Self-Assembly of a Novel Pentapeptide into Hydrogelated Dendritic Architecture: Synthesis, Properties, Molecular Docking and Prospective Applications.

Currently, ultrashort oligopeptides consisting of fewer than eight amino acids represent a cutting-edge frontier in materials science, particularly in the realm of hydrogel formation. By employing solid-phase synthesis with the Fmoc/tBu approach, a novel pentapeptide, FEYNF-NH2, was designed, inspired by a previously studied sequence chosen from hen egg-white lysozyme (FESNF-NH2). Qualitative peptide analysis was based on reverse-phase high performance liquid chromatography (RP-HPLC), while further purification was accomplished using solid-phase extraction (SPE). Exact molecular ion confirmation was achieved by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-ToF MS) using two different matrices (HCCA and DHB). Additionally, the molecular ion of interest was subjected to tandem mass spectrometry (MS/MS) employing collision-induced dissociation (CID) to confirm the synthesized peptide structure. A combination of research techniques, including Fourier-transform infrared spectroscopy (FTIR), fluorescence analysis, transmission electron microscopy, polarized light microscopy, and Congo red staining assay, were carefully employed to glean valuable insights into the self-assembly phenomena and gelation process of the modified FEYNF-NH2 peptide. Furthermore, molecular docking simulations were conducted to deepen our understanding of the mechanisms underlying the pentapeptide's supramolecular assembly formation and intermolecular interactions. Our study provides potential insights into amyloid research and proposes a novel peptide for advancements in materials science. In this regard, in silico studies were performed to explore the FEYNF peptide's ability to form polyplexes.

Michael Przybylski (1948-2023) Devoted Half a Century to Mass Spectrometry.

Michael Przybylski (1948-2023) was a Polymer Chemist by training and devoted nearly his entire scientific life, almost 50 years, to mass spectrometry and its biomedical applications. After earning his PhD in Chemistry, there followed a Postdoc stay at the National Cancer Institute, Bethesda, MD, USA, and his habilitation at the University of Mainz, Germany. Soon thereafter, Michael Przybylski took the Chair for Analytical Chemistry at the University of Konstanz, Germany, where he served as Director of the Analytical Chemistry and Biopolymer Structure Analysis Laboratory. As Emeritus Michael Przybylski moved the Steinbeis Centre for Biopolymer Analytics and Biomedical Mass Spectrometry to Rüsselsheim, Germany. Michael Przybylski's research was from the beginning interdisciplinary-oriented and in many ways groundbreaking: leading to over 400 scientific papers published in internationally renowned journals and to about 25 patents. Michael Przybylski gave approximately 150 invited lectures and was awarded several scientific prizes. In recognition of his outstanding achievements and fruitful collaboration, he received the Doctorate of honor from the "Alexandru Ioan Cuza" University of Iasi, Romania. Michael Przybylski was the Director of the by him founded "Biopolymer Analytics and Biomedical Mass Spectrometry" research center until his sudden and unexpected death.

An update on multiplexed mass spectrometry-based lysosomal storage disease diagnosis.

Lysosomal storage disorders (LSDs) are a type of inherited metabolic disorders in which biomolecules, accumulate as a specific substrate in lysosomes due to specific individual enzyme deficiencies. Despite the fact that LSDs are incurable, various approaches, including enzyme replacement therapy, hematopoietic stem cell transplantation, or gene therapy are now available. Therefore, a timely diagnosis is a critical initial step in patient treatment. The-state-of-the-art in LSD diagnostic uses, in the first stage, enzymatic activity determination by fluorimetry or by mass spectrometry (MS) with the aid of dry blood spots, based on different enzymatic substrate structures. Due to its sensitivity, high precision, and ability to screen for an unprecedented number of diseases in a single assay, multiplexed tandem MS-based enzyme activity assays for the screening of LSDs in newborns have recently received a lot of attention. Here, (i) we review the current approaches used for simultaneous enzymatic activity determination of LSDs in dried blood spots using multiplex-LC-MS/MS; (ii) we explore the need for designing novel enzymatic substrates that generate different enzymatic products with distinct molecular masses in multiplexed-MS studies; and (iii) we give examples of the relevance of affinity-MS technique as a basis for reversing undesirable immune-reactivity in enzyme replacement therapy.

Applications of MALDI-MS/MS-Based Proteomics in Biomedical Research.

