and characterised by modified enzyme-linked immunosorbent assays using resin-bound peptides. The antibodies reacted to two epitopes: CREACLQGWTE for SSI-HYB 385-01, 385-02, 385-03, 385-04, 385-07, 385-08, and 385-09 and CLQGWT for SSI-HYB 385-06. For the majority of antibodies, the residues Cys1, Trp9, and Glu11 were essential for reactivity. SSI-HYB 385-06, with the highest affinity, recognised recombinant CRTfs produced in yeast and the MARIMO cell line expressing CRTfs when examined in Western immunoblotting. Moreover, SSI-HYB 385-06 occasionally reacted to CRTfs from MPN patients when analysed by flow cytometry. The characterized antibodies may be used to understand the role of CRTfs in the pathogenesis of MPNs and to design and develop new diagnostic assays and therapeutic targets.

Tyrosine-sulfated dermatopontin shares multiple binding sites and recognition determinants on triple-helical collagens with proteins implicated in cell adhesion and collagen folding, fibrillogenesis, cross-linking, and degradation.

Dermatopontin (DPT), a small extracellular matrix protein that stimulates collagen fibrillogenesis, contains sulfotyrosine residues but neither its level of sulfation nor its binding sites on fibrillar collagens are known. Here, we discovered that DPT is present in a relatively high mass concentration (~ 0.02%) in porcine corneal stroma, from which we purified five DPT charge variants (A-E) containing up to six sulfations. The major variant (C), containing four sulfotyrosine residues, was used to locate binding sites for DPT on triple-helical collagens II and III using the Collagen Toolkits. DPT-binding loci included the triple helix crosslinking sites and collagenase cleavage site. We find that strong DPT-binding sites on triple-helical collagen comprise an arginine-rich, positively-charged sequence that also contains hydrophobic residues. This collagen-binding signature of DPT is similar to that of the chaperone HSP47. Thus, we propose that DPT assumes the role of HSP47 as a collagen chaperone during and after the secretion. Peptide II-44, harbouring the conserved collagenase cleavage site, shows the strongest DPT-binding of the Collagen Toolkit II peptides. Substituting any of the three arginine residues (R) with alanine in the sequence GLAGQRGIVGLOGQRGER of II-44 resulted in almost complete loss of DPT binding. Since osteogenesis imperfecta, spondyloepiphyseal dysplasia, and spondyloepimetaphyseal dysplasia congenita are associated with missense mutations that substitute the corresponding arginine residues in collagens alpha-1(I) and alpha-1(II), we suggest that disrupted DPT binding to fibrillar collagens may contribute to these connective tissue disorders. In conclusion, the present work provides a cornerstone for further elucidation of the role of DPT.

Comparative Analysis of Type 1 and Type Z Protein Phosphatases Reveals D615 as a Key Residue for Ppz1 Regulation.

Type 1 Ser/Thr protein phosphatases are represented in all fungi by two enzymes, the ubiquitous PP1, with a conserved catalytic polypeptide (PP1c) and numerous regulatory subunits, and PPZ, with a C-terminal catalytic domain related to PP1c and a variable N-terminal extension. Current evidence indicates that, although PP1 and PPZ enzymes might share some cellular targets and regulatory subunits, their functions are quite separated, and they have individual regulation. We explored the structures of PP1c and PPZ across 57 fungal species to identify those features that (1) are distinctive among these enzymes and (2) have been preserved through evolution. PP1c enzymes are more conserved than PPZs. Still, we identified 26 residues in the PP1 and PPZ catalytic moieties that are specific for each kind of phosphatase. In some cases, these differences likely affect the distribution of charges in the surface of the protein. In many fungi, Hal3 is a specific inhibitor of the PPZ phosphatases, although the basis for the interaction of these proteins is still obscure. By in vivo co-purification of the catalytic domain of ScPpz1 and ScHal3, followed by chemical cross-linking and MS analysis, we identified a likely Hal3-interacting region in ScPpz1 characterized by two major and conserved differences, D566 and D615 in ScPpz1, which correspond to K210 and K259 in ScPP1c (Glc7). Functional analysis showed that changing D615 to K renders Ppz1 refractory to Hal3 inhibition. Since ScHal3 does not regulate Glc7 but it inhibits all fungal PPZ tested so far, this conserved D residue could be pivotal for the differential regulation of both enzymes in fungi.

Mapping human calreticulin regions important for structural stability.

