Are Internal Fragments Observable in Electron Based Top-Down Mass Spectrometry?

Protein tandem mass spectrometry (MS/MS) often generates sequence-informative fragments from backbone bond cleavages near the termini. This lack of fragmentation in the protein interior is particularly apparent in native top-down mass spectrometry (MS). Improved sequence coverage, critical for reliable annotation of posttranslational modifications and sequence variants, may be obtained from internal fragments generated by multiple backbone cleavage events. However, internal fragment assignments can be error prone due to isomeric/isobaric fragments from different parts of a protein sequence. Also, internal fragment generation propensity depends on the chosen MS/MS activation strategy. Here, we examine internal fragment formation in electron capture dissociation (ECD) and electron transfer dissociation (ETD) following native and denaturing MS, as well as LC/MS of several proteins. Experiments were undertaken on multiple instruments, including quadrupole time-of-flight, Orbitrap, and high-field Fourier-transform ion cyclotron resonance (FT-ICR) across four laboratories. ECD was performed at both ultrahigh vacuum and at similar pressure to ETD conditions. Two complementary software packages were used for data analysis. When feasible, ETD-higher energy collision dissociation MS3 was performed to validate/refute potential internal fragment assignments, including differentiating MS3 fragmentation behavior of radical versus even-electron primary fragments. We show that, under typical operating conditions, internal fragments cannot be confidently assigned in ECD or ETD. On the other hand, such fragments, along with some b-type terminal fragments (not typically observed in ECD/ETD spectra) appear at atypical ECD operating conditions, suggesting they originate from a separate ion-electron activation process. Furthermore, atypical fragment ion types, e.g., x ions, are observed at such conditions as well as upon EThcD, presumably due to vibrational activation of radical z-type ions.

Unrevealing the Nitrogen Elusive Chirality of 3-Sulfanyl and 3-Sulfinyl N-Tosyl Isoindolinones by ECD Spectra: An Experimental and Theoretical Investigation.

The nitrogen-hybridization/pyramidalization of two solvated N-tosylisoindolinone derivatives having chiral residues in adjacent (I) or adjacent and distal (II) position has been investigated by a theoretical-computational procedure based on Molecular Dynamics simulations and Quantum-Chemical calculations. After validation of our methodology in providing a reliable repertory of conformations by modeling the electronic circular dichroism (EDC) spectra, the electronic features associated with N-pyramidalization were further characterized through Natural Bond Order (NBO) analysis. Comparing against the N-geometry observed in crystal structures as a reference, our findings reveal that the presence of neighbouring chiral centers induces a more pronounced N-pyramidalization in solution than in the solid state, both in I and II. Furthermore, NBO analysis confirms that the N-lactam mostly retains the sp2 character but exhibits slight configurational distortion (ξI=13°; ξII=21°), which significantly influences the chiroptical activities observed in ECD spectra of I and II. This substantiates the N-lactams as configurationally stable chiral centers.

Controlling Chirogenic Effects in Porphyrin Based Supramolecular Systems: Theoretical Analysis Versus Experimental Observations.

Electronic circular dichroism (ECD) spectroscopy is a widely employed method for studying chiral analysis, requiring the presence of a chromophore close to a chiral centre. Porphyrinoids are found to be one of the best chromophoric systems serving for this purpose and enabling the application of ECD spectroscopy for chirality determination across diverse classes of organic compounds. Consequently, it is crucial to understand the induction mechanisms of ECD in the porphyrin-based complexes. The present study explores systematically the influence of secondary chromophores, bonded to an achiral zinc porphyrin or to chiral guest molecules, on the B-region of ECD spectra using the time-dependent density functional theory (TD-DFT) calculations. The study analyses the impact of change in both the conformation of achiral porphyrin (host) and change in position and conformation of chiral organic molecule (guest) on the B-band of ECD spectra (energy, intensity, sign of Cotton effect). Finally, conclusions made on model complexes are applied to published experimental data, contributing to a deeper understanding of various factors influencing ECD spectra in chiral systems. In addition, a computer program aimed to help rationalise ECD spectra by visualizing corresponding orbital energies, rotatory strengths, electric and magnetic transition moments, and angles between them, is presented.

