Endgroup analysis of polyethylene glycol polymers by matrix-assisted laser desorption/ionization Fourier-transform ion cyclotron resonance mass spectrometry

Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) by external injection of matrix-assisted laser desorbed and ionized (MALDI) polymers offers good possibilities for characterization of low molecular weight homopolymers (MW range up to 10 kDa). The molecular masses of the molecular weight distribution (MWD) components of underivatized and derivatized (dimethyl, dipropyl, dibutyl and diacetyl) polyethylene glycol (PEG) 1000 and 4000 were measured by MALDI-FTICR-MS. These measurements have been performed using a commercial FTICR spectrometer with a home-built external ion source. MALDI of the samples with a 2,5-dihydroxybenzoic acid matrix in a 1000:1 matrix-to-analyte molar ratio produces sodiated molecules in a sufficient yield to trap the ions in the ICR cell. The masses of the molecular weight distribution of PEG components were measured in broad-band mode with a mass accuracy of < 5 ppm in the mass range around 1000 u and within 40 ppm accuracy around 4000 u. From these measurements, the endgroup mass of the polymer was determined by correlation of the measured component mass with the degree of polymerization. The masses of the PEG endgroups have been determined within a deviation of 3-10 millimass units for the PEG1000 derivatives and 10-100 millimass units for the PEG4000 derivatives, thus confirming the identity of the distal parts of the model compounds.     

ESI/ion trap/ion mobility/time-of-flight mass spectrometry for rapid and sensitive analysis of biomolecular mixtures

An ion trap/ion mobility/time-of-flight mass spectrometry technique is shown to be a rapid and sensitive means of analyzing peptide/protein mixtures. In this approach, an ion trap is used to accumulate ions that have been electrosprayed from a mixture into concentrated packets. The ion packets are injected into a drift tube where components of the mixture are separated based on differences in mobility through a buffer gas. Ions that exit the drift tube are dispersed in a time-of-flight mass spectrometer for mass-to-charge (m/z) determination. The gas-phase separation strategy reduces congestion in the mass spectrum, and experimental mobilities complement m/z measurements in assigning peaks. Examples of the application of the approach to identification of peptides (from tryptic digests) and to separation of charge-state distributions from electrospray of a mixture containing ubiquitin and myoglobin are presented. Most peptides that are observed from tryptic digests of proteins such as cytochrome c and myoglobin can be identified from data that are acquired in under 1 min; studies of mixtures with known compositions indicate that detection limits are approximately 0.5-3 pmol for individual components. Factors that may influence the distributions that are observed, such as storage time in the trap, injection voltages used for the mobility experiment, and variations in ion cross section with charge state, are discussed.     

Detection of designer drugs in human hair by ion mobility spectrometry (IMS)

The objective of this study was to investigate cyclic-adenosinemonophosphate (cAMP)-dependent phosphorylation in murine erythroleukemia (MEL) cells and to identify either direct substrates of cAMP-dependent kinase or downstream effectors of cAMP dependent phosphorylation with a potential function in growth and differentiation. MEL-cells rendered deficient in cAMP-dependent protein kinase (A-kinase) activity by stable transfection with DNA encoding for either a mutant regulatory subunit or a specific peptide inhibitor of A-Kinase (PKI) are unable to differentiate normally in response to chemical inducers. We have identified by 2-D Western blotting 2 phosphorylated forms of p19, a highly conserved 18-19 kDa cytosolic protein that is frequently upregulated in transformed cells and undergoes phosphorylation in mammalian cells upon activation of several signal transduction pathways. The phosphorylation of the more acidic phosphorylated form is increased in a cAMP-dependent fashion and impaired in cells deficient in cAMP-dependent kinase (A-kinase). Treatment of MEL-cells with the chemical inducer of differentiation hexamethylene-bisacetamide (HMBA) led to dephosphoryation of this phosphoform. Our data are compatible with previous observations which imply that phosphorylation of Ser 38 in p19 by p34cdc2-kinase leads to a more basic phosphoform and simultaneous phosphorylation by mitogen-activated kinase of Ser 25 in response to protein kinase C and the cAMP-dependent kinase creates the more acidic species.     