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) is one of the most widely used techniques in proteomics to achieve structural identification and characterization of proteins and peptides, including their variety of proteoforms due to post-translational modifications (PTMs) or protein-protein interactions (PPIs). MALDI-MS and MALDI tandem mass spectrometry (MS/MS) have been developed as analytical techniques to study small and large molecules, offering picomole to femtomole sensitivity and enabling the direct analysis of biological samples, such as biofluids, solid tissues, tissue/cell homogenates, and cell culture lysates, with a minimized procedure of sample preparation. In the last decades, structural identification of peptides and proteins achieved by MALDI-MS/MS helped researchers and clinicians to decipher molecular function, biological process, cellular component, and related pathways of the gene products as well as their involvement in pathogenesis of diseases. In this review, we highlight the applications of MALDI ionization source and tandem approaches for MS for analyzing biomedical relevant peptides and proteins. Furthermore, one of the most relevant applications of MALDI-MS/MS is to provide "molecular pictures", which offer in situ information about molecular weight proteins without labeling of potential targets. Histology-directed MALDI-mass spectrometry imaging (MSI) uses MALDI-ToF/ToF or other MALDI tandem mass spectrometers for accurate sequence analysis of peptide biomarkers and biological active compounds directly in tissues, to assure complementary and essential spatial data compared with those obtained by LC-ESI-MS/MS technique.

Proteomics-Based Identification of Dysregulated Proteins in Breast Cancer.

Immunohistochemistry (IHC) is still widely used as a morphology-based assay for in situ analysis of target proteins as specific tumor antigens. However, as a very heterogeneous collection of neoplastic diseases, breast cancer (BC) requires an accurate identification and characterization of larger panels of candidate biomarkers, beyond ER, PR, and HER2 proteins, for diagnosis and personalized treatment, without the limited availability of antibodies that are required to identify specific proteins. Top-down, middle-down, and bottom-up mass spectrometry (MS)-based proteomics approaches complement traditional histopathological tissue analysis to examine expression, modification, and interaction of hundreds to thousands of proteins simultaneously. In this review, we discuss the proteomics-based identification of dysregulated proteins in BC that are essential for the following issues: discovery and validation of new biomarkers by analysis of solid and liquid/non-invasive biopsies, cell lines, organoids and xenograft models; identification of panels of biomarkers for early detection and accurate discrimination between cancer, benign and normal tissues; identification of subtype-specific and stage-specific protein expression profiles in BC grading and measurement of disease progression; characterization of new subtypes of BC; characterization and quantitation of post-translational modifications (PTMs) and aberrant protein-protein interactions (PPI) involved in tumor development; characterization of the global remodeling of BC tissue homeostasis, diagnosis and prognostic information; and deciphering of molecular functions, biological processes and mechanisms through which the dysregulated proteins cause tumor initiation, invasion, and treatment resistance.

Applications of Tandem Mass Spectrometry (MS/MS) in Protein Analysis for Biomedical Research.

Mass Spectrometry (MS) allows the analysis of proteins and peptides through a variety of methods, such as Electrospray Ionization-Mass Spectrometry (ESI-MS) or Matrix-Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-MS). These methods allow identification of the mass of a protein or a peptide as intact molecules or the identification of a protein through peptide-mass fingerprinting generated upon enzymatic digestion. Tandem mass spectrometry (MS/MS) allows the fragmentation of proteins and peptides to determine the amino acid sequence of proteins (top-down and middle-down proteomics) and peptides (bottom-up proteomics). Furthermore, tandem mass spectrometry also allows the identification of post-translational modifications (PTMs) of proteins and peptides. Here, we discuss the application of MS/MS in biomedical research, indicating specific examples for the identification of proteins or peptides and their PTMs as relevant biomarkers for diagnostic and therapy.

Structural Characterization of a New Collagen Biomimetic Octapeptide with Nanoscale Self-Assembly Potential: Experimental and Theoretical Approaches.

Bioinspired peptides are attractive biomolecules which can improve our understanding of self-assembly processes for rational design of new peptide-based materials. Herein, a new amidated peptide FRSAPFIE (FRS), based on a sequence present in human collagen, was synthesized, characterized by mass spectrometry and subjected to self-assembling investigations. The optimal conditions for self-assembly were disclosed by dynamic light scattering at 32 °C and a peptide concentration of 0.51 %. In addition, AFM studies revealed ellipsoidal FRS shapes with an area between 0.8 and 3.1 µm2 . The ability of self-assembly was also proved using FAD dye as extrinsic fluorescence reporter. According to the theoretical analysis, the FRS peptide tends to form a bundle-type association, with a type of fibrillary tangles particle. Altogether, our findings address new challenges regarding the FRS peptide which can be used in further self-assembly studies to design biocompatible drug-delivery platforms.

A Proteomic Approach to Identify Zein Proteins upon Eco-Friendly Ultrasound-Based Extraction.