Calreticulin (CALR) is a highly conserved multifunctional chaperone protein primarily present in the endoplasmic reticulum, where it regulates Ca2+ homeostasis. Recently, CALR has gained special interest for its diverse functions outside the endoplasmic reticulum, including the cell surface and extracellular space. Although high-resolution structures of CALR exist, it has not yet been established how different regions and individual amino acid residues contribute to structural stability of the protein. In the present study, we have identified key residues determining the structural stability of CALR. We used a Saccharomyces cerevisiae expression system to express and purify 50 human CALR mutants, which were analysed for several parameters including secretion titer, melting temperature (Tm), stability and oligomeric state. Our results revealed the importance of a previously identified small patch of conserved surface residues, amino acids 166-187 ("cluster 2") for structural stability of the human CALR protein. Two residues, Tyr172 and Asp187, were critical for maintaining the native structure of the protein. Mutant D187A revealed a severe drop in secretion titer, it was thermally unstable, prone to degradation, and oligomer formation. Tyr172 was critical for thermal stability of CALR and interacted with the third free Cys163 residue. This illustrates an unusual thermal stability of CALR dominated by Asp187, Tyr172 and Cys163, which may interact as part of a conserved structural unit. Besides structural clusters, we found a correlation of some measured parameter values in groups of CALR mutants that cause myeloproliferative neoplasms (MPN) and in mutants that may be associated with sudden unexpected death (SUD).

Epitope Mapping of Monoclonal Antibodies to Calreticulin Reveals That Charged Amino Acids Are Essential for Antibody Binding.

Calreticulin is a chaperone protein, which is associated with myeloproliferative diseases. In this study, we used resin-bound peptides to characterize two monoclonal antibodies (mAbs) directed to calreticulin, mAb FMC 75 and mAb 16, which both have significantly contributed to understanding the biological function of calreticulin. The antigenicity of the resin-bound peptides was determined by modified enzyme-linked immunosorbent assay. Specific binding was determined to an 8-mer epitope located in the N-terminal (amino acids 34-41) and to a 12-mer peptide located in the C-terminal (amino acids 362-373). Using truncated peptides, the epitopes were identified as TSRWIESK and DEEQRLKEEED for mAb FMC 75 and mAb 16, respectively, where, especially the charged amino acids, were found to have a central role for a stable binding. Further studies indicated that the epitope of mAb FMC 75 is assessable in the oligomeric structure of calreticulin, making this epitope a potential therapeutic target.

Structural Analysis of Calreticulin, an Endoplasmic Reticulum-Resident Molecular Chaperone.

Calreticulin (Calr) is an endoplasmic reticulum (ER) chaperone involved in protein quality control, Ca2+ regulation and other cellular processes. The structure of Calr is unusual, reflecting different functions of the protein: a proline-rich ß-hairpin arm and an acidic C-terminal tail protrude from a globular core, composed of a ß-sheet sandwich and an α-helix. The arm and tail interact in the presence of Ca2+ and cover the upper ß-sheet, where a carbohydrate-binding site gives the chaperone glycoprotein affinity. At the edge of the carbohydrate-binding site is a conserved, strained disulphide bridge, formed between C106 and C137 of human Calr, which lies in a polypeptide-binding site. The lower ß-sheet has several conserved residues, comprised of a characteristic triad, D166-H170-D187, Tyr172 and the free C163. In addition to its role in the ER, Calr translocates to the cell surface upon stress and functions as an immune surveillance marker. In some myeloproliferative neoplasms, the acidic Ca2+-binding C-terminal tail is transformed into a polybasic sequence.

Cannabidiol is an effective helper compound in combination with bacitracin to kill Gram-positive bacteria.

The cannabinoid cannabidiol (CBD) is characterised in this study as a helper compound against resistant bacteria. CBD potentiates the effect of bacitracin (BAC) against Gram-positive bacteria (Staphylococcus species, Listeria monocytogenes, and Enterococcus faecalis) but appears ineffective against Gram-negative bacteria. CBD reduced the MIC value of BAC by at least 64-fold and the combination yielded an FIC index of 0.5 or below in most Gram-positive bacteria tested. Morphological changes in S. aureus as a result of the combination of CBD and BAC included several septa formations during cell division along with membrane irregularities. Analysis of the muropeptide composition of treated S. aureus indicated no changes in the cell wall composition. However, CBD and BAC treated bacteria did show a decreased rate of autolysis. The bacteria further showed a decreased membrane potential upon treatment with CBD; yet, they did not show any further decrease upon combination treatment. Noticeably, expression of a major cell division regulator gene, ezrA, was reduced two-fold upon combination treatment emphasising the impact of the combination on cell division. Based on these observations, the combination of CBD and BAC is suggested to be a putative novel treatment in clinical settings for treatment of infections with antibiotic resistant Gram-positive bacteria.

Citrullinome of Porphyromonas gingivalis Outer Membrane Vesicles: Confident Identification of Citrullinated Peptides.