Visualization of the hydrogen desorption process from ferrite,pearlite, and graphite by secondary ion mass spectrometry

The distribution and desorption processes of hydrogen and deuterium have been visualized by secondary ion mass spectrometry (SIMS). The present article deals with four principal points: (1) visualizing the hydrogen distribution, (2) visualizing the hydrogen desorption process from each metallurgical microstructure under various holding times at 25 °C, (3) visualizing the hydrogen desorption process during heating, and (4) determining the correspondence between desorption profiles and desorption sites. A spheroidal graphite cast iron specimen was prepared for visualizing hydrogen, since it consists of basic microstructures of steels such as ferrite and pearlite. Hydrogen and deuterium were occluded into the cast iron. The amount of hydrogen and the existing states of hydrogen in the cast iron were analyzed by thermal desorption spectrometry (TDS). The TDS analyses show that the hydrogen desorption has two peaks, namely, the low- and high-temperature peaks corresponding to trap activation energies of 21.6 and 105.8 kJ/mol, respectively. The SIMS analyses of the specimen cooled after heating to 100 °C, 200 °C, and 300 °C reveal that the hydrogen desorbs from the ferrite after heating to 100 °C, from the pearlite and the interfaces between the ferrite and the graphite after heating to 200 °C, and from the pearlite after heating to 300 °C. The graphite remains, trapping hydrogen after heating to 300 °C. On the basis of TDS and SIMS results, the relationship between the desorption profile and desorption sites was identified; that is, the low-temperature peak corresponds to ferrite, pearlite, and graphite/ferrite interfaces, while the high-temperature peak corresponds to graphite.     

Matrix-assisted laser desorption/ionization mass spectrometry of noncovalent protein-transition metal ion complexes

Transition metal ion complexes with proteins and peptides are important in many areas of analytical and biological chemistry. We used positive and negative ion MALDI-MS to detect complexes with Cu and Ni ions, and show that the specific and non-specific transition metal ion-peptide complexes can be distinguished by the use of different analytical protocols. The pH dependent stability of these complexes is also reflected in the MALDI data. We further show that triple complexes of peptides or protein with chelated metal ions can be detected efficiently and rapidly by MALDI mass spectrometry. Such triple complexes play an important role in metal chelate affinity chromatography, where histidine containing biopolymers in particular are thought to bind metal-ligand complexes, depending on the oxidation state of the metal and the number of unoccupied coordination sites of the ligand.     

Lipid analysis by matrix-assisted laser desorption and ionization mass spectrometry: A methodological approach

It has been hypothesized that protein factors may protect CpG islands from methyltransferase during development and that demethylation may involve protein-DNA interactions at demethylated sites. However, direct evidence has been lacking. In this study, demethylation at the EBNA-1 binding sites of the Epstein-Barr virus latent replication origin, oriP, was investigated by using human cells. Several novel findings are discussed. First, there are specific preferential demethylation sites within the oriP region. Second, the DNA sequence of oriP alone is not the target of an active demethylation process. Third, EBNA-1 binding is required for the site-specific demethylation in oriP. Interestingly, CpG sites adjacent to and between the EBNA-1 sites do not become demethylated. Fourth, demethylation of the first DNA strand in oriP at the EBNA-1 binding sites involves a passive (replication-dependent) mechanism. The second-strand demethylation appears to occur through an active mechanism. That is, EBNA-1 protein binding prevents the EBNA-1 binding sites from being remethylated after one round of DNA replication, and it appears that an active demethylase then demethylates these hemimethylated sites. This study provides clear evidence that protein binding specifies sites of DNA demethylation and provides insights into the sequence of steps and the mechanism of demethylation.     

Enhanced mass resolving power, sensitivity, and selectivity in laser desorption Fourier transform ion cyclotron resonance mass spectrometry by ion axialization and cooling

Ion cooling and axialization produced by azimuthal quadrupolar excitation in the presence of ion-neutral collisions are applied to laser desorption Fourier transform ion cyclotron resonance mass spectrometry (LD/FT/ICR-MS). With this technique, the large kinetic and internal energies of ions generated by laser desorption processes can be cooled effectively by collisions of ions with neutral argon atoms (at > 5 x 10(-7) Torr). After sufficient cooling in the source compartment of a dual ion trap, the axialized ions may be transferred to the analyzer compartment for detection at much lower pressure (and thus much higher mass resolving power). Enhancements in both FT/ICR mass resolving power and sensitivity are observed; moreover, ion isolation with high selectivity at high pressure is also demonstrated.     

