Elucidation of O-Phosphoryl and N-Phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

Mass spectroscopic characteristics of phosphoryl amino acids were studied in detail by positive and negative electrospray ionization mass spectrometry (ESI-MS) in conjunction with tandem mass spectrometry (MS/MS). Besides N-diisopropyloxyphosphoryl amino acids (N-DIPP-AA), O-phospho- and O-diisopropyloxyphosphoryl amino acids (O-DIPP-AA) were studied and compared to N-DIPP-AA. The fragmentation pathways of [M H]^ ,[M Na]^ and [M-H]^- ions of phosphoryl amino acids were summarized. In addition to several similar patterns, each of them showed its characteristic fragmention.     

Characterization of Oil by Micro-Solid-Phase Extraction and Gas Chromatography–Mass Spectrometry

Micro-solid-phase extraction is reported for the preparation of Bohai crude oil for the determination of hydrocarbons by gas chromatography-mass spectrometry. The operational parameters were optimized. Micro-solid-phase extraction provided higher quantities of low-molecular weight components than conventional liquid chromatography. The concentrations of high-molecular weight n-alkanes, polycyclic aromatic hydrocarbons, and their alkylated homologs obtained were comparable by micro-solid-phase extraction and liquid chromatography. The diagnostic ratios also indicated that there were no significant differences between these methods. Therefore, micro-solid-phase extraction with gas chromatography–mass spectrometry is recommended for the characterization of spilled oil.     

Sequence Analysis of Polypeptide by Mass Spectrometry

The method described in this work provides a sensitive and fast technique for investigating the primary structure of peptides with molecular weight up to 3340 amu. Usually, the metastable ion kinetic energy spectra (MIKES) and collisional activated decomposition (CAD) spectra provide complementary information for the FAB mass spectra, the MIKES and CAD spectra generally contain high-mass sequence ions.     

Pyrolysis Gas Chromatography–Mass Spectrometry for the Characterization of Tire Marks for Forensic Analysis

Pyrolysis–gas chromatography–mass spectrometry was applied to the quantitative and comparative analysis of rubber samples collected from tire marks left on different types of road surfaces. Ten types of tires were used to create skid marks. It was observed that factors such as surface type, sampling method, and the place where the sample was taken revealed no influence on chemical composition of pyrolysate. Chemical analysis of the traces left on different types of substrates allowed one to distinguish between these traces, allocate samples to different rubber classes, and to assign the trace to a particular type of tire. Application of a simple, semi-quantitative method developed to describe the composition of the samples facilitated the distinction between samples. Furthermore, in most cases, tire marks showed great consistency with the rubber taken from the respective tires.     

Gas-Phase Binding of Noncovalent Complexes Between α-cyclodextrin and Amino Acids Investigated by Mass Spectrometry

Noncovalent complexes between cyclodextrins and small molecules have been extensively studied recently because of their widespread application in the pharmaceutical industry for chiral and molecular recognition. To date, gas phase noncovalent binding affinities between α-cyclodextrin and amino acids have not been widely investigated. In this study, gas-phase binding of noncovalent complexes between α-CD and amino acids was investigated by electrospray ionization mass spectrometry (ESI-MS), demonstrating the formation of 1:1 stoichiometric noncovalent complexes. The binding of the complexes were further confirmed by collision-induced dissociation by tandem mass spectrometry. Mass spectrometric titrations between α-cyclodextrin and phenylalanine, glutamic acid, and arginine were performed to provide binding constants (lgK_a) as references for competitive ESI-MS. Calibration curves for the complexes of α-cyclodextrin with phenylalanine, glutamic acid, and arginine were plotted. Through competitive ESI-MS, the lgK_a for the complexes of α-CD with aspartic acid, lysine, proline, glycine, alanine, asparagine, cystine, glutamine, histidine, leucine, isoleucine, methionine, serine, threonine, and valine were measured directly. By comparison, it is seen that the measured binding constants for the complexes of α-cyclodextrin with basic amino acids such as arginine and lysine are lower than those for most complexes of neutral amino acids. The chiral selectivity of α-cyclodextrin for L- and D-isomers of methionine, threonine, asparagine, and phenylalanine determined by ESI-MS revealed its application as a chiral selector.     

