Electrospray ionization Fourier transform ion cyclotron resonance mass spectrometric analysis of the recombinant human macrophage colony stimulating factor β and derivatives

The potential of electrospray ionization (ESI) Fourier transform ion cyclotron mass spectrometry (FTICR-MS) to assist in the structural characterization of monomeric and dimeric derivatives of the macrophage colony stimulating factor beta (rhM-CSF beta) was assessed. Mass spectrometric analysis of the 49 kDa protein required the use of sustained off-resonance irradiation (SORI) in-trap cleanup to reduce adduction. High resolution mass spectra were acquired for a fully reduced and a fully S-cyanylated monomeric derivative (approximately 25 kDa). Mass accuracy for monomeric derivatives was better than 5 ppm, after applying a new calibration method (i.e., DeCAL) which eliminates space charge effects upon high accuracy mass measurements. This high mass accuracy allowed the direct determination of the exact number of incorporated cyanyl groups. Collisionally induced dissociation using SORI yielded b- and y-fragment ions within the N- and C-terminal regions for the monomeric derivatives, but obtaining information on other regions required proteolytic digestion, or potentially the use of alternative dissociation methods.     

Reactions of O−. with methyl benzoate: A negative ion chemical ionization and Fourier transform ion cyclotron resonance study

The reactions of O*- with methyl benzoate have been examined by the measurement of negative ion chemical ionization (NICI) mass spectra using a CI source, with confirmatory studies carried out on a Fourier transform ion cyclotron resonance mass spectrometer. Reaction mechanisms have been elucidated using isotopically labeled esters. Nucleophilic attack at the carbonyl carbon and the aromatic ring were important reaction pathways. Nucleophilic attack at the carbonyl carbon was followed by the production of products (C6HsCO2- and CH3OCO2-) characteristic of radical, beta-fragmentation. Using 18O-labeled methyl benzoate, the SN2 reaction was found to account for a smaller percentage, 21(+/-1)%, of the benzoate product. Aromatic ring attack resulted in formation of [M + O - H]- and [M - 2H]*- ions. Although aryl hydrogens accounted for most H2*+ abstracted by O*-, evidence for abstraction of HarylH*+alkyl and HalkylH*+alkyl was also found. Although present at much lower abundance, dehydrobenzoate, dehydrophenoxy, and C7H6*- ([M - 2H - CO2]*-) radical anions were also observed. An Haryl/Halkyl exchange associated with formation of the benzoate anion was attributed to an Halkyl abstraction that occurred within the methanol/dehydrobenzoate ion-dipole complex. The [M - 2H]*-, dehydrobenzoate, dehydrophenoxy, and [M - 2H - CO2]*- ion signals were quenched by reaction with O2. Conditions required for production of O*- spectra under NICI conditions were also examined.     

Asymmetric polymerase chain reaction improves streptavidin–biotin based purification of polymerase chain reaction products prior to matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis

Asymmetric polymerase chain reaction (PCR) simplifies the application of biotin–streptavidin based purification formats for PCR products in matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. The use of an excess of non-biotinylated primer improves the turnover of biotinylated primer. Thereby performance deficiencies caused by non-extended biotinylated primer are eliminated. © 1998 John Wiley & Sons, Ltd.     

Alkaloid profiling of the Chinese herbal medicine Fuzi by combination of matrix-assisted laser desorption ionization mass spectrometry with liquid chromatography–mass spectrometry

A matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) method was developed for the high throughput and robust qualitative profiling of alkaloids in Fuzi—the processed lateral roots of the Chinese herbal medicine Aconitum carmichaeli Debx (A. carmichaeli). After optimization, powdered roots – without any further sample preparation – could be used to screen for the presence of Aconitum alkaloids. Furthermore, the semi-quantitative potential of MALDI-MS was confirmed using liquid chromatography–mass spectrometry (LC–MS) as reference. In total over sixty alkaloids were detected by LC–MS and fifteen of them were tentatively identified. Both MALDI-MS and LC–MS analysis revealed significant variation in alkaloid content in different (commercial) samples. LC–MS analysis of three toxic alkaloids in 14 batches of Fuzi resulted in a variation of their concentrations expressed as RSDs of 138%, 99% and 221% for aconitine, hypaconitine and mesaconitine, respectively. The variation in concentrations (expressed as RSD) of about the ninety constituents detected were classified as follows: 13 constituents showed an RSD of 77–100%, 46 with an RSD of 100–150%, 21 with an RSD of 150–200% and 9 constituents with an RSD in concentration of 200–235%. These results demonstrate a strong difference in chemical composition of the various Fuzi and illustrate the necessity of adequate QA/QC procedures for both safety and efficiency of herbal medicine. The described analytical procedures for alkaloid profiling could play a role in these procedures.     