Zein is a type of prolamin storage protein that has a variety of biomedical and industrial applications. Due to the considerable genetic variability and polyploidity of the starting material, as well as the extraction methods used, the characterization of the protein composition of zein requires a combination of different analytical processes. Therefore, we combined modern analytical methods such as mass spectrometry (MS), Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), atomic force microscopy (AFM), or Fourier transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) for a better characterization of the extracted zein. In this study, we present an enhanced eco-friendly extraction method, including grinding and sieving corn seeds, for prolamins proteins using an ultrasonic extraction methodology. The use of an ultrasonic homogenizer, 65% ethanol extraction buffer, and 710 µm maize granulation yielded the highest protein extraction from all experimental conditions we employed. An SDS PAGE analysis of the extracted zein protein mainly revealed two intense bands of approximatively 20 and 23 kDa, suggesting that the extracted zein was mostly α-zein monomer. Additionally, MS analysis revealed as a main component the α-zein PMS2 (Uniprot accession no. P24450) type protein in the maize flour extract. Moreover, AFM studies show that extracting zein with a 65% ethanol and a 710 µm granulation yields a homogeneous content that could allow these proteins to be employed in future medical applications. This research leads to a better understanding of zeins content critical for developing new applications of zein in food and pharmaceutical industries, such as biocompatible medical vehicles based on polyplexes complex nanoparticles of zein with antimicrobial or drug delivery properties.

Mass Spectrometric (MS) Analysis of Proteins and Peptides.

The human genome is sequenced and comprised of ~30,000 genes, making humans just a little bit more complicated than worms or flies. However, complexity of humans is given by proteins that these genes code for because one gene can produce many proteins mostly through alternative splicing and tissue-dependent expression of particular proteins. In addition, post-translational modifications (PTMs) in proteins greatly increase the number of gene products or protein isoforms. Furthermore, stable and transient interactions between proteins, protein isoforms/proteoforms and PTM-ed proteins (protein-protein interactions, PPI) add yet another level of complexity in humans and other organisms. In the past, all of these proteins were analyzed one at the time. Currently, they are analyzed by a less tedious method: mass spectrometry (MS) for two reasons: 1) because of the complexity of proteins, protein PTMs and PPIs and 2) because MS is the only method that can keep up with such a complex array of features. Here, we discuss the applications of mass spectrometry in protein analysis.

Fluorescence spectroscopy and molecular modeling of anthocyanins binding to bovine lactoferrin peptides.

This work was aimed to obtain lactoferrin peptides, with anthocyanins-binding capabilities, by using eggplant peels extract as a source of anthocyanins. The chromatographic analysis of the extract evidenced the presence of five individual anthocyanins, with delfinidin-3-rutinoside being identified as the predominant. 20 small peptides were identified, from which four are containing Trp at C-terminal. By estimating the thermodynamic parameters, van der Waals and hydrogen bonding were found to have important roles in binding of anthocyanins to LF and LF-derived peptides. In order to complement the experimental results, the in silico methods were further employed to add single molecule level details on the potential interactions between different peptides and the main anthocyanins from eggplant peels. The docking tests indicated that the Trp containing peptides can bind, with different affinities either delphynidine-3-glycoside or delphynidine-3-rutinoside, therefore explaining the fluorescence quenching results. Our results have indicated a mechanism for the interactions between anthocyanins and LF and its small molecular weight peptides, whereas providing insights for formulating ingredients and foods with enhanced bioactives-binding properties.

Direct evidence for binding of aluminum to NAP anti-amyloid peptide and its analogs.

NAP (NAPVSIPQ) is a small peptide derived from the activity-dependent neuroprotective protein (ADNP), which provides neuroprotection against amyloid-ß peptide toxicity associated with Alzheimer disease. Several metal ions are able to promote the formation of amyloid-ß peptide oligomers and protofibrils in human brain tissue. Although the relationship between metal ions and amyloid-ß peptide peptides is extensively investigated, that with the NAP peptide is less understood. Nevertheless, our previous research revealed unexpected iron binding to NAP peptide and its analogs. However, a link between aluminum ions, Alzheimer disease and amyloid-ß peptide or NAP peptides still remains controversial. Therefore, we have investigated the possible binding of aluminum ions to NAP peptide and its four analogs. Indeed, MALDI-ToF mass spectrometry (MS), including MS/MS study, and Fourier transform infrared (FT-IR) spectroscopy revealed an unexpected pattern of aluminum ion binding to both NAP peptide and its analogs. Our results have been discussed with respect to NAP protection against Alzheimer disease-related neurotoxicity.

Immuno-Affinity Mass Spectrometry: A Novel Approaches with Biomedical Relevance.