Preliminary Characterization of the Composition and Phenolic Fragmentation of Olive Byproducts by Gas Chromatography–Mass Spectrometry and High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Olive waste is a potential resource due to its rich variety of biologically active compounds. To investigate chemical components of olive waste, the selected samples were extracted using ultrasound assisted enzyme hydrolysis and petroleum ether, ethyl acetate and n-butyl alcohol were used to obtain a series of solvent extracts. Gas chromatography–mass spectrometry analysis showed hydrocarbons, esters, acids, alcohols, and ketones present in the extracts. Some fatty acids were considered to be predominant; it is noteworthy that phenolic compounds were detected in the ethyl acetate extract fraction. Furthermore, the primary phenolic compounds were also determined by high-performance liquid chromatography coupled with electrospray ionization tandem mass spectrometry. The possible fragmentation patterns have been proposed in positive and negative ion modes; the main fragment ions were observed from the loss of methyl, hydroxyl, or carboxyl groups. The compounds showed different fragmentation ions types in both positive and negative ionization modes and gave structural information on the main phenolic compounds in olive waste. The results of this study may be used to identify valuable active compounds and guide commercial applications of olive waste.     

Rapid Characterization of Microorganisms by Mass Spectrometry—What Can Be Learned and How?

Strategies for the rapid and reliable analysis of microorganisms have been sought to meet national needs in defense, homeland security, space exploration, food and water safety, and clinical diagnosis. Mass spectrometry has long been a candidate technique because it is extremely rapid and can provide highly specific information. It has excellent sensitivity. Molecular and fragment ion masses provide detailed fingerprints, which can also be interpreted. Mass spectrometry is also a broad band method—everything has a mass—and it is automatable. Mass spectrometry is a physiochemical method that is orthogonal and complementary to biochemical and morphological methods used to characterize microorganisms.      

Comprehensive Proteoform Characterization of Plasma Complement Component C8αβγ by Hybrid Mass Spectrometry Approaches

The human complement hetero-trimeric C8αβγ (C8) protein assembly (~?150 kDa) is an important component of the membrane attack complex (MAC). C8 initiates membrane penetration and coordinates MAC pore formation. Here, we charted in detail the structural micro-heterogeneity within C8, purified from human plasma, combining high-resolution native mass spectrometry and (glyco)peptide-centric proteomics. The intact C8 proteoform profile revealed at least ~?20 co-occurring MS signals. Additionally, we employed ion exchange chromatography to separate purified C8 into four distinct fractions. Their native MS analysis revealed even more detailed structural micro-heterogeneity on C8. Subsequent peptide-centric analysis, by proteolytic digestion of C8 and LC-MS/MS, provided site-specific quantitative profiles of different types of C8 glycosylation. Combining all this data provides a detailed specification of co-occurring C8 proteoforms, including experimental evidence on N-glycosylation, C-mannosylation, and O-glycosylation. In addition to the known N-glycosylation sites, two more N-glycosylation sites were detected on C8. Additionally, we elucidated the stoichiometry of all C-mannosylation sites in all the thrombospondin-like (TSP) domains of C8α and C8β. Lastly, our data contain the first experimental evidence of O-linked glycans located on C8γ. Albeit low abundant, these O-glycans are the first PTMs ever detected on this subunit. By placing the observed PTMs in structural models of free C8 and C8 embedded in the MAC, it may be speculated that some of the newly identified modifications may play a role in the MAC formation.

Open image in new window

Graphical Abstract ?