Metabolic study of grapevine leaves infected by downy mildew using negative ion electrospray – Fourier transform ion cyclotron resonance mass spectrometry

Grapevine is of worldwide economic importance due to wine production. However, this culture is often affected by pathogens causing severe harvest losses. Understanding host–pathogen relationships may be a key to solve this problem. In this paper, we evaluate the direct flow injection by electrospray – Fourier transform ion cyclotron resonance mass spectrometry (MS) of leaf extracts as a rapid method for the study of grapevine response to downy mildew (Plasmopara viticola) attack. The comparison of MS profiles obtained from control and infected leaves of different levels of resistant grapevines highlights several classes of metabolites (mainly saccharides, acyl lipids, hydroxycinnamic acids derivatives and flavonoids) which are identified using high resolution MS and tandem MS (MS/MS). Statistical analyses of 19 markers show a clear segregation between inoculated and healthy samples. This study points out relative high levels of disaccharides, acyl lipids and glycerophosphoinositol in inoculated samples. Sulfoquinovosyl diacylglycerols also emerge as possible metabolites involved in plant defense.     

Studies on extracting solutions of endohedral rare-earth metallofullerenes by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Thirteen extracting solutions of rare-earth metallofullerenes containing La,Ce,Pr,Nd Sm,Eu,Gd,Tb,Dy,Ho,Er,Tm and Yb respectively have been investigated by means of matrix-assisted laser desorpuon/ ionization time-of-flight mass spectrometry.The influences of the positive-ion/negative-ion mode,laser intensity,ma trix and mass discrimination to the analytical results are studied,based on which the optimal analytical conditions have been determined.The results show that the extracting solutions contain large quantities of rare-earth metallofullerenes besides empty fullerenes.On the basis of comparing their relative intensities,the different structure stabilities and solubilities of metallofullerenes with different rare-earth metals encapsulated into the fullerene cages,as well as some possible reasons to those differences,are discussed.     

A novel sample preparation method of matrix-assisted laser desorption/ionization time of flight mass spectrometry for polystyrene

A novel sample preparation method of matrix-assisted laser desorption/ionization mass spectrometry for polystyrene was reported. Compared to the conventional dried-droplet method, the efficiency of ionization and signal intensity of mass spectra were improved. The mechanism was also analyzed.     

A novel type of matrix for surface-assisted laser desorption–ionization mass spectrometric detection of biomolecules using metal-organic frameworks

A 3D metal-organic framework (MOF) nanomaterial as matrix for surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) and tandem mass spectrometry (MS/MS) was developed for the analysis of complex biomolecules. Unlike other nanoparticle matrices, this MOF nanomaterial does not need chemical modification prior to use. An exceptional signal reproducibility as well as very low background interferences in analyzing mono-/di-saccharides, peptides and complex starch digests demonstrate its high potential for biomolecule assays, especially for small molecules.     

Determination of 3,3′-Dichlorobenzidine and its degradation products in environmental samples with a small low-field Fourier transform ion cyclotron resonance mass spectrometer

3,3'-Dichlorobenzidine (DCB) and its degradation products, 3-chlorobenzidine (MCB) and benzidine, are of environmental concern because of their carcinogenic nature. The suitability of a small Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer for the analysis of these environmental contaminants in different types of matrices was explored. All the measurements were carried out by depositing the sample solution directly on a disk that was introduced into the mass spectrometer. This approach is very fast and simple because it requires no prior chromatographic separation or derivatization. Calibration curves determined by collecting 70-eV electron ionization mass spectra of neat samples yielded lower limits of detection of 29 and 17 pg (total amount on the solids probe) for DCB and benzidine, respectively (based on a signal to noise ratio of > or = 2:1), while chemical ionization with ammonia resulted in lower limits of detection of 21 pg for DCB and 9 pg for benzidine (total amount on the solids probe). FT-ICR analysis of sediments collected from Lake Macatawa (Holland, MI) verified the presence of DCB in this complex, environmentally significant sample matrix. Laboratory experiments designed to probe biodegradation and photodegradation pathways showed that DCB undergoes sequential dehalogenation to yield MCB and then benzidine under exposure to microorganisms and under simulated tropospheric solar radiation. The ability of the FT-ICR to determine elemental compositions of compounds introduced as described above was demonstrated for one of the degradation products.     

