De novo sequencing of a 21-kDa cytochrome c4 from Thiocapsa roseopersicina by nanoelectrospray ionization ion-trap and Fourier-transform ion-cyclotron resonance mass spectrometry

We have determined the primary structure of cytochrome c(4) from Thiocapsa roseopersicina by de novo protein sequencing using the 'bottom up' approach. Three different enzymes (trypsin, endoproteinase Lys-C, and endoproteinase Glu-C) were employed to prepare four different sets of proteolytic digests. The digestion strategy was designed to permit a gradual buildup of smaller peptides into larger ones that were overlapped to yield the complete protein sequence. In this way we countered the main problem: peptides larger than about 1500 Da were difficult to sequence fully by tandem mass spectrometry. Direct infusion and online liquid chromatography were used on a linear ion trap Fourier-transform ion-cyclotron resonance hybrid instrument. The high resolving power, high mass accuracy and the availability of electron capture dissociation and collision-induced dissociation were essential to achieve full sequence coverage. The software DeNovoX complemented by manual interpretation was used to generate sequence information from tandem mass spectra. The predominantly automated nature of data acquisition and handling allowed for a relatively straightforward and fast procedure, which could compete with the mainstream alternative of nucleotide sequence determination.     

CABS‐NMR—De novo tool for rapid global fold determination from chemical shifts,residual dipolar couplings and sparse methyl‐methyl noes

Recent development of nuclear magnetic resonance (NMR) techniques provided new types of structural restraints that can be successfully used in fast and low‐cost global protein fold determination. Here, we present CABS‐NMR, an efficient protein modeling tool, which takes advantage of such structural restraints. The restraints are converted from original NMR data to fit the coarse grained protein representation of the C‐Alpha‐Beta‐Side‐group (CABS) algorithm. CABS is a Monte Carlo search algorithm that uses a knowledge‐based force field. Its versatile structure enables a variety of protein‐modeling protocols, including purely de novo folding, folding guided by restraints derived from template structures or, structure assembly based on experimental data. In particular, CABS‐NMR uses the distance and angular restraints set derived from various NMR experiments. This new modeling technique was successfully tested in structure determination of 10 globular proteins of size up to 216 residues, for which sparse NMR data were available. Additional detailed analysis was performed for a S100A1 protein. Namely, we successfully predicted Nuclear Overhauser Effect signals on the basis of low‐energy structures obtained from chemical shifts by CABS‐NMR. It has been observed that utility of chemical shifts and other types of experimental data (i.e. residual dipolar couplings and methyl‐methyl Nuclear Overhauser Effect signals) in the presented modeling pipeline depends mainly on size of a protein and complexity of its topology. In this work, we have provided tools for either post‐experiment processing of various kinds of NMR data or fast and low‐cost structural analysis in the still challenging field of new fold predictions. © 2010 Wiley Periodicals, Inc. J Comput Chem, 2011     

Characterization and de novo sequencing of snow crab tropomyosin enzymatic peptides by both electrospary ionization and matrix‐assisted laser desorption ionization QqToF tandem mass spectrometry

The protein tropomyosin (TM) is a known major allergen present in shellfish causing frequent food allergies. TM is also an occupational allergen generated in the working environment of snow crab (Chionoecetes opilio) processing plants. The TM protein was purified from both claw and leg meats of snow crab and analyzed by electrospray ionization and matrix‐assisted laser desorption/ionization (MALDI) using hybrid quadruple time‐of‐flight tandem mass spectrometry (QqToF‐MS). The native polypeptide molecular weight of TM was determined to be 32 733 Da. The protein was further characterized using the ‘bottom‐up’ MS approach. A peptide mass fingerprinting was obtained by two different enzymatic digestions and de novo sequencing of the most abundant peptides performed. Any post‐translational modifications were identified by searching their calculated and predicted molecular weights in precursor ion spectra. The immunological reactivity of snow crab extract was evaluated using specific antibodies and allergenic reactivity assessed with serum of allergic patients. Subsequently, a signature peptide for TM was identified and evaluated in terms of identity and homology using the basic local alignment search tool (BLAST). The identification of a signature peptide for the allergen TM using MALDI‐QqToF‐MS will be critical for the sensitive and specific quantification of this highly allergenic protein in the work place. Copyright © 2010 John Wiley & Sons, Ltd.     