CPL of Mellein and Related Natural Compounds: Analysis of the ESIPT Phenomenon.

(R)-(-)-Mellein, (3R,4R)-4-hydroxymellein and (3R,4S)-4-hydroxymellein obtained from fungi, i. e. from Diplodia globulosa, were investigated as a class of natural products presenting ESIPT (excited state intramolecular proton transfer) phenomenon, through fluorescence and CPL (circularly polarized luminescence). The study was preceded by the assessment of the absolute configuration through ECD and VCD (electronic and vibrational circular dichroism) spectroscopies in addition to NMR spectra. It is found that ESIPT takes place in these systems very rapidly, and no dual fluorescence has been observed. The experimental study is backed up by TD-DFT calculations of ECD and CPL spectra, plus MD calculations to follow proton transfer in the excited state and careful analysis of the puckering dynamics of the lactone ring. Deprotonated forms of the three compounds were also investigated by the same chiroptical experimental and theoretical methods, showing how one can find in natural compounds not only biological activity but also biologically compatible sensing probes.

The discovery of an anti-inflammatory monoterpenoid, neoroseoside from the Zea mays.

A new monoterpenoid, neoroseoside (1), along with two previously reported compounds, 2″-O-α-l-rhamnosyl-6-C-fucosylluteolin (2) and farobin A (3) were isolated from the Zea mays. The structure of compound 1 was determined through the analysis spectroscopic data, including mass spectrometry (MS), infrared (IR) spectroscopy, and nuclear magnetic resonance (NMR) data. The absolute configurations of 1 were deduced from the comparing the values of optical rotations and from the interpretation of electronic circular dichroism (ECD) spectra. Compounds 2 and 3 displayed moderate antibacterial activity against Streptococcus mutans ATCC 25175 (inhibition rates 24 % and 28 %, respectively) and Streptococcus sobrinus ATCC 33478 (inhibition rate of 26 %), at a concentration of 100 µg/mL, whereas compound 1 did not have any significant antibacterial activities. The compounds 1-3 also showed anti-inflammatory activity on cytokine IL-6 and TNF-α.

Penicimides A and B, two novel diels-alder [4 + 2] cycloaddition ergosteroids from Penicillium herquei.

Two novel naturally occurring [4 + 2] Diels-Alder cycloaddition ergosteroids (1 and 2), three undescribed oxidized ergosteroids (3-5), and eleven known analogs (6-16) were isolated from Penicillium herquei. Compounds 1 and 2 represent the first reported cycloadducts of a steroid with 1,4,6-trimethyl-1,6-dihydropyridine-2,5-dione or 4,6-dimethyl-1,6-dihydropyridine-2,5-dione to date. Compound 3 is the C-15 epimer of (22E,24R)-9α,11ß-dihydroxyergosta-4,6,8(14),22-tetraen-3-one (14). The chemical structures of these compounds were elucidated through widespread spectroscopic analyses, mainly including HRESIMS and 1D and 2D NMR data, calculated 13C NMR-DP4+ analysis, and electronic circular dichroism (ECD) data analyses. Biological evaluations of Compounds 1-16 revealed that 3, 9-11, and 15 inhibited the production of NO in LPS-induced RAW264.7 cells with an IC50 value from 7.37 ± 0.69 to 38.9 ± 2.25 µM (the positive control dexamethasone IC50: 9.54 ± 0.71 µM). In addition, Compound 3 exhibited a potent inhibitory effect on the secretion of the proinflammatory cytokines TNF-α and IL-6, the transcription level of the proinflammatory macrophage markers TNF-α, and the expression of the iNOS protein.

Induced Chirality in Sulfasalazine by Complexation With Albumins: Theoretical and Experimental Study.