Sequence and modified group analysis on C-terminal modified analogs of endomorphin-2 using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometer

In this paper, a series of C-terminal modified analogs of endomorphin-2 is investigated using ESI-FT-ICR-MS. Some b, y″, a, and internal ions are found in the CID spectra and slight mass differ- ences between the calculated and observed results are obtained. Moreover, if the C-terminal modified group is t-butyloxy, it can lose butene through McLafferty rearrangement. FT-ICR MS shows its power in peptide sequencing successfully helping us obtain the structure of peptide analogs.     

Characterization of ethylene oxide–propylene oxide block copolymers by combination of different chromatographic techniques and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy

Block copolymers of ethylene oxide (EO) and propylene oxide (PO) are characterized by combination of two-dimensional chromatography and MALDI-TOF-MS. Liquid chromatography under critical conditions (LCCC) is used as first dimension and fractions are collected, mobile phase evaporated and diluted in the mobile phase used in second dimension (SEC or LAC). This two-dimensional chromatography in combination of MALDI-TOF-MS gives information about purity of reaction products, presence of the byproducts, chemical composition and molar mass distribution of all the products.     

Material-enhanced laser desorption/ionization (MELDI)—A new protein profiling tool utilizing specific carrier materials for time of flight mass spectrometric analysis

Over the past couple of years, proteomics pattern analysis has emerged as an effective method for the early diagnosis of diseases such as ovarian, breast, or prostate cancer, without identification of single biomarkers. MALDI-TOF MS, for example, offers a simple approach for fast and reliable protein profiling, especially by using carrier materials with various physical and chemical properties, in combination with a MALDI matrix. This approach is referred to as material-enhanced laser desorption/ionization (MELDI). In this paper, we report the development and application of derivatized carrier materials [cellulose, silica, poly(glycidyl methacrylate/divinylbenzene) (GMA/DVB) particles, and diamond powder] for fast and direct MALDI-TOF MS protein profiling. The applicability of MELDI for rapid protein profiling was evaluated with human serum samples. These carriers, having various hydrophobicities, resulted in characteristic mass fingerprints, even if all materials were derivatized with iminodiacetic acid (IDA) to yield an immobilized metal affinity chromatography (IMAC) functionality. Our study demonstrates that analyzing complex biological samples, such as human serum, by employing different MELDI carrier materials yielded type- and size-dependent performance variation.     

Bioconversion of red ginseng saponins in the gastro-intestinal tract in vitro model studied by high-performance liquid chromatography–high resolution Fourier transform ion cyclotron resonance mass spectrometry

A high-performance liquid chromatography–high resolution Fourier transform ion cyclotron resonance mass spectrometry (HPLC–FTICR-MS) method was developed to investigate the metabolism of ginsenosides in in vitro models of the gastro-intestinal tract. The metabolites were identified by high-resolution tandem mass spectrometry. Degradation and bioconversion routes of the different ginsenosides at acidic (gastric) conditions and in the presence of intestinal microbiota were elaborated. Besides hydrolysis (deglycosylation) also hydration reactions occurred at acidic conditions. The results illustrate the value of metabolite profiling by HPLC–FTICR-MS for understanding of the mechanisms in bioavailability of herbal drugs and their metabolites.     

Study of three-dimensional configurations of (γ-methacryloxypropyl)-silsesquioxanes by ultraviolet laser matrix-assisted desorption/ionization time-of-flight mass spectrometry and quantum chemical calculation

[(3-Methacryloxy)propyl]silsesquioxanes (MSSO) were prepared from the hydrolytic condensation of [(3-methacryloxy)-propyl]trimethoxysilane (MPMS) in the presence of an acid catalyst (HCOOH). The proposed MSSO structures were characterized with Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) ((1)H, (13)C and (29)Si), and were assigned by ultraviolet laser matrix-assisted desorption/ionization time-of-flight mass spectrometry (UV-MALDI-TOF MS). The large organic group connected to silicon was simplified for the quantum chemical calculation (QCC), and the correlation of the calculated total energies (E(T)) before and after simplification was analyzed by multiple linear regression, verifying no significant influence on the final conclusions of the research of structural formulas by a correlation coefficient (r). The geometric parameters (Si-O bond length and Si-O-Si, O-Si-O bond angles) and E(T) of the simplified MSSO were calculated by QCC to determine the relative stability of various MSSO structures. The structural geometry (silicon ring), the fraction of intramolecular cycles (f) and the number of the silicon rings (F) were also employed to qualitatively determine the relative stability. The results of the calculation showed that almost all of the cage structures had a lower E(T) than the isomeric ladder structures; therefore, most MSSO structures are of the cage type.     