Structure analysis of trivalent glycoclusters by post-source decay matrix-assisted laser desorption/ionization mass spectrometry

Post-source decay (PSD) matrix-assisted laser desorption/ionization time-of-flight mass spectra were found to be useful for the structural elucidation of a series of tris [2-(glycosylthiourylene)ethyl]amines. The reported fragmentation behaviours of [M + H]+, [M + Na]+ and [M - H]- ions differ from each other significantly; however, they can be compared to tree pruning in every case. Whereas detailed structural information on unprotected glycoclusters is obtained from all PSD experiments, only the positive-ion mode can be used to gain relevant information about the acetylated glycoclusters.     

Characterisation of proteins from two-dimensional electrophoresis gels by matrix-assisted laser desorption mass spectrometry and amino acid compositional analysis

Amino acid compositional analysis and peptide mass fingerprinting by matrix assisted laser desorption mass spectrometry have been used to characterise proteins obtained from two-dimensional electrophoresis (2-DE) separations of human cardiac proteins. A group of twelve protein spots was selected for analysis. The identities of eight of the proteins had been determined by conventional protein characterisation methods, two were unknown proteins and two had putative identities from protein database spot comparison. Amino acid analysis and peptide mass fingerprinting gave corresponding identities for seven of the twelve proteins, which also agreed with our initial identifications. Three proteins which had been identified previously were not confirmed in this study and putative identities were obtained for the two unknown proteins. The advantages, problems and use of amino acid analysis and peptide mass fingerprinting for the analysis of proteins from 2-DE are discussed. The data highlight the need to use orthogonal techniques for the unequivocal identification of proteins from 2-DE gels.     

热脱附-气相色谱质谱法测定电子电气产品聚合物材料中多溴联苯和多溴联苯醚

多溴联苯(PBBs)和多溴联苯醚(PBDEs)为持久性的有机污染物,因此对电子电气产品聚合物材料中PBBs和PBDEs含量的测定和控制具有重要意义。以往在对其进行测定前常需先对样品进行前处理,操作较为繁琐或需样品量较大。实验采用热脱附方式将目标化合物从样品中脱附出来进入气质联用仪进行分析,只需mg级别样品量就可实现直接固体进样-气相色谱质谱法(GC-MS)对电子电气产品聚合物材料中PBBs和PBDEs的测定。对热脱附条件包括热脱附温度、热脱附时间、热脱附升温速率等进行了优化,结果表明,在优化的实验条件下,方法线性范围为100～2 000mg/kg,相关系数为0.996以上,方法检出限在5.4～24.8mg/kg之间,定量限在18.0～82.6mg/kg之间。按照实验方法对聚丙烯标准物质ERM-EC591中PBBs和PBDEs进行7次平行测定,结果表明,对于含量高于定量限的PBBs和PBDEs,测定值与认定值基本吻合,测定结果的相对标准偏差(RSD)均小于15%。采用实验方法对20批电子电气产品聚合物材料中PBBs和PBDEs进行定性定量分析,并与标准方法GB/T 26125—2011附...     

Protein conformational changes determined by matrix-assisted laser desorption mass spectrometry