Through molecular recognition, drugs can interact and complex with macromolecules circulating in the body. The serum albumin transport protein, found in several mammals, has several interaction sites where these molecules can be located. The drug sulfasalazine (SSZ) is known in the literature to complex at drug site 1 (DS1) in human serum (HSA) and bovine serum (BSA) proteins. This complexation can be studied using various spectroscopic techniques. With the techniques used in this work, absorption in the ultraviolet and visible regions (UV-Vis) and electronic circular dichroism (ECD), a significant difference was observed in the results involving HSA and BSA. The application of theoretical methodologies, such as TD-DFT and molecular docking, suggests that the conformation that SSZ assumes in DS1 of the two proteins is different, which exposes it to different amino acid residues and different hydrophobicities. This difference in conformation may be related to the location of DS1 where the drug interacts or to the possibility of SSZ moving in the BSA site, due to its larger size, and moving less freely in HSA.

Exploratory analysis of serum HER2 extracellular domain for HER2 positive gastric cancer treated with SOX plus trastuzumab.

BACKGROUND: The aim of this study was to explore the clinical utility of serum HER2 extracellular domain (sHER2 ECD) using data from a clinical trial evaluating trastuzumab combined S-1 plus oxaliplatin (SOX) in HER2 positive gastric cancer. METHODS: sHER2 ECD were prospectively measured at baseline and subsequent treatment courses. Based on each quantile point of baseline sHER2 ECD levels and its early changes, patients were divided into two groups and compared clinical outcomes. RESULTS: 43 patients were enrolled, and 17 patients (39.5%) were positive for baseline sHER2 ECD. Higher baseline sHER2 ECD levels tended to have lower hazard ratios (HRs). When divided into two groups by baseline sHER2 ECD of 19.1 ng/ml, median progression-free survival (PFS) and overall survival (OS) was longer in the higher group (mPFS: 16.8 vs 8.7 months, p = 0.359. mOS: 35.5 vs 20.6 months, p = 0.270), respectively. After initiation of treatment, sHER2 ECD significantly decreased up until the third cycle. Higher reduction rates of sHER2 ECD within 3 cycles also tended to have lower HRs. When divided into two groups by reduction rate of 42.5%, mPFS and mOS was longer in the higher reduced group (mPFS: 17.2 vs 8.7 months, p = 0.095. mOS: 65.0 vs 17.8 months, p = 0.047), respectively. Furthermore, higher reduction rates could surrogate higher objective response rates (ORR) (ORR: 90% vs 63.2% for 29.5%, p = 0.065. 100% vs 70% for 42.5%, p = 0.085), respectively. CONCLUSIONS: Baseline sHER2 ECD levels and its early decline may be useful biomarkers for SOX plus trastuzumab efficacy in HER2 positive gastric cancer.

Clinical results with a multifocal intraocular lens with a novel optical design.

BACKGROUND: To evaluate the optical performance and safety of a new multifocal lens with a novel optical design featuring two additional foci (or intensifiers) in patients with cataract and presbyopia. METHODS: In this single-center, non-randomized prospective observational study, 31 patients underwent implantation of the new multifocal IOL between March 2020 and November 2021 at a tertiary clinical center in Buenos Aires and Ramos Mejia, Argentina. Postoperative examinations with emphasis on uncorrected and corrected visual acuity at distance and near and at two different intermediate distances (80 cm and 60 cm) were performed during the 3 postoperative months. RESULTS: Of the 31 patients who underwent implantation of the new IOL, 30 underwent bilateral surgery (61 eyes in total). At 3 months, all 61 eyes had an uncorrected distance visual acuity (UCDVA) of at least 0.15 logMAR; 57 eyes (93%) had an uncorrected distance visual acuity (UCDVA) of 0.1 logMAR and 27 eyes (44%) had an UCDVA of 0.0 logMAR. At 80 cm, 60 eyes (98%) had an uncorrected intermediate visual acuity (UCIVA) of at least 0.1 log MAR and 48 eyes (79%) had an UCIVA of 0.0 logMAR. CONCLUSION: The new multifocal IOL with a novel optical concept (5 foci) showed a wide range of visual acuity especially at intermediate and near distances in patients undergoing cataract surgery. Uncorrected visual acuity was excellent at all tested distances, monocularly and binocularly, spectacle independence and patient satisfaction were high.

Clinical observations of EVO-ICL implantation with single incision without viscoelastic agent.