A method to detect metal–drug complexes and their interactions with pathogenic bacteria via graphene nanosheet assist laser desorption/ionization mass spectrometry and biosensors

A new method was proposed to probe the interactions between transition metals of Fe(II), Fe(III), Cu(II) with a non steroidal anti-inflammatory drug (NSAID), flufenamic acid (FF) using graphene as a matrix for Graphene assisted laser desorption ionization mass spectrometry (GALDI-MS). Metal–drug complexation was confirmed via UV absorption spectroscopy, fluorescence spectroscopy, pH meter, and change in solution conductivity. The optimal molar ratios for these complexation interactions are stoichiometry 1:2 in both Cu(II) and Fe(II) complexes, and 1:3 in Fe(III) complexes at physiological pH (7.4). Metal complexation of the drug could enhance fluorescence for 20 fold which is due to the charge transfer reaction or increase rigidity of the drug. The main interaction between graphene and flufenamic acid is the П–П interaction which allows us to probe the metal–drug complexation. The GALDI-MS could sensitively detect the drug at m/z 281.0 Da (protonated molecule) with detection limit 2.5 pmol (1.0 μM) and complexation at m/z 661.0, 654.0 and 933.0 Da corresponding to [Cu(II)(FF)₂(H₂O)₂ + H]⁺, [Fe(II)(FF)₂(H₂O)₂ + H]⁺ and [Fe(III) (FF)₃(H₂O)₂ + H]⁺, respectively (with limit of detection (LOD) 2.0 pmol (10.0 μM). Matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) spectra show change in the protein profile of intact pathogenic bacteria (Pseudomonas aeroginosa, Staphylococcus aureus). The change in the ionization ability (mainly proton affinity) of pathogenic bacteria may be due to the interactions between the bacteria with the drug (or its complexes). Shielding carboxylic group by metals and increase the hydrophilicity could enhance the biocompatibility of complexes toward the pathogenic bacteria which can be used as biosensors with high sensitivity and lowest detectable concentrations are in the range of 3.3 × 10³–3.9 × 10⁴ cfu mL⁻¹ with large linear dynamic range.     

Rapid analysis of six phthalate esters in wine by ultrasound-vortex-assisted dispersive liquid–liquid micro-extraction coupled with gas chromatography-flame ionization detector or gas chromatography–ion trap mass spectrometry

An Ultrasound-Vortex-Assisted Dispersive Liquid–Liquid Micro-Extraction (USVADLLME) procedure coupled with Gas Chromatography-Flame Ionization Detector (GC-FID) or Gas Chromatography-Ion Trap Mass Spectrometry (GC-IT/MS) is proposed for rapid analysis of six phthalate esters in hydroalcoholic beverages (alcohol by volume, alc vol⁻¹, ≤40%). Under optimal conditions, the enrichment factor of the six analytes ranges from 220- to 300-fold and the recovery from 85% to 100.5%. The limit of detection (LOD) and limit of quantification (LOQ) are ≥0.022 μg L⁻¹ and ≥0.075 μg L⁻¹, respectively. Intra-day and inter-day precisions expressed as relative standard deviation (RSD), are ≤8.2% and ≤7.0%, respectively. The whole proposed methodology has demonstrated to be simple, reproducible and sensible for the determination of trace phthalate esters in red and white wine samples.     

Specific cleavage at peptide backbone Cα-C and CO-N bonds during matrix-assisted laser desorption/ionization in-source decay mass spectrometry with 5-nitrosalicylic acid as the matrix

The use of 5-nitrosalicylic acid (5-NSA) as a matrix for in-source decay (ISD) of peptides during matrix-assisted laser desorption/ionization (MALDI) is described herein. Mechanistically, the decay process is initiated by a hydrogen abstraction from a peptide backbone amide nitrogen by 5-NSA. Hydrogen abstraction results in formation of an oxidized peptide containing a radical amide nitrogen. Subsequently, the C(α)-C bond N-terminal to the peptide bond is cleaved to form an a·/x fragment pair. The C(α)-C bonds C-terminal to Gly residues were less susceptible to cleavage than were those of other residues. C(α)-C bonds N-terminal to Pro and Sar residues were not cleaved by the aforementioned mechanism; instead, after hydrogen abstraction from a Pro or Sar C(α)-H bond, the peptide bond N-terminal to the Pro was cleaved yielding b- and y-series ions. We also show that fragments produced by MALDI 5-NSA-induced ISD were formed independently of the ionization process.