Identifying antigen-antibody interactions have been shown as a critical step in understanding the proteins biological functions and their involvement in various pathological conditions. While many techniques have been developed to characterize antigen-antibody interactions, one strategy that has gained considerable momentum over the last decade for the identification and quantification of antigen-antibody interactions, is immune affinity-chromatography followed by mass spectrometry. Moreover, the combination of enzymatic digestion of antigens and mass spectrometric identification of specific binding peptide(s) to the corresponding anti-antigen antibody has become a versatile and clinical relevant method for mapping epitopes by mass spectrometry. In this chapter, the development and applications of novel immunoaffinity mass spectrometric methodologies for elucidating biomedical aspects will be presented. First, a simplified mass spectrometric approach that maps an epitope from a digested antigen solution without immobilizing the anti-antigen antibody on a solid support will be reported. iMALDI (from immunoaffinity and MALDI, matrix-assisted laser desorption/ionization), a technique that involves immunoaffinity capture of specific peptides and direct MALDI measurements was used for absolute quantification of serine/threonine-specific protein kinase (AKT) peptides from breast cancer and colon cancer cell lines and flash-frozen tumor lysates. The intact transition epitope mapping (ITEM) was shown as a rapid and accurate epitope mapping method by using Ion mobility mass spectrometry (IMS-MS) for analysing the antigen peptide-containing immune complex previously generated by in solution epitope extraction/excision procedures.

Lactuca capensis reverses memory deficits in Aß1-42-induced an animal model of Alzheimer's disease.

We investigated the neuropharmacological effects of the methanolic extract from Lactuca capensis Thunb. leaves (100 and 200 mg/kg) for 21 days on memory impairment in an Alzheimer's disease (AD) rat model produced by direct intraventricular delivery of amyloid-ß1-42 (Aß1-42). Behavioural assays such as Y-maze and radial arm maze test were used for assessing memory performance. Aß1-42 decreased cognitive performance in the behavioural tests which were ameliorated by pre-treatment with the methanolic extract. Acetylcholinesterase activity and oxidant-antioxidant balance in the rat hippocampus were abnormally altered by Aß1-42 treatment while these deficits were recovered by pre-treatment with the methanolic extract. In addition, rats were given Aß1-42 exhibited in the hippocampus decreased brain-derived neurotrophic factor (BDNF) mRNA copy number and increased IL-1ß mRNA copy number which was reversed by the methanolic extract administration. These findings suggest that the methanolic extract could be a potent neuropharmacological agent against dementia via modulating cholinergic activity, increasing of BDNF levels and promoting antioxidant action in the rat hippocampus.

Protein expression profile of HT-29 human colon cancer cells after treatment with a cytotoxic daunorubicin-GnRH-III derivative bioconjugate.

Targeted delivery of chemotherapeutic agents is a new approach for the treatment of cancer, which provides increased selectivity and decreased systemic toxicity. We have recently developed a promising drug delivery system, in which the anticancer drug daunorubicin (Dau) was attached via oxime bond to a gonadotropin-releasing hormone-III (GnRH-III) derivative used as a targeting moiety (Glp-His-Trp-Lys(Ac)-His-Asp-Trp-Lys(Da  = Aoa)-Pro-Gly-NH2; Glp = pyroglutamic acid, Ac = acetyl; Aoa = aminooxyacetyl). This bioconjugate exerted in vitro cytostatic/cytotoxic effect on human breast, prostate and colon cancer cells, as well as significant in vivo tumor growth inhibitory effect on colon carcinoma bearing mice. In our previous studies, H-Lys(Dau = Aoa)-OH was identified as the smallest metabolite produced in the presence of rat liver lysosomal homogenate, which was able to bind to DNA in vitro. To get a deeper insight into the mechanism of action of the bioconjugate, changes in the protein expression profile of HT-29 human colon cancer cells after treatment with the bioconjugate or free daunorubicin were investigated by mass spectrometry-based proteomics. Our results indicate that several metabolism-related proteins, molecular chaperons and proteins involved in signaling are differently expressed after targeted chemotherapeutic treatment, leading to the conclusion that the bioconjugate exerts its cytotoxic action by interfering with multiple intracellular processes.

