Laser Desorption-time Of Flight Mass Spectrometry Research Articles

Shifted Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65 results in formation of high mannose N-glycans in aggressive prostate cancer cells

BackgroundThere is a pressing need for biomarkers that can distinguish indolent from aggressive prostate cancer to prevent over-treatment of patients with indolent tumor. MethodsGolgi targeting of glycosyltransferases was characterized by confocal microscopy after knockdown of GM130, giantin, or both. N-glycans on a trans-Golgi enzyme β4galactosyltransferase-1 isolated by immunoprecipitation from androgen-sensitive and independent prostate cancer cells were determined by matrix-assisted laser desorption-time of flight-mass spectrometry. In situ proximity ligation assay was employed to determine co-localization of (a) α-mannosidase IA, an enzyme required for processing Man8GlcNAc2 down to Man5GlcNAc2 to enable synthesis of complex-type N-glycans, with giantin, GM130, and GRASP65, and (b) trans-Golgi glycosyltransferases with high mannose N-glycans terminated with α3-mannose. ResultsDefective giantin in androgen-independent prostate cancer cells results in a shift of Golgi targeting of glycosyltransferases and α-mannosidase IA from giantin to GM130-GRASP65. Consequently, trans-Golgi enzymes and cell surface glycoproteins acquire high mannose N-glycans, which are absent in cells with functional giantin. In situ proximity ligation assays of co-localization of α-mannosidase IA with GM130 and GRASP65, and trans-Golgi glycosyltransferases with high mannose N-glycans are negative in androgen-sensitive LNCaP C-33 cells but positive in androgen-independent LNCaP C-81 and DU145 cells, and LNCaP C-33 cells devoid of giantin. ConclusionIn situ proximity ligation assays of Golgi localization of α-mannosidase IA at giantin versus GM130-GRASP65 site, and absence or presence of N-glycans terminated with α3-mannose on trans-Golgi glycosyltransferases may be useful for distinguishing indolent from aggressive prostate cancer cells.

Possibility of production of amino acids by impact reaction using a light-gas gun as a simulation of asteroid impacts.

In order to investigate impact production of carbonaceous products by asteroids on Titan and other satellites and planets, simulation experiments were carried out using a 2-stage light gas gun. A small polycarbonate or metal bullet with about 6.5km/s was injected into a pressurized target chamber filled with 1atm of nitrogen gas, to collide with a ice + iron target or an iron target or a ice + hexane + iron target. After the impact, black soot including fine particles was deposited on the chamber wall. The soot was carefully collected and analyzed by High Performance Liquid Chromatography (HPLC), Fourier Transform Infrared Spectroscopy (FT-IR), and Laser Desorption Time-of-Flight Mass Spectrometry (LD-ToF-MS). As a result of the HPLC analysis, about 0.04-8pmol of glycine, and a lesser amount of alanine were found in the samples when the ice + hexane + iron target was used. In case of the ice + iron target and the iron target, less amino acids were produced. The identification of the amino acids was also supported by FTIR and LD-ToF-MS analysis.

Identification of Henosepilachna vigintioctomaculata (Coleoptera: Coccinellidae) midgut putative receptor for Bacillus thuringiensis insecticidal Cry7Ab3 toxin

Bt WZ-9 strain, containing a single Cry7Ab3 toxin, had effective insecticidal activity against larvae of Henosepilachna vigintioctomaculata. By incubation with larvae midgut homogenate and trypsin in vitro, 130kDa Cry7Ab3 protoxin was degraded into the ∼75kDa proteinase-resistant fragments. In vivo analysis, 130kDa Cry7Ab3 protoxin was also processed into ∼75kDa fragment. Histopathological observations indicated that Cry7Ab3 ingestion by H. vigintioctomaculata larvae causes acceleration in the blebbing of the midgut epithelium cells into the gut lumen and eventual lysis of the epithelium cells resulting in larval death. A ligand blotting experiment demonstrated that Cry7Ab3 toxin bound a 220kDa BBMV protein. This receptor protein was identified as cadherin by matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS). The cadherin protein may be the receptor of Cry7Ab3. The data obtained may contribute to a better understanding of the mechanism of Cry7Ab3 toxin against H. vigintioctomaculata larvae.