Caldesmon is an actin/calmodulin/tropomyosin protein located in the thin filaments of smooth muscle cells and microfilaments of nonmuscle cells. Two isoforms of caldesmon, h- and l-types, shown to exist in vertebrate smooth and nonmuscle cells respectively, are produced by alternative splicing of the caldesmon mRNA encoded by a single gene. To study the expression of smooth muscle specific h-caldesmon during the differentiation of mesenchymal cells into smooth muscle cells, soluble protein and total RNA from the gizzard primordium in the gut region of 5-day and gizzards of 7-, 9-, 13-, 17- and 21-day embryos and 2-days post-hatch chicks were extracted and analyzed for caldesmon expression at both protein and mRNA levels. Western blot analysis of proteins and immunofluorescence microscopy of tissue section were carried out using an antibody specific for h-caldesmon. Total RNA was analyzed by Northern blotting using a caldesmon cDNA probe, and h- and l-caldesmon cDNAs were identified due to the difference in their molecular sizes (4.8 and 4.1 kb respectively). The mRNA was also analyzed by reverse transcribed-polymerase chain reaction (RT-PCR) and Southern blot analysis. Our results show that the I-caldesmon mRNA was expressed at higher levels in the gizzard primordium during the early stages of development, and decreased gradually during growth. The h-caldesmon protein and mRNA, not expressed at day 5, is minimally expressed at day 7 and is fully turned on by day 9. Additionally, sequence analyses of the RT-PCR products of I-caldesmon showed that it lacked the spacer region, as predicted. RT-PCR analysis of total RNA gave two h-caldesmon fragments. These two fragments were identified as two different isoforms of h-caldesmon since they both contained the spacer region. They also showed homology in the region of exon 4 had differences in the region of exon 3b.     

Direct analysis of aqueous samples by matrix-assisted laser desorption ionization mass spectrometry using membrane targets precoated with matrix

To determine bone mineral density in patients with differentiated thyroid carcinoma receiving thyroxine replacement therapy in suppressive doses, we studied 65 patients (47 women and 18 men; age 25-83 years, mean+/-SD 52.5+/-15.4 years). Patients were free of thyroid cancer in clinical and laboratory examinations at the time of the study. Bone mineral density of the lumbar spine and both hips was measured by dual-energy X-ray absorptiometry. There was no decrease in bone density in either 32 postmenopausal or 15 premenopausal women compared with an age- and sex-matched control group, nor was any decrease in bone density found in men. Our data suggest that thyroxine treatment in suppressive doses in patients with differentiated thyroid carcinoma is not a risk factor for the development of osteoporosis.     

The quantitative analysis of inhalational anaesthetics in forensic samples by gas chromatography/mass spectrometry/selected ion monitoring

In 1990 Kapolowé was, without a doubt, the site of the only surgical centre in Zaire dealing with handicaps which developed in as an after-effect of leprosy. It would be useful to explain the hazards involved in such a venture for reasons which do not pertain to medicine but, rather, to particularly trying socio-political circumstances. The best surgical expertise was thrown out for political reasons. Insecurity and economic hardships practically halted movement and, consequently, the wider application of such expertise. During a mission in 1994, there was a partial resumption of activities. The surgical team was reinstalled and made operational. It had been possible to state that multidrug therapy (MDT) had always ensured that the disabled leprosy patients, living in groups, and treated before 1990 under regular supervision, did not experience serious relapses. That fact corroborates earlier information relating particularly to surgical decompression. Although most of them were able to resume a certain measure of professional activity, social factors must still be borne in mind and the concept of partial permanent disability must be applied.     

Structure analysis of acetylated and non-acetylated O-linked MUC1-glycopeptides by post-source decay matrix-assisted laser desorption/ionization mass spectrometry

We have investigated the potential of structural elucidation of O-linked glycopeptides by post-source decay matrix-assisted laser desorption ionization mass spectrometry (PSD-MALDI-MS). In order to establish detailed fragmentation patterns and to dissect fragment ions with and without carbohydrate content, the same O-linked MUC1-derived glycopeptides with acetylated and non-acetylated sugars were analysed and compared. Furthermore, we were interested to examine possible differences in the fragmentation between glycopeptides with acetylated and non-acetylated sugars. The obtained PSD-MALDI-MS spectra showed a rather complete set of fragmentation data which allows to localize the glycan on the peptide, in parallel with sequencing a short glycan and the backbone peptide. Fragmentations of the sugars were dominated by inter-ring cleavages at the glycosidic bond. Intra-ring cleavage did also occur from the non-reducing end, but to a much lower extent. The fragmentation of the peptide backbone was not changed either by acetylated or non-acetylated sugars. Glycosylated peptide fragments occurred as fully glycosylated fragment ions, partially deglycosylated ions and completely deglycosylated ions, and was not influenced by the acetylation of sugars. However, differences occurred in the quality and quantity of fragment ions from the non-reducing end of the glycan part when comparing acetylated with non-acetylated glycopeptides.     