Top‐down mass spectrometry of intact phosphorylated β‐casein: Correlation between the precursor charge state and internal fragments

Phosphorylated proteins play essential roles in many cellular processes, and identification and characterization of the relevant phosphoproteins can help to understand underlying mechanisms. Herein, we report a collision‐induced dissociation top‐down approach for characterizing phosphoproteins on a quadrupole time‐of‐flight mass spectrometer. β‐casein, a protein with two major isoforms and five phosphorylatable serine residues, was used as a model. Peaks corresponding to intact β‐casein ions with charged states up to 36⁺ were detected. Tandem mass spectrometry was performed on β‐casein ions of different charge states (12⁺, and 15⁺ to 28⁺) in order to determine the effects of charge state on dissociation of this protein. Most of the abundant fragments corresponded to y, b ions, and internal fragments caused by cleavage of the N‐terminal amide bond adjacent to proline residues (Xxx‐Pro). The abundance of internal fragments increased with the charge state of the protein precursor ion; these internal fragments predominantly arose from one or two Xxx‐Pro cleavage events and were difficult to accurately assign. The presence of abundant sodium adducts of β‐casein further complicated the spectra. Our results suggest that when interpreting top‐down mass spectra of phosphoproteins and other proteins, researchers should consider the potential formation of internal fragments and sodium adducts for reliable characterization.     

Advances in native high‐performance liquid chromatography and intact mass spectrometry for the characterization of biopharmaceutical products

The characterization of biotherapeutics represents a major analytical challenge. This review discusses the current state‐of‐the‐art in analytical technologies to profile biopharma products under native conditions, i.e., the protein three dimensional conformation is maintained during liquid chromatographic analysis. Native liquid‐chromatographic modes that are discussed include aqueous size‐exclusion chromatography, hydrophobic interaction chromatography, and ion‐exchange chromatography. Infusion conditions and the possibilities and limitations to hyphenate native liquid chromatography to mass spectrometry are discussed. Furthermore, the applicability of native liquid‐chromatography methods and intact mass spectrometry analysis for the characterization of monoclonal antibodies and antibody–drug conjugates is discussed.     

Bis(4‐pyridylamino)triazine‐stabilized magnetite nanoparticles: preparation,characterization and application as a retrievable catalyst for the green synthesis of 4H‐pyran, 4H‐thiopyran and 1,4‐dihydropyridine derivatives

Synthesis of bis(4‐pyridylamino)triazine stabilized on silica‐coated nano‐Fe₃O₄ particles, and their feasibility as a reusable heterogeneous basic catalyst are reported. The catalytic performance of this novel material was studied for the green synthesis of highly functionalized 4H ‐pyran, 4H ‐thiopyran and 1,4‐dihydropyridine derivatives via one‐pot multicomponent reactions. Eco‐friendly method, high yield and purity of desired products, short reaction time along with ease of workup procedure outline the advantages of these new methodologies over earlier ones. Surface and magnetic properties of the core–shell hybrid nanoparticles were characterized via transmission and scanning electron microscopies, X‐ray diffraction, energy‐dispersive X‐ray and Fourier transform infrared spectroscopies and vibrating sample magnetometry. Copyright © 2016 John Wiley & Sons, Ltd.     

HPLC with quadrupole TOF‐MS and chemometrics analysis for the characterization of Folium Turpiniae from different regions

Folium Turpiniae has been used as a traditional Chinese medicine for the treatment of abscesses, fevers, gastric ulcers, and inflammations. This paper describes a strategy of combining HPLC with photodiode array detection and quadrupole TOF‐MS, as well as phytochemical and chemometrics analysis for the characterization, isolation, and simultaneous quantification of the chemical constituents of Folium Turpiniae. 19 constituents were identified, namely, 11 flavonoids, seven gallic acid derivates, and quinic acid. Among them, 15 compounds were identified in this herbal medicine for the first time; compound 10 appears to be novel and was isolated and confirmed as ellagic acid‐3‐O‐α‐l ‐rhamnopyranoside by NMR spectroscopy and MS. In addition, nine marker compounds, namely gallic acid ( 2 ), ellagic acid‐3‐O‐α‐l ‐rhamnopyranoside ( 10 ), apigenin‐7‐O‐(2′′‐rhamnosyl)gentiobioside ( 11 ), ellagic acid ( 12 ), luteolin‐7‐O‐β‐d ‐neohesperidoside ( 13 ), ligustroflavone ( 14 ), 4′‐O‐methylellagic acid‐3‐O‐α‐l ‐rhamnopyranoside ( 16 ), rhoifolin ( 17 ), and neobudofficide ( 18 ), were quantified simultaneously in ten batches of Folium Turpiniae collected from different regions. Moreover, hierarchical clustering analysis and principal component analysis indicated that both samples from Hubei ( S1 ) and Guangxi ( S3 ) provinces showed apparent differences from the others. Samples from Jiangxi province ( S2 , S4 , and S7–10 ) possessed similar properties and therefore belong to the same group.     

