Characterization of a new qQq-FTICR mass spectrometer for post-translational modification analysis and top-down tandem mass spectrometry of whole proteins

The use of a new electrospray qQq Fourier transform ion cyclotron mass spectrometer (qQq-FTICR MS) instrument for biologic applications is described. This qQq-FTICR mass spectrometer was designed for the study of post-translationally modified proteins and for top-down analysis of biologically relevant protein samples. The utility of the instrument for the analysis of phosphorylation, a common and important post-translational modification, was investigated. Phosphorylation was chosen as an example because it is ubiquitous and challenging to analyze. In addition, the use of the instrument for top-down sequencing of proteins was explored since this instrument offers particular advantages to this approach. Top-down sequencing was performed on different proteins, including commercially available proteins and biologically derived samples such as the human E2 ubiquitin conjugating enzyme, UbCH10. A good sequence tag was obtained for the human UbCH10, allowing the unambiguous identification of the protein. The instrument was built with a commercially produced front end: a focusing rf-only quadrupole (Q0), followed by a resolving quadrupole (Q1), and a LINAC quadrupole collision cell (Q2), in combination with an FTICR mass analyzer. It has utility in the analysis of samples found in substoichiometric concentrations, as ions can be isolated in the mass resolving Q1 and accumulated in Q2 before analysis in the ICR cell. The speed and efficacy of the Q2 cooling and fragmentation was demonstrated on an LCMS-compatible time scale, and detection limits for phosphopeptides in the 10 amol/muL range (pM) were demonstrated. The instrument was designed to make several fragmentation methods available, including nozzle-skimmer fragmentation, Q2 collisionally activated dissociation (Q2 CAD), multipole storage assisted dissociation (MSAD), electron capture dissociation (ECD), infrared multiphoton induced dissociation (IRMPD), and sustained off resonance irradiation (SORI) CAD, thus allowing a variety of MS(n) experiments. A particularly useful aspect of the system was the use of Q1 to isolate ions from complex mixtures with narrow windows of isolation less than 1 m/z. These features enable top-down protein analysis experiments as well structural characterization of minor components of complex mixtures.     

Synthesis of monodisperse polymers from proteins

Proteins are functional biopolymers; viewed as molecules, they are also monodisperse polyamides with chemically reactive side chains. This paper describes the use of proteins as starting materials for the synthesis of monodisperse polymers with nonbiological functionalities attached to the side chains. It demonstrates the complete derivatization of amine groups (lysine side chains and N-termini) on three different proteins by addition of activated carboxylate reagents in aqueous solutions containing sodium dedecyl sulfate (SDS), under denaturing conditions. Several different acylating reagents were used to generate derivatized proteins; the resulting compounds constitute a new class of monodisperse, semisynthetic polymers, having the potential for wide variation in the structure of the backbone and of the side chains. Modification of lysozyme on a gram scale demonstrated that the method can generate useful quantities of material.     

Site-specific qualitative and quantitative analysis of the N- and O-glycoforms in recombinant human erythropoietin

Recombinant human erythropoietin (rhEPO) has been extensively used as a pharmaceutical product for treating anemia. Glycosylation of rhEPO affects the biological activity, immunogenicity, pharmacokinetics, and in-vivo clearance rate of rhEPO. Characterization of the glycosylation status of rhEPO is of great importance for quality control. In this study, we established a fast and comprehensive approach for reliable characterization and relative quantitation of rhEPO glycosylation, which combines multiple-enzyme digestion, hydrophilic-interaction chromatography (HILIC) enrichment of glycopeptides, and tandem mass spectrometry (MS) analysis. The N-linked and O-linked intact glycopeptides were analyzed with high-resolution and high-accuracy (HR–AM) mass spectrometry using an Orbitrap. In total, 74 intact glycopeptides from four glycosylation sites at N₂₄, N₃₈, N₈₃, and O₁₂₆ were identified, with the simultaneous determination of peptide sequences and glycoform compositions. The extracted ion chromatograms based on the HR–AM data enabled relative quantification of glycoforms. Our results could be extended to quality control of rhEPO or could help establish detection approaches for glycosylation of other proteins. Graphical Abstract

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Synthesis,Antibacterial, and Antioxidant Evaluation of Novel Series of Condensed Thiazoloquinazoline with Pyrido,Pyrano, and Benzol Moieties as Five- and Six-Membered Heterocycle Derivatives