Porphyromonas gingivalis is a key pathogen in chronic periodontitis and has recently been mechanistically linked to the development of rheumatoid arthritis via the activity of peptidyl arginine deiminase generating citrullinated epitopes in the periodontium. In this project the outer membrane vesicles (OMV) from P. gingivalis W83 wild-type (WT), a W83 knock-out mutant of peptidyl arginine deiminase (ΔPPAD), and a mutant strain expressing PPAD with the active site cysteine mutated to alanine (C351A), have been analyzed using a two-dimensional HFBA-based separation system combined with LC-MS. For optimal and positive identification and validation of citrullinated peptides and proteins, high resolution mass spectrometers and strict MS search criteria were utilized. This may have compromised the total number of identified citrullinations but increased the confidence of the validation. A new two-dimensional separation system proved to increase the strength of validation, and along with the use of an in-house build program, Citrullia, we establish a fast and easy semi-automatic (manual) validation of citrullinated peptides. For the WT OMV we identified 78 citrullinated proteins having a total of 161 citrullination sites. Notably, in keeping with the mechanism of OMV formation, the majority (51 out of 78) of citrullinated proteins were predicted to be exported via the inner membrane and to reside in the periplasm or being translocated to the bacterial surface. Citrullinated surface proteins may contribute to the pathogenesis of rheumatoid arthritis. For the C351A-OMV a single citrullination site was found and no citrullinations were identified for the ΔPPAD-OMV, thus validating the unbiased character of our method of citrullinated peptide identification.

Immunoglobulin G structure and rheumatoid factor epitopes.

Antibodies are important for immunity and exist in several classes (IgM, IgD, IgA, IgG, IgE). They are composed of symmetric dimeric molecules with two antigen binding regions (Fab) and a constant part (Fc), usually depicted as Y-shaped molecules. Rheumatoid factors found in patients with rheumatoid arthritis are autoantibodies binding to IgG and paradoxically appear to circulate in blood alongside with their antigen (IgG) without reacting with it. Here, it is shown that rheumatoid factors do not react with native IgG in solution, and that their epitopes only become accessible upon certain physico-chemical treatments (e.g. heat treatment at 57 °C), by physical adsorption on a hydrophobic surface or by antigen binding. Moreover, chemical cross-linking in combination with mass spectrometry showed that the native state of IgG is a compact (closed) form and that the Fab parts of IgG shield the Fc region and thereby control access of rheumatoid factors and presumably also some effector functions. It can be inferred that antibody binding to pathogen surfaces induces a conformational change, which exposes the Fc part with its effector sites and rheumatoid factor epitopes. This has strong implications for understanding antibody structure and physiology and necessitates a conceptual reformulation of IgG models.

Enhanced trypsin on a budget: Stabilization, purification and high-temperature application of inexpensive commercial trypsin for proteomics applications.

Trypsin is by far the most commonly used protease in proteomics. Even though the amount of protease used in each experiment is very small, digestion of large amounts of protein prior to enrichment can be rather costly. The price of commercial trypsin is highly dependent on the quality of the enzyme, which is determined by its purity, activity, and chemical modifications. In this study we evaluated several strategies for improving the quality of crude trypsin by reductive methylation and affinity purification. We present a protocol applicable to most proteomics laboratories for obtaining a highly stable and pure trypsin preparation using reductive methylation and purification by benzamidine-sepharose. The entire workflow can be performed within a day and yields ~4 mg per batch but is completely scalable. The methylated product was benchmarked against sequencing grade trypsin from Promega and they were found to be comparable for one hour digestions at elevated temperatures, where residual chymotryptic activity was found to be negligible.

An IgY-based immunoassay to evaluate the biomarker potential of the Tannerella forsythia virulence factor karilysin in human saliva.

Tannerella forsythia is a gram-negative anaerobic bacterium that is associated with the development of destructive periodontal disease. T. forsythia secretes the metalloprotease-like enzyme karilysin. Using in vitro systems karilysin has been shown to modulate the host immune response by degradation of complement system proteins and by inactivation of the antimicrobial peptide LL-37 by proteolytic cleavage. This makes karilysin a highly interesting virulence factor to study in the framework of drug development and diagnostics. However, to date the presence of karilysin in clinical samples has not been demonstrated due to the lack of specific probes. In the present work, a high titer and stable affinity-purified avian IgY antibody against karilysin was developed. By surface plasmon resonance imaging the IgY affinity was found to be in the low nanomolar range. The antibody could be used to detect karilysin in saliva samples by immuno-blotting and was specific when tested towards human MMP-3. Furthermore, an avian IgY-based immunoassay was developed, which demonstrated low intra- and interday assay variability (CV's below 10%). Application of the immunoassay on a well-characterized set of saliva samples from adolescents with or without signs of periodontitis showed that it was possible to detect karilysin in saliva. A significant difference in karilysin concentration was found between saliva from participants with signs of periodontitis and saliva from healthy controls (p = .0024). The median of karilysin levels among periodontitis cases was 957 pg/ml (IQR, 499-2132 pg/ml) and the median for controls was 569 pg/ml (IQR, 210-1343 pg/ml). Collectively our data confirm the presence of karilysin in clinical samples. The described IgY-based immunoassay may prove useful as part of protein-based biomarker screenings in the clinic or in point-of care settings.