Molecular mass ranges of coal tar pitch fractions by mass spectrometry and size‐exclusion chromatography

A coal tar pitch was fractionated by solvent solubility into heptane-solubles, heptane-insoluble/toluene-solubles (asphaltenes), and toluene-insolubles (preasphaltenes). The aim of the work was to compare the mass ranges of the different fractions by several different techniques. Thermogravimetric analysis, size-exclusion chromatography (SEC) and UV-fluorescence spectroscopy showed distinct differences between the three fractions in terms of volatility, molecular size ranges and the aromatic chromophore sizes present. The mass spectrometric methods used were gas chromatography/mass spectrometry (GC/MS), pyrolysis/GC/MS, electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry (ESI-FTICRMS) and laser desorption time-of-flight mass spectrometry (LD-TOFMS). The first three techniques gave good mass spectra only for the heptane-soluble fraction. Only LDMS gave signals from the toluene-insolubles, indicating that the molecules were too involatile for GC and too complex to pyrolyze into small molecules during pyrolysis/GC/MS. ESI-FTICRMS gave no signal for toluene-insolubles probably because the fraction was insoluble in the methanol or acetonitrile, water and formic acid mixture used as solvent to the ESI source. LDMS was able to generate ions from each of the fractions. Fractionation of complex samples is necessary to separate smaller molecules to allow the use of higher laser fluences for the larger molecules and suppress the formation of ionized molecular clusters. The upper mass limit of the pitch was determined as between 5000 and 10,000 u. The pitch asphaltenes showed a peak of maximum intensity in the LDMS spectra at around m/z 400, in broad agreement with the estimate from SEC. The mass ranges of the toluene-insoluble fraction found by LDMS and SEC (400-10,000 u with maximum intensity around 2000 u by LDMS and 100-9320 u with maximum intensity around 740 u by SEC) are higher than those for the asphaltene fraction (200-4000 u with maximum intensity around 400 u by LDMS and 100-2680 u with maximum intensity around 286 u by SEC) and greater than values considered appropriate for petroleum asphaltenes (300-1200 u with maximum intensity near 700 u).

Mass spectrometric analysis of large PAH in a fuel-rich ethylene flame

Mass spectrometric analysis by laser desorption-time of flight-mass spectrometry (LDI-TOF-MS) was exploited to extend the detection of flame-formed polycyclic aromatic hydrocarbons (PAH) up to the mass limit of the first soot particles (>2000 Da) in the soot formation region of a premixed fuel-rich (C/O = 1) ethylene flame. The typical decreasing intensity of PAH ion peaks with increasing mass was found in the mass range m/ z 500–1700 although a slight enrichment in the heavier part of PAH could be observed to occur along the flame axis. The separation by means of size exclusion chromatography (SEC) into two different classes of PAH followed by UV-visible spectroscopy corroborated the mass spectral identification of large mass PAH. Critical examination of mass spectral features and SEC separation was the starting point for speculation about the changes occurring in PAH growth from planar to concave structures which could be important for soot inception mechanisms.

The search for fullerenes in rocks from the Ries impact crater

Since their discovery, fullerenes have been reported from various geological environments. One group of these findings has been related to bolide impacts, e.g., the Sudbury crater and the K-T and P-T boundaries. Impact rocks of the Ries crater, Germany, including samples of suevites, metamorphosed crystalline clasts, and glass bombs, have been collected in the Otting, Altebrg, and Seelbronn quarries. No fullerenes in concentrations above 1 ppb have been found in analyzed samples. Laser desorption time-of-flight mass spectrometry (LD-TOF-MS) confirmed the absence of fullerenes in the analyzed samples. These results support the concept that the Ries impactor was a stony meteorite.