BACKGROUND: To investigate the safety and effectiveness of non-viscoelastic agent technique for EVO-ICL implantation. METHODS: A total of 181 myopia eyes that underwent non-toric ICL without viscoelastic agent through single incision from Beijing Tongren Hosipital were included. An analysis was conducted on the quantity of haptics that were initially implanted intraoperatively into the posterior chamber. Intraocular pressure (IOP) was evaluated at before and 2 h,24 h,1week,6month after surgery. Anterior chamber volume(ACV), anterior chamber depth(ACD), anterior chamber angle(ACA), pupil diameter(PD) and corneal densitometry density (ECD) were evaluated at before and 24 h postoperatively. Refractive outcomes were investigated at before, 24 h ,7 days and 6months. Vault was evaluated at 24 h ,7 days and 6months. RESULTS: The efficacy and safety indices were 1.30 ± 0.32 and 1.31 ± 0.32, respectively. Of 181 eyes, 99 eyes received 4 haptics on the first attempt without any adjustment, and 72 eyes received lens alignment without an viscoelastic agent. The success rate of the viscoelastic agent free procedure was 94.5%. Two hours postoperatively, IOP was 17.41 ± 3.77 mmHg, which was significantly higher than baseline value (t = 8.930, P < 0.000), however there was no significant difference between preoperative IOP and IOP at 1 day ,1 week and 6 months postoperatively. The ECD changed from 2895.52 ± 253.73 cells/mm2 preoperatively to 2873.66 ± 244.17 cells/mm2 at 1 day and 2882.63 ± 239.97 postoperatively, and the difference was not statistically significant (t = 1.811, P = 0.072). The ACA was narrowed by 42% on the first day. CONCLUSION: The pure viscoelastic agent free technique is an efficient and safe way for ICL implantation. It can be a safer method of ICL implantation because of it reduces the risk of complications associated with ocular hypertension at the early postoperative stages. TRIAL REGISTRATION: Chinese Clinical Trial Registry (ChiCTR2000036335) at August 20, 2020.

Peptide-Based Mass Spectrometry for the Investigation of Protein Complexes.

In the last two decades, biological mass spectrometry has become the gold standard for the identification of proteins in biological samples. The technological advancement of mass spectrometers and the development of methods for ionization, gas phase transfer, peptide fragmentation as well as for acquisition of high-resolution mass spectrometric data marked the success of the technique. This chapter introduces peptide-based mass spectrometry as a tool for the investigation of protein complexes. It provides an overview of the main steps for sample preparation starting from protein fractionation, reduction, alkylation and focus on the final step of protein digestion. The basic concepts of biological mass spectrometry as well as details about instrumental analysis and data acquisition are described. Finally, the most common methods for data analysis and sequence determination are summarized with an emphasis on its application to protein-protein complexes.

New Zosteropenillines and Pallidopenillines from the Seagrass-Derived Fungus Penicillium yezoense KMM 4679.

Ten new decalin polyketides, zosteropenilline M (1), 11-epi-8-hydroxyzosteropenilline M (2), zosteropenilline N (3), 8-hydroxyzosteropenilline G (4), zosteropenilline O (5), zosteropenilline P (6), zosteropenilline Q (7), 13-dehydroxypallidopenilline A (8), zosteropenilline R (9) and zosteropenilline S (10), together with known zosteropenillines G (11) and J (12), pallidopenilline A (13) and 1-acetylpallidopenilline A (14), were isolated from the ethyl acetate extract of the fungus Penicillium yezoense KMM 4679 associated with the seagrass Zostera marina. The structures of isolated compounds were established based on spectroscopic methods. The absolute configurations of zosteropenilline Q (7) and zosteropenilline S (10) were determined using a combination of the modified Mosher's method and ROESY data. The absolute configurations of zosteropenilline M (1) and zosteropenilline N (3) were determined using time-dependent density functional theory (TD-DFT) calculations of the ECD spectra. A biogenetic pathway for compounds 1-14 is proposed. The antimicrobial, cytotoxic and cytoprotective activities of the isolated compounds were also studied. The significant cytoprotective effects of the new zosteropenilline M and zosteropenillines O and R were found in a cobalt chloride (II) mimic in in vitro hypoxia in HEK-293 cells. 1-Acetylpallidopenilline A (14) exhibited high inhibition of human breast cancer MCF-7 cell colony formation with IC50 of 0.66 µM and its anticancer effect was reduced when MCF-7 cells were pretreated with 4-hydroxitamoxifen. Thus, we propose 1-acetylpallidopenilline A as a new xenoestrogen with significant activity against breast cancer.