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

Tyrosine nitration in proteins occurs under physiologic conditions and is increased at disease conditions associated with oxidative stress, such as inflammation and Alzheimer's disease. Identification and quantification of tyrosine-nitrations are crucial for understanding nitration mechanism(s) and their functional consequences. Mass spectrometry (MS) is best suited to identify nitration sites, but is hampered by low stabilities and modification levels and possible structural changes induced by nitration. In this insight, we discuss methods for identifying and quantifying nitration sites by proteolytic affinity extraction using nitrotyrosine (NT)-specific antibodies, in combination with electrospray-MS. The efficiency of this approach is illustrated by identification of specific nitration sites in two proteins in eosinophil granules from several biological samples, eosinophil-cationic protein (ECP) and eosinophil-derived neurotoxin (EDN). Affinity extraction combined with Edman sequencing enabled the quantification of nitration levels, which were found to be 8 % and 15 % for ECP and EDN, respectively. Structure modeling utilizing available crystal structures and affinity studies using synthetic NT-peptides suggest a tyrosine nitration sequence motif comprising positively charged residues in the vicinity of the NT- residue, located at specific surface- accessible sites of the protein structure. Affinities of Tyr-nitrated peptides from ECP and EDN to NT-antibodies, determined by online bioaffinity- MS, provided nanomolar K(D) values. In contrast, false-positive identifications of nitrations were obtained in proteins from cystic fibrosis patients upon using NT-specific antibodies, and were shown to be hydroxy-tyrosine modifications. These results demonstrate affinity- mass spectrometry approaches to be essential for unequivocal identification of biological tyrosine nitrations.

Epitope motif of an anti-nitrotyrosine antibody specific for tyrosine-nitrated peptides revealed by a combination of affinity approaches and mass spectrometry.

Nitration of tyrosine residues has been shown to be an important oxidative modification in proteins and has been suggested to play a role in several diseases such as atherosclerosis, asthma, lung and neurodegenerative diseases. Detection of nitrated proteins has been mainly based on the use of nitrotyrosine-specific antibodies. In contrast, only a small number of nitration sites in proteins have been unequivocally identified by MS. We have used a monoclonal 3-NT-specific antibody, and have synthesized a series of tyrosine-nitrated peptides of prostacyclin synthase (PCS) in which a single specific nitration site at Tyr-430 had been previously identified upon reaction with peroxynitrite17. The determination of antibody-binding affinity and specificity of PCS peptides nitrated at different tyrosine residues (Tyr-430, Tyr-421, Tyr-83) and sequence mutations around the nitration sites provided the identification of an epitope motif containing positively charged amino acids (Lys and/or Arg) N-terminal to the nitration site. The highest affinity to the anti-3NT-antibody was found for the PCS peptide comprising the Tyr-430 nitration site with a K(D) of 60 nM determined for the peptide, PCS(424-436-Tyr-430NO(2) ); in contrast, PCS peptides nitrated at Tyr-421 and Tyr-83 had substantially lower affinity. ELISA, SAW bioaffinity, proteolytic digestion of antibody-bound peptides and affinity-MS analysis revealed highest affinity to the antibody for tyrosine-nitrated peptides that contained positively charged amino acids in the N-terminal sequence to the nitration site. Remarkably, similar N-terminal sequences of tyrosine-nitration sites have been recently identified in nitrated physiological proteins, such as eosinophil peroxidase and eosinophil-cationic protein.

Structural characterisation of tyrosine-nitrated peptides by ultraviolet and infrared matrix-assisted laser desorption/ionisation Fourier transform ion cyclotron resonance mass spectrometry.

Nitration of tyrosine residues in proteins may occur in cells upon oxidative stress and inflammation processes mediated through generation of reactive nitroxyl from peroxynitrite. Tyrosine nitration from oxidative pathways may generate cytotoxic species that cause protein dysfunction and pathogenesis. A number of protein nitrations in vivo have been reported and some specific Tyrosine nitration sites have been recently identified using mass spectrometric methods. High-resolution Fourier transform ion cyclotron resonance mass spectrometry (MALDI) FT-ICR-MS) is shown here to be a highly efficient method in the determination of protein nitrations. Following the identification of nitration of the catalytic site Tyr-430 residue of bovine prostacyclin synthase, we synthesised several model peptides containing both unmodified tyrosine and 3-nitro-tyrosine residues, using solid-phase peptide synthesis (SPPS). The structures of the nitrotyrosine peptides were characterised both by ESI- and by matrix-assisted laser desorption/ionisation (MALDI)-FT-ICR-MS, using a standard ultraviolet (UV) nitrogen nitrogen laser and a 2.97 microm Nd-YAG infrared laser. Using UV-MALDI-MS, 3-nitrotyrosyl-peptides were found to undergo extensive photochemical fragmentation at the nitrophenyl group, which may hamper or prevent the unequivocal identification of Tyr-nitrations in cellular proteins. In contrast, infrared-MALDI-FT-ICR-MS did not produce fragmentation of molecular ions of Tyr-nitrated peptides.