     

Structural Characterization and Disulfide Assignment of Spider Peptide Phα1β by Mass Spectrometry

Native Phα1β is a peptide purified from the venom of the armed spider Phoneutria nigriventer that has been shown to have an extensive analgesic effect with fewer side effects than ω-conotoxin MVIIA. Recombinant Phα1β mimics the effects of the native Phα1β. Because of this, it has been suggested that Phα1β may have potential to be used as a therapeutic for controlling persistent pathological pain. The amino acid sequence of Phα1β is known; however, the exact structure and disulfide arrangement has yet to be determined. Determination of the disulfide linkages and exact structure could greatly assist in pharmacological analysis and determination of why this peptide is such an effective analgesic. Here, we used biochemical and mass spectrometry approaches to determine the disulfide linkages present in the recombinant Phα1β peptide. Using a combination of MALDI-MS, direct infusion ESI-MS, and nanoLC-MS/MS analysis of the undigested recombinant Phα1β peptide and digested with AspN, trypsin, or AspN/trypsin, we were able to identify and confirm all six disulfide linkages present in the peptide as Cys1-2, Cys3-4, Cys5-6, Cys7-8, Cys9-10, and Cys11-12. These results were also partially confirmed in the native Phα1β peptide. These experiments provide essential structural information about Phα1β and may assist in providing insight into the peptide’s analgesic effect with very low side effects.

Open image in new window

Graphical Abstract ?

     

Aliphatic Polyesters for Advanced Technologies – Structural Characterization of Biopolyesters with the Aid of Mass Spectrometry

Application of the ESI- mass spectrometry technique for the structural studies of biopoliesters is reported. The structural studies were performed on two selected commercially available biopolyesters i.e. poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), contained up to 87% of PHB repeat units, with the aid of ESI-MS analysis of the respective low molar mass oligomers. The molecular architecture of individual macromolecules, including the chemical structure of the end groups, composition and sequence distribution were determined based on mass-resolved signals of ESI-mass spectra of PHBH and PHBV oligomers. The general utility of MS characterization of biopoliester macromolecules at the molecular level has been demonstrated and supported by NMR analysis.     

Improved Accuracy of Low Affinity Protein–Ligand Equilibrium Dissociation Constants Directly Determined by Electrospray Ionization Mass Spectrometry

There is continued interest in the determination by ESI-MS of equilibrium dissociation constants (K_D) that accurately reflect the affinity of a protein–ligand complex in solution. Issues in the measurement of K_D are compounded in the case of low affinity complexes. Here we present a K_D measurement method and corresponding mathematical model dealing with both gas-phase dissociation (GPD) and aggregation. To this end, a rational mathematical correction of GPD (f_sat) is combined with the development of an experimental protocol to deal with gas-phase aggregation. A guide to apply the method to noncovalent protein–ligand systems according to their kinetic behavior is provided. The approach is validated by comparing the K_D values determined by this method with in-solution K_D literature values. The influence of the type of molecular interactions and instrumental setup on f_sat is examined as a first step towards a fine dissection of factors affecting GPD. The method can be reliably applied to a wide array of low affinity systems without the need for a reference ligand or protein.     

Synthesis of Dendritic-Linear Block Copolymers by Atom Transfer Radical Polymerization

Polymers and copolymers with complex, yet well-defined architectures are drawing significant attentions in the search for materials with excellent properties. Of these macromolecular structures, dendritic-linear block copolymers consisting of covalently bound linear and dendritic segments have shown interesting solution, solid-state, and interfacial properties. As a novel polymerization approach, atom transfer radical polymerization (ATRP) has been attracting increasing interest recently, sin…     

Studies on Fragmentation Pathways of N-Ethoxy （phenyl） phosphoryl Amino Acids by Electrospray Ionization Tandem Mass Spectrometry

IntroductionPhosphorylation often acts as a molecular switchcontrolling the protein activity in different pathways asin metabolism,signal transduction,cell division,andso on.Therefore,N-phosphoryl amino acids play aspecial and important role in biological…     

Detection and Characterization of Non-covalent Complex between Lappaconitine and β-Cyclodextrin by Electrospray ionization Tandem Mass Spectrometry

Lappaconitine(LA), exhibits antiarrhythm1 and analgesic actions2-5, and has been used to release pain for cancer patients. However, its application is limited due to poor water solubility. But its non-covalent complexes with CDs can solve this problem, th…     

Mapping a Noncovalent Protein–Peptide Interface by Top-Down FTICR Mass Spectrometry Using Electron Capture Dissociation