Aberrant profiles of native and oxidized glycoproteins in Alzheimer plasma.

A proteomic approach was employed to elucidate possible differential expression of native and oxidized glycoproteins using pooled plasma samples derived from ten patients with sporadic Alzheimer's disease (AD) and pooled plasma samples from nine normal elderly control (NEC) subjects. The plasma samples were fractionated by sequential affinity chromatography on heparin-agarose (HepA) and concanavalin A-agarose (ConA) columns followed by separation on one-dimensional and two-dimensional polyacrylamide gels. Carbonylation (oxidation) of proteins was monitored by in-strip derivatization with 2,4-dinitrophenylhydrazine (DNP) and anti-DNP immunoblotting. Nine spots representing glycoproteins which showed enrichment or high specific oxidation indices in AD HepA-ConA 2-D gels relative to NEC samples were analyzed by matrix-assisted laser desorption-time of flight-mass spectrometry and identified with high probability (p < 0.001) as isoforms of human transferrin (Tf), hemopexin (Hpx) and alpha-1-antitrypsin (alpha-1-AT). These glycoproteins were concentrated, respectively, 5-, 6.5- and 107-fold in HepA-ConA eluates derived from AD plasma relative to the NEC samples. Specific oxidation indices of the identified Tf and Hpx isoforms in AD plasma were respectively, 7.4 and 2.8 relative to NEC. Our findings provide further evidence for systemic derangements in heme/iron/redox homeostasis and activation of the acute phase response in sporadic AD. Moreover, the data implicate isoforms of Tf, Hpx and alpha-1-AT as potential biological markers of this condition.

Separation of basic drug enantiomers by capillary electrophoresis using chicken alpha1-acid glycoprotein: insight into chiral recognition mechanism.

Recombinant chicken alpha(1)-acid glycoprotein (alpha(1)-AGP) was prepared by the Escherichia coli expression system and completely deglycosylated alpha(1)-AGP (cd-alpha(1)-AGP) was obtained by treatments of native alpha(1)-AGP with a mixture of endoglycosidase and N-glycosidase. The average molecular masses of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP were estimated to be about 29 200, 21 700 and 20 700, respectively, by matrix-assisted laser desorption-time of flight-mass spectrometry. We compared the chiral recognition ability of chicken alpha(1)-AGP, cd-alpha(1)-AGP and recombinant alpha(1)-AGP using them as chiral selectors in capillary electrophoresis. The chicken alpha(1)-AGP showed higher resolution for eperisone, pindolol and tolperisone than cd-alpha(1)-AGP or recombinant alpha(1)-AGP. Recombinant alpha(1)-AGP still showed chiral recognition for three basic drugs tested. By addition of propranolol as a competitor in the separation solution in CE, no enantioseparations of three basic drugs were observed with chicken alpha(1)-AGP, cd-alpha(1)-AGP or recombinant alpha(1)-AGP. These results reveal that the protein domain of the chicken alpha(1)-AGP is responsible for the chiral recognition ability, and that the chiral recognition site(s) for basic drugs exists on the protein domain.

Proteomics Methods for Probing Molecular Mechanisms in Signal Transduction

mRNA splicing and various posttranslational modifications to proteins result in a larger number of proteins than genes. Assessing the dynamic nature of this proteome is the challenge of modern proteomics. Recent advances in high throughput methods greatly facilitate the analysis of proteins involved in signal transduction, their production, posttranslational modifications and interactions. Highly reproducible two dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods, coupled with matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS) allow rapid separation and identification of proteins. These methods, alone or in conjunction with other techniques such as immunoprecipitation, allow identification of various critical posttranslational modifications, such as phosphorylation. High throughput identification of important protein-protein interactions is accomplished by yeast two hybrid approaches. In vitro and in vivo pulldown assays, coupled with MALDI-TOF-MS, provide an important alternative to two hybrid approaches. Emerging advances in production of protein-based arrays promise to further increase throughput of proteomics-based approaches to signal transduction.