New Alpiniamide-Type Polyketide with Antibiofilm Activities from the Marine-Derived Streptomyces sp. ZS-A65.

Two new alpiniamide-type polyketides, alpiniamides H-I (1-2), in addition to four recognized compounds, were discovered in Streptomyces sp. ZSA65 derived from the marine sediments. The planar structure and absolute configuration of alpiniamides H-I were elucidated using a combination of 1D, 2D NMR, HRESIMS data analysis, Mosher's method and ECD calculations. The antibiofilm and antibacterial activities against P. aeruginosa were evaluated using the microdilution method. Notably, Compound 2 exhibited strong antibiofilm property.

Structurally Diverse Coumarins from Peucedanum praeruptorum and their Anti-Inflammatory Activities via NF-κB Signaling Pathway.

The phytochemical study of Peucedanum praeruptorum led to the isolation of twenty-five coumarins (1-25). Of which, (±) praeruptol A (±1), one pair of previous undescribed seco-coumarin enantiomers were obtained. Their structures were established according to HR-ESI-MS, NMR, X-ray single crystal diffraction analysis, as well as ECD calculation. All compounds were tested for anti-inflammatory activity in the RAW264.7 macrophage model, and eight compounds (7-10, and 13-16) exhibited significant inhibitory effects with IC50 values ranging from 9.48 to 34.66 µM. Among them, compound 7 showed the strongest inhibitory effect, which significantly suppressed the production of IL-6, IL-1ß, and TNF-α, as well as iNOS and COX-2 in a concentration-dependent manner. Further investigated results showed that compound 7 exerted an anti-inflammatory effect via the NF-κB signaling pathway.

Retrobisabolane A, a Novel Bisabolane-Derived Sesquiterpenoid Isolated from Deep-Sea-Derived Fungus Retroconis fusiformis MCCC 3A00792.

One novel bisabolane-derived sesquiterpenoid retrobisabolane A (1), featuring a methyl group location at the C-4 position instead of C-3 in the bisabolanes, and a known ester-substituted eremophilane-type sesquiterpenoid cryptosphaerolide (2), along with three known indole alkaloids (3-5) were discovered from the fermented cultures of a deep-sea-derived fungus Retroconis fusiformis MCCC 3A00792. The planar structure of new compound 1 was determined by extensive analysis of the NMR and HRESIMS spectra. The relative and absolute configurations of 1 were resolved by the coupling constant (J), calculation of ECD and NMR spectra, and the DP4+ probability analysis of the 1H and 13C NMR data. Interestingly, retrobisabolane A was the new subclass of bisabolanes bearing a methyl group linkage at C-4 instead of C-3 position. Three human cancer cell lines (Hela, AGS, and BIU-87) were subjected to evaluate the cytotoxic activities of compounds 1-5. As a result, compound 2 exhibited significant inhibitory activities against three cell lines with IC50 values ranging from 9.95 to 18.77 µM.

Chemical Composition of Achillea millefolium L. and Their Anti-Inflammatory Activity.

A new monoterpene, (-)-10-hydroxydihydroactinidiolide (1), along with two known monoterpenes, loliolide (2) and (+)-isololiolide (3), three known megastigmanes, 3α-hydroxy-5ß,6ß-epoxy-ß-ionone (4), 3α-hydroxy-5α,6α-epoxy-ß-ionone (5), and (+)-dehydrovomifoliol (6), a eudesmane-type sesquiterpene, 4α-hydroxy-4ß-methyldihydrocostol (7), a monoterpene, 8-hydroxycarvotanacetone (8), two flavonoids, chrysoeriol (9) and apigenin (10), and a phenylpropanoid, 3-(4-hydroxyphenyl)-1-propanol (11), were isolated from the whole plant of Achillea millefolium. The structure of compound 1 was identified according to spectroscopic data of HRMS and NMR, and its absolute configuration was assigned by 13C NMR calculations with DP4+ probability analyses and ECD calculations. The absolute configuration of compound 6 was determined by ECD calculations. Compounds 3, 6, 9 and 10 could dose-dependently inhibit the NO release in LPS-induced RAW264.7 cells.