A novel synthesis of thiazolo[2,3-b]quinazolines 4(a–e), pyrido[2′,3′:4,5]thiazolo[2,3-b]quinazolines {5(a–e), 6(a–e), and 7(a–e)}, pyrano[2′,3′:4,5]thiazolo[2,3-b]quinazolines 8(a–e), and benzo[4,5]thiazolo[2,3-b]quinazoloine9(a–e) derivatives starting from 2-(Bis-methylsulfanyl-methylene)-5,5-dimethyl-cyclohexane-1,3-dione 2 as efficient α,α dioxoketen dithioacetal is reported and the synthetic approaches of these types of compounds will provide an innovative molecular framework to the designing of new active heterocyclic compounds. In our study, we also present optimization of the synthetic method along with a biological evaluation of these newly synthesized compounds as antioxidants and antibacterial agents against the bacterial strains, like S. aureus, E. coli, and P. aeruginosa. Among all the evaluated compounds, it was found that some showed significant antioxidant activity at 10 μg/mL while the others exhibited better antibacterial activity at 100 μg/mL. The results of this study showed that compound 6(c) possessed remarkable antibacterial activity, whereas compound 9(c) exhibited the highest efficacy as an antioxidant. The structures of the new synthetic compounds were elucidated by elemental analysis, IR, ¹H-NMR, and ¹³C-NMR.     

Thiaminase I (42 kDa) heterogeneity,sequence refinement,and active site location from high-resolution tandem mass spectrometry

Thiaminase I (E.C. 2.5. 1.2) from Bacillus thiaminolyticus catalyzes the degradation of thiamin (vitamin B₁). Unexpected mass heterogeneity (MW 42,127, 42,197, and 42,254; 1:2:1) in recombinant thiaminase I from Escherichia coli was detected by electrospray ionization Fourier-transform mass spectrometry, resolving power 7×10⁴. Nozzle-skimmer fragmentation data reveal an extra Ala (+71.02; 71.04=theory) and GlyAla (+128.04; 128.06=theory) on the N-terminus, in addition to the fully processed enzyme. However, the fragment ion masses were consistent only with this sequence through 330 N-terminal residues; resequencing of the last 150 bps of the thiaminase I gene yields a sequence consistent with the molecular weight values and all 61 fragment ion masses. Covalently labeling the active site with a 108-Da pyrimidine moiety via mechanism-based inhibition produces a corresponding molecular weight increase in all three thiaminase I components, which indicates that they are all enzymatically active. Inspection of the fragment ions that do and do not increase by 108 Da indicates that the active site nucleophile is located between Pro⁷⁹ and Thr¹⁷⁷ in the 379 amino acid enzyme.     

Chitosan loaded with silver nanoparticles,CS‐AgNPs,using thymus syriacus,wild mint,and rosemary essential oil extracts as reducing and capping agents

The present study describes the green method for the preparation of chitosan loaded with silver nanoparticles (CS‐AgNPs) in the presence of 3 different extracted essential oils. The essential oils play dual roles as reductant and capping agents. The reducing power and DPPH (2,2‐diphenyl‐1‐picrylhydrazyl) assay for the 3 essential oils—Thymus syriacus (T), wild mint (M), and rosemary (R)—have been reported. The preparation of CS‐AgNPs was performed by 2 steps. The 3 previously extracted essential oils have been used as reducing and capping agent in the first step, while in the second step, silver nanoparticles were integrated in chitosan. The integration of AgNPs in the structure of chitosan was confirmed by ultraviolet‐visible, Fourier transform infrared spectroscopy, scanning electron microscopy techniques, and energy dispersive X‐ray. Surface plasmon resonance confirmed the formation of CS‐AgNPs with maximum absorbance at λ_max between 405 ‐ 410 and 410 ‐ 430 nm for colloidal and films of CS‐AgNPs, respectively. The intensity of bands at 3408 cm^?1 in the fourier transform infrared spectroscopy measurements was decreased substantially and shifted slightly to lower frequency (?υ = 43 cm^?1). Scanning electron microscopy shows a spherical morphology of AgNPs with size of 62 nm for both colloidal and film samples, and energy dispersive X‐ray analysis shows peaks confirming AgNPs formation.     

An Identification Problem for First-Order Degenerate Differential Equations

We study a first-order identification problem in a Banach space. We discuss the nondegenerate and mainly the degenerate case. As a first step, suitable hypotheses on the involved closed linear operators are made in order to obtain unique solvability after reduction to a nondegenerate case; the general case is then handled with the help of new results on convolutions. Some applications to partial differential equations motivate this abstract approach.Communicated by I. GalliganiWork partially supported by MIUR (Ministero dell’ Istruzione, dell’ Università e dalla Ricerca), Project PRIN 2004011204 “Analisi Matematica nei Problemi Inversi,” and by the University of Bologna Funds for Selected Research Topics.