Laser desorption time-of-flight mass spectrometry of fluorocarbon films synthesized by C4F8/H2 plasmas

Molecular structures of fluorocarbon films synthesized by C4F8/H2 plasmas were analyzed by laser desorption time-of-flight mass spectrometry (LD-TOFMS). Mass peaks of positive and negative ions with m/z up to 250 were observed in the mass spectra. Most of these ions were assigned as atomic hydrogen, atomic fluorine, carbon clusters, hydrocarbon clusters, fluorocarbon clusters, and hydrofluorocarbon clusters. The mass spectra changed sensitively with the partial pressure of H2. The LD-TOFMS analysis showed that the films changed from a polytetrafluoroethylene-like film into crosslinked carbon- and hydrogen-rich one by the addition of H2. The change in the chemical composition of the mass spectrum was consistent with the analyses by conventional x-ray photoelectron spectroscopy and Fourier transform infrared absorption spectroscopy. It is emphasized that LD-TOFMS can be used for the analysis of the degree of polymerization, which is an advantage of LD-TOFMS over the conventional analysis methods.

Proteomics methods for probing molecular mechanisms in signal transduction.

mRNA splicing and various posttranslational modifications to proteins result in a larger number of proteins than genes. Assessing the dynamic nature of this proteome is the challenge of modern proteomics. Recent advances in high throughput methods greatly facilitate the analysis of proteins involved in signal transduction, their production, posttranslational modifications and interactions. Highly reproducible two dimensional polyacrylamide gel electrophoresis (2D-PAGE) methods, coupled with matrix assisted laser desorption-time of flight-mass spectrometry (MALDI-TOF-MS) allow rapid separation and identification of proteins. These methods, alone or in conjunction with other techniques such as immunoprecipitation, allow identification of various critical posttranslational modifications, such as phosphorylation. High throughput identification of important protein-protein interactions is accomplished by yeast two hybrid approaches. In vitro and in vivo pulldown assays, coupled with MALDI-TOF-MS, provide an important alternative to two hybrid approaches. Emerging advances in production of protein-based arrays promise to further increase throughput of proteomics-based approaches to signal transduction.

Laser-Desorption Time-of-Flight Mass-Spectrometry Analysis of Soot from Various Hydrocarbon Fuels

Laser-desorption time-of-flight mass spectrometry (LDMS) was used to analyze soot collected from seven different hydrocarbon fuels (methane, n-hexane, n-heptane, n-octane, n-decane, benzene and toluene) burning with air under normal atmospheric pressure. To evaluate the technique, samples of two different types of furnace blacks, two types of graphite, a synthetic diamond and a C70 fullerene also were analyzed. With proper experimental design, the C70 produced a single peak of mass 840 Da, thereby showing the ability of LDMS to remove and singly ionize large intact molecular species from the sample for the purpose of chemical analysis. The LDMS soot analysis showed that each mature soot has a unique chemical fingerprint. A family of fuels (e.g., normal alkanes) produces soot that is similar in appearance when mature and that therefore might be expected to yield similar mass spectra, but it is found instead that each spectrum has unique features that are different for each parent fuel. These initially surprising results point to the need for further study of fuel pyrolysis in flames.

Synthetic metal-binding protein surface domains for metal ion-dependent interaction chromatography: I. Analysis of bound metal ions by matrix-assisted UV laser desorption time-of-flight mass spectrometry