Morushalunin D, a tri-O-bridged Diels-Alder type adduct from Morus alba var. shalun root cultures and the related precursors.

Three Diels-Alder type adducts (1-3) along with their precursors, including one 2-arylbenzofuran (4) and one stilbene (5), were isolated from the MeOH extract of M. alba var. shalun root cultures. Among them, 1 is a new Diels-Alder type adduct named morushalunin D. The molecular structures of 1-5 were elucidated based on spectroscopic data and comparison with the literatures. Cytotoxic properties of compounds 1-5 were evaluated against murine leukemia P-388 cells. Morushalunin D (1), mulberrofuran T (2), sorocein A (3), moracin M (4), and oxyresveratrol (5) were active, significantly inhibiting the growth of P-388 cells with IC50 values of 0.5, 1.0, 0.6, 2.0, and 3.3 µg/ml, respectively.

Analysis of Receptor-Type Protein Tyrosine Phosphatase Extracellular Regions with Insights from AlphaFold.

The receptor-type protein tyrosine phosphatases (RPTPs) are involved in a wide variety of physiological functions which are mediated via their diverse extracellular regions. They play key roles in cell-cell contacts, bind various ligands and are regulated by dimerization and other processes. Depending on the subgroup, they have been described as everything from 'rigid rods' to 'floppy tentacles'. Here, we review current experimental structural knowledge on the extracellular region of RPTPs and draw on AlphaFold structural predictions to provide further insights into structure and function of these cellular signalling molecules, which are often mutated in disease and are recognised as drug targets. In agreement with experimental data, AlphaFold predicted structures for extracellular regions of R1, and R2B subgroup RPTPs have an extended conformation, whereas R2B RPTPs are twisted, reflecting their high flexibility. For the R3 PTPs, AlphaFold predicts that members of this subgroup adopt an extended conformation while others are twisted, and that certain members, such as CD148, have one or more large, disordered loop regions in place of fibronectin type 3 domains suggested by sequence analysis.

Quantum Size-Driven Spectral Variations in Pillar[n]arene Systems: A Density Functional Theory and Wave Function Assessment.

This study explores the quantum size effects on the optical properties of pillar[n]arene (n = 5, 6, 7, 8) utilizing density functional theory (DFT) and wave function analysis. The mechanisms of electron transitions in one-photon absorption (OPA) and two-photon absorption (TPA) spectra are investigated, alongside the calculation of electron circular dichroism (ECD) for these systems. Transition Density Matrix (TDM) and electron-hole pair density maps are employed to study the electron excitation characteristics, unveiling a notable size dependency. Analysis of the transition electric dipole moment (TEDM) and the transition magnetic dipole moment (TMDM) reveals the electromagnetic interaction mechanism within pillar[n]arene. Raman spectra computations further elucidate vibrational modes, while interactions with external environments are studied using electrostatic potential (ESP) analysis, and electron delocalization is assessed under an external magnetic field, providing insights into the magnetically induced current phenomena within these supramolecular structures. The thermal stability of pillar[n]arene was investigated by ab initio molecular dynamics (AIMD).

A new illudane sesquiterpene from the edible fungus Pholiota nameko.

Fungi have different genetic expression abilities and biosynthetic pathways under different cultivation conditions, which can produce various secondary metabolites. The "one strain many compounds" strategy is used to activate silent biosynthetic genes of fungi to produce various compounds, which is an effective method. In order to discover various new compounds in the edible fungus Pholiota nameko, a fermentation strategy involving precursor feeding and enzyme inhibitor addition has been employed. A new illudane sesquiterpene (1), along with one known indole diterpenoid alkaloid, cladosporine A (2) were isolated from the extracts of liquid culture of P. nameko. The new compound was identified by combination of 1D and 2D NMR, MS, optical rotation, and ECD calculations. We conducted experiments on the cytotoxicity of all isolated compounds on three cancer cell lines, but we did not observe any significant cytotoxicity (IC50 > 40 µM).