Qualitative and quantitative analysis of pyrrolizidine alkaloids for the entire process quality control from Senecio scandens to Senecio scandens‐containing preparations by high performance liquid chromatography‐tandem mass spectrometry

Senecio scandens as a commonly used traditional Chinese medicine that is used alone or in combination with other herbs in preparations such as QianBai BiYan tablets has attracted much attention because of its hepatotoxic pyrrolizidine alkaloids. Nowadays, most studies for pyrrolizidine alkaloids are only performed on herbs or a preparation, however, production of preparations is a dynamic process, control of toxic impurities for raw materials, or finished products cannot monitor the production process dynamically. Thus, in this study, qualitative and quantitative analysis of pyrrolizidine alkaloids for the entire process quality control from S. scandens to its preparations was carried out with HPLC‐MS/MS for the first time, which was more comprehensive and dynamic than the previous single‐layer analysis. First, the species of pyrrolizidine alkaloids in S. scandens were analyzed, and the characteristic fragmentation rules of pyrrolizidine alkaloids containing common parent nucleus were found, which can be used to identify these components rapidly in the future. Then, a quantitative method for S. scandens to QianBai BiYan tablets and other nine S. scandens‐containing preparations was established, and after the medication safety speculation, all of them met the relevant safety requirements. After that, in order to ensure the stability and controllable of drug quality, the limit of pyrrolizidine alkaloids in preparations was determined according to the safe dosage that is stipulated to be the same as raw materials. Finally, the factors causing the content change of pyrrolizidine alkaloids in S. scandens from different source were studies, which can provide theoretical basis for selecting suitable raw materials for production.     

High‐throughput ultra high performance liquid chromatography combined with mass spectrometry approach for the rapid analysis and characterization of multiple constituents of the fruit of Acanthopanax senticosus (Rupr. et Maxim.) Harms

Acanthopanax senticosus (Rupr. et Maxim.) Harms, a traditional Chinese medicine, has been widely used to improve the function of skeleton, heart, spleen and kidney. This fruit is rich in nutrients, but the chemical constituents of Acanthopanax senticosus fruit are still unclear. A rapid method based on ultra high performance liquid chromatography with time‐of‐flight mass spectrometry was developed for the compound analysis of Acanthopanax senticosus fruit in vitro and in vivo. In this study, the Acanthopanax senticosus fruit could significantly increase the weight of immune organs, promote the proliferation of lymphatic T cells, regulate the lymphatic B cell function, and decrease the ability of natural killer cells. A total of 104 compounds of Acanthopanax senticosus fruit including lignans, flavones, triterpenoidsaponins, phenolic acids, and other constituents were identified. Among them, seven chemical compounds were reported for the first time in the Acanthopanax senticosus fruit. Compared with the serum sample of blank and dosed samples, 24 prototype compositions were characterized. The results of our experiment could be helpful to understand the complex compounds of Acanthopanax senticosus fruit in vitro and in vivo for further pharmacological activity studies.     

Proteomic analysis of cellular soluble proteins from human bronchial smooth muscle cells by combining nondenaturing micro 2DE and quantitative LC‐MS/MS. 2. Similarity search between protein maps for the analysis of protein complexes

Human bronchial smooth muscle cell soluble proteins were analyzed by a combined method of nondenaturing micro 2DE, grid gel‐cutting, and quantitative LC‐MS/MS and a native protein map was prepared for each of the identified 4323 proteins [1]. A method to evaluate the degree of similarity between the protein maps was developed since we expected the proteins comprising a protein complex would be separated together under nondenaturing conditions. The following procedure was employed using Excel macros; (i) maps that have three or more squares with protein quantity data were selected (2328 maps), (ii) within each map, the quantity values of the squares were normalized setting the highest value to be 1.0, (iii) in comparing a map with another map, the smaller normalized quantity in two corresponding squares was taken and summed throughout the map to give an “overlap score,” (iv) each map was compared against all the 2328 maps and the largest overlap score, obtained when a map was compared with itself, was set to be 1.0 thus providing 2328 “overlap factors,” (v) step (iv) was repeated for all maps providing 2328 × 2328 matrix of overlap factors. From the matrix, protein pairs that showed overlap factors above 0.65 from both protein sides were selected (431 protein pairs). Each protein pair was searched in a database (UniProtKB) on complex formation and 301 protein pairs, which comprise 35 protein complexes, were found to be documented. These results demonstrated that native protein maps and their similarity search would enable simultaneous analysis of multiple protein complexes in cells.