To extend the analytical capabilities of immobilized metal ion affinity chromatography (IMAC) for evaluation of biologically relevant peptide-metal ion interactions, we have prepared synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein known as histidine-rich glycoprotein (HRG). Three synthetic peptides, representing multiples of a 5-residue repeat sequence (Gly-His-His-Pro-His) from within the histidine- and proline-rich region of the C-terminal domain were prepared. Prior to immobilization, the synthetic peptides were evaluated for identity and sample homogeneity by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), a method developed recently for the mass determination of high-molecular-mass biopolymers. 2,5-Dihydroxybenzoic acid was evaluated as a matrix to facilitate the laser desorption and ionization of intact peptides and was found to be ideally suited for determinations of mass within the low-mass region of interest (641.7 to 1772.8 dalton). We observed minimal chemical noise from photochemically generated peptide-matrix adduct signals, clustering, and multiply-charged peptide species. Peptides with bound sodium and potassium ions were observed; however, these signal intensities were reduced by immersion of the sample probe tip in water. Mixtures of the three different synthetic peptides were also evaluated by LDTOF-MS after their elution through a special immobilized peptide-metal ion column designed to investigate metal ion transfer. We found LDTOF-MS to be a useful new method to verify the presence of peptide-bound metal ions. Thus, LDTOF-MS is ideally suited for the rapid (3−5 min), sensitive (<1 pmol), accurate (±0.05%), and relatively high resolution ( m/Δ m = 300−500, full width at half maximum, where m = mass) evaluation of synthetic peptides. Further, LDTOF-MS was found to be an important tool for the characterization of peptide mixtures and peptide-metal ion interactions.

Recognition of transition metal ions by peptides Identification of specific metal-binding peptides in proteolytic digest maps by UV laser desorption time-of-flight mass spectrometry

Metal-binding peptides in proteolytic digest maps have been identified by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS). The plasma and milk metal transport protein chosen to demonstrate this process, histidine-rich glycoprotein (HRG), was purified and then digested with trypsin; the cleavage products were analyzed by LDTOF-MS with dihydroxbenzoic acid as the matrix. The selective interaction of specific peptides with one or more Cu atoms was observed when Cu(II) ions were added to the digest mixture. At least one specific metal-binding peptide was identified by computerized sequence analysis using the molecular mass data and available cDNA sequence. These results demonstrate the first direct observation by mass spectrometry of differential peptide—metal ion interactions in protein digest maps. The ability to evaluate peptide—metal ion interactions, including stoichiometry, with less than 1 pmol of sample improves significantly our ability to identify metal binding domains in metal-binding proteins.

Frontiers in elemental analysis by mass spectrometry

New developments in inductively coupled plasma mass spectrometry (ICP-MS) and laser desorption-time-of-flight mass spectrometry (LD-TOF-MS) for inorganic analysis are described. These include fundamental studies of the ion extraction, process in ICP-MS, development of a highly efficient nebulizer, isotope tracer studies, measurement of elemental speciation by liquid chromatography with ICP-MS detection and characterization of the structures of solids by ion association. The possibilities of determining inorganic species in solutions by electrospray MS are also described.

Evaluation of peptide/metal ion interactions by UV laser desorption time‐of‐flight mass spectrometry

A relatively recent method developed to determine the molecular weights of intact peptides and proteins, matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), has been evaluated as a new means to investigate the metal ion-binding properties of model synthetic peptides. A contiguous sequence of 25 residues on the surface of the 74 kDa human plasma metal-binding transport protein histidine-rich glycoprotein (HRG) has been identified as a bioactive metal-binding domain. The peptide, (GHHPH)5G, was synthesized and evaluated by LDTOF-MS before and after the addition of Cu(II) in solution with 2,5-dihydroxybenzoic acid as the matrix. In the absence of added Cu(II), the major protonated molecular ion (M + H)+ was observed to have a mass equal to its calculated mass (2904.0 Da). In the presence of Cu(II), however, five additional peaks were observed at mass increments of approximately 63.9 Da. The maximum Cu(II)-binding capacity observed for the 26-residue peptide (5 g-atoms/mol) suggested that up to 1 Cu(II) may be bound per 5-residue internal repeat unit (GHHPH) within this peptide; several other monovalent and divalent metal cations were not bound under identical conditions of analysis. The Cu(II)-binding stoichiometry was verified by spectrophotometric titration and by frontal analyses of the immobilized peptide with a solution of Cu(II) ions. These results demonstrate the ability to verify directly the solution-phase binding capacity of metal-binding peptides by LDTOF-MS.