Identification of urinary oligosaccharides by matrix-assisted laser desorption ionization time-of-flight mass spectrometry

A new method of urinary oligosaccharides identification by matrix-assisted laser desorption time-of-flight mass spectrometry is presented. The method involves three steps: coupling of the urinary oligosaccharides with 8-aminonaphthalene-1,3,6-trisulfonic acid; fast purification over a porous graphite carbon extraction column; and mass spectrometric analysis. Identification of urinary oligosaccharides is based on the patterns and values of the pseudomolecular ions observed. We report here the patterns in urines from patients with Pompe disease, alpha and beta mannosidoses, galacto-sialidosis, and GM1 gangliosidosis. The protocols described here allowed facile and sensitive identification of the pathognomonic oligosacchariduria present in lysosomal diseases and can be extended to any pathological oligosacchariduria.     

Peptide sequencing by partial acid hydrolysis and high resolution plasma desorption mass spectrometry

A method of deriving peptide sequence information using partial acid hydrolysis in combination with accurate mass measurements and immonium ion analysis provided by high-resolution plasma desorption mass spectrometry has been developed. The technique is very simple in terms of the chemistry and involves a short-time (3-30 min) incubation of the peptide in 1N-6N HCl at 100-110 degrees C with subsequent mass spectrometric analysis. Partial acid hydrolysis is found to produce sequence-specific segments, often ladder-like, although not always a complete set. Two application examples of the method are given: the linear peptide bradykinin and desmopressin, a peptide with an internal S-S bond and a non-amino-acid constituent. The technique has proved to be particularly useful in cases where some a priori information on the peptide structure was already known or where the automated Edman degradation technique might yield erratic results or not work at all.     

Investigation of spectral reproducibility in direct analysis of bacteria proteins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry (TOFMS) can be used for rapid detection of bacteria proteins in a crude mixture. It can potentially be used as a tool for bacterial identification based on the mass spectral patterns or the appearance of some characteristic mass peaks. However, there are many experimental parameters that can potentially have a strong effect on the observed mass spectra. The objective of this work is to address the mass spectral reproducibility issue. Several experimental parameters that may affect the MALDI spectra are systematically investigated. Results of spectral comparison from two laboratories with different operators and instrumentation are presented. It is demonstrated that minor variations in the sample/matrix preparation procedures for MALDI and in the experimental conditions used for bacterial protein extraction can result in a significant change in the observed spectra, though a number of peaks are conserved in the spectra obtained under different experimental conditions from the same bacterial sample. These conserved peaks may potentially be used as the biomarkers for bacterial identification. It is stressed that this type of investigation on spectral reproducibility should be carried out for different bacterial species in order to identify the mass spectral peaks that are consistently detected regardless of operator and nominal variations in sample preparation approach.     

Preliminary evaluation of an SF5+ polyatomic primary ion beam for analysis of organic thin films by secondary ion mass spectrometry

Organic vapor deposited thin films of pure biomolecules, polymer films and biomolecules dispersed in gelatin and biological tissue have been analyzed in a magnetic sector secondary ion mass spectrometer using an SF5+ primary ion beam at keV impact energies. In comparison to Ar+ bombardment under identical conditions, bombardment with SF5+ gives a 10 to 50 fold enhancement in the secondary ion yields for characteristic molecular ions. The SF5+ primary ion beam can be focussed to a small spot allowing molecular ion images to be obtained at micrometer spatial resolution with enhanced sensitivity. More importantly, the decay in molecular ion signal as a function of primary ion dose commonly observed in SIMS using monoatomic primary ions is either eliminated or greatly reduced, allowing molecular depth profiles to be obtained of organic thin films. By continuing to sample intact molecules as sputtering proceeds into the sample, the total number of detected characteristic secondary ions is increased by as much as a factor of approximately 700 for SF5+ bombardment as compared to Ar+ bombardment under identical analytical conditions. This effect is thought to be a result of the high erosion rate and the low penetration depth inherent in the use of a polyatomic primary projectile.