Noncovalent protein–ligand and protein–protein complexes are readily detected using electrospray ionization mass spectrometry (ESI MS). Furthermore, recent reports have demonstrated that careful use of electron capture dissociation (ECD) fragmentation allows covalent backbone bonds of protein complexes to be dissociated without disruption of noncovalent protein–ligand interactions. In this way the site of protein–ligand interfaces can be identified. To date, protein–ligand complexes, which have proven tractable to this technique, have been mediated by ionic electrostatic interactions, i.e., ion pair interactions or salt bridging. Here we extend this methodology by applying ECD to study a protein–peptide complex that contains no electrostatics interactions. We analyzed the complex between the 21 kDa p53-inhibitor protein anterior gradient-2 and its hexapeptide binding ligand (PTTIYY). ECD fragmentation of the 1:1 complex occurs with retention of protein–peptide binding and analysis of the resulting fragments allows the binding interface to be localized to a C-terminal region between residues 109 and 175. These finding are supported by a solution-phase competition assay, which implicates the region between residues 108 and 122 within AGR2 as the PTTIYY binding interface. Our study expands previous findings by demonstrating that top-down ECD mass spectrometry can be used to determine directly the sites of peptide–protein interfaces. This highlights the growing potential of using ECD and related top-down fragmentation techniques for interrogation of protein–protein interfaces.     

Exact Mass Measurements for a-Allenic Alcohol by Atmospheric Pressure Chemical Ionization／Time-of-flight Mass Spectrometry

The atmospheric pressure chemical ionization/time,of-flight mass speetrmtry (APEI/TOF-MS) was applied to determine the mass of five a.aIIenic alcohols via their vrotonated molecu.lar ions nslna Imsifive ion mode. Polyethylene Idycol (PEG) was used as the hlternal reference. All results were obtained under the resolution of about 5000 FWHM (full width at the half maximum). Solvent effects were studied and the satired results were obtained in acetonitrile. Comvared with the theoreflcal values, nun absolute errors were less thRn 1.0 mmu. The efTeets Of nozzle pote.Jldal, push pulse potential, pug pulse potentlai, puO bias potential and ic(lulsltion rate on exact mass determina/lon were also discussed. APCI/TOF.MS is proven to be a very semi/ire analytical technique and an alternative ionizafion mode in analytical technique lablle compounds with relatively weak polarity, such as a-allenic alcohol.     

Sequence pattern Correlation of Amino Acid in Collision—induced Dissociation Electrospray Ionization Mass Spectrometry

A novel approach of seuqence pattern correlation has been applied to predict an expected amino acid sequence from CID ESI-MS spectra.The proposed approach deduces sequence patterns with no help form known protein database such that it is useful to identify an unknown petide or new protein.The algorithm applies a cross-correlation to match an experimental CID spectrum with predicted sequence pattern generated form fragmentation information.The fragmentation knowledge of both y-series and other non y-series are utilized to generate the predicted sequence patterns.In contrast to the normal de novo approach,the proposed approach is insensitive to mass tolerance and non-sussceptive to spectral integrality with no need for selection of a starting point.     

Detection of Radical Adducts with Small Molecular Weights by Matrix-Assisted Laser Desorption/Ionization with Fourier Transform Mass Spectrometry

As an alternative method, matrix-assisted laser desorption/ionization with Fourier transform mass spectrometry （MALDI-FTMS） has been successfully used to detect and identify free radical adducts with small molecular weights of hydroxyl and 2-cyano-2-propyl radicals trapped with 5,5-dimethylpyrroline N-oxide （DMPO）. The detection and identification by MS/MS experiments using sustained offresonance irradiation collision-induced dissociation （SORI-CID） of [（DMPO＋·OH-·H）＋H^＋] （m/z 130.0868） and [DMPO＋2 ·CH（CH3）2CN＋H^＋] （m/z 250.1917） have demonstrated that MALDI-FTMS could be an effective method for detection and identification of free radical adducts. Other radical adducts have been also detected and identified. The approach of MALDI-FTMS is simple, fast, and sensitive which has potential for high-throughput analysis.