Structural analysis and quantitative evaluation of the modifications produced in human hemoglobin by methyl bromide using mass spectrometry and Edman degradation

The present study reports a procedure developed for the identification and quantitative analysis of the adducts formed by interaction of methyl bromide with human hemoglobin, based on combined analysis by electrospray mass spectrometry and automated Edman degradation of either intact globin chains or tryptic peptides of globin chains. The procedure has allowed identification of the reactive sites in human hemoglobin, and has been applied to the analysis of samples modified in vitro by methyl bromide. The results obtained represent the basis for the complete structural characterization of the modified hemoglobin and demonstrate the usefulness of the proposed analytical approach for the evaluation of the degree of alkylation and the identification of modified amino acids in proteins.     

Structural characterization by mass spectrometry of hemoglobin adducts formed after in vivo exposure to methyl bromide

A mass spectrometric procedure is described for the structural study of the adducts formed in human hemoglobin by in vitro exposure of erythrocytes to the alkylating agent methyl bromide using different protein to reagent ratios. Peptide mapping by HPLC and tandem mass spectrometry allowed location of methylated amino acids within the protein sequence. A prominent reactivity of several nucleophilic side chains in human hemoglobin subunits was observed, which was modulated by the concentration of the alkylating agent. Cysteine residues, the main reactive sites, were fully methylated in hemoglobin exposed to a 10-fold excess of methyl bromide, differently from other residues, including histidines, showing a heterogeneous pattern of methylation that was largely directed by their environment. No evidence of methylation was found at the heme proximal histidines beta92 and alpha87. A more selective methylation was obtained when the ratio methyl bromide: hemoglobin was lowered to about 1:1. In this last case only specific residues were reactive. Among them, the N-terminal amino group of both alpha- and beta-globins, cysteine 104 in the alpha-chain and cysteine 93 (not cysteine 112) in the beta-chain, indicating a different accessibility to reaction of the sulfhydryl groups on the protein chain. Thus hemoglobin side chains are selectively modified and the degree of modification at each site is a function of the position of the single amino acid residue within the protein quaternary structure, raising the possibility that alterations of structure and functional properties of human hemoglobin following exposure to alkylating agents may be mediated through such covalent protein modifications. The results obtained demonstrate the usefulness of the analytical approach for the characterization of hemoglobin adducts with methyl bromide or similar compounds, which can constitute the basis for biomonitoring of human exposure.     

Macrofollicular variant of papillary thyroid carcinoma

The first identification of haemoglobin Le Lamentin (alpha 20 (B1) His-->Gln) in a British patient is described. The patient was a 69 year old asymptomatic white male being screened for diabetes, whose blood was undergoing its first ever analysis for glycated haemoglobin using a Glycomat II analyser. Identification of the haemoglobin variant was by electrospray mass spectrometry.     

Professors Antoni Dziatdowiak and Andrzej Nowak: honorary members of the Calisien Medical Society

There have been over 300 cases of methyl bromide poisoning reported in the literature. The first objective of this case report was to bring out an experience with the false belief that work in a closed space is safe when accompanied by the use of a cartridge respirator with activated charcoal. The second objective of this article was to demonstrate the marked toxicity of methyl bromide with the potential to cause long-term neurological damage. Two experienced fumigation workers (equipped with rapidly saturable respiratory cartridges) entered a building where the concentration of methyl bromide was 17g x m-3 instead of the advised 20mg x m-3. They felt rapidly unwell and complained of nausea and shortness of breath, followed for one them by generalized convulsions. Five months later this last man was still bedridden. The other worker had almost no after-effects. The highest bromide level was found in the blood and also in the activated charcoal cartridge of the most injured worker. There was a relationship between methyl bromide level exposure and neurological damage importance.     

火花源原子发射光谱法鉴别汽车板表面线状缺陷

根据汽车面板线状缺陷区域与无缺陷区域的碳、硅、镁、铝、钙、氧等元素的单火花强度在时序上存在差异特性,提出了汽车板表面线状缺陷火花源原子发射光谱定性与定量识别方法。在对锌埚样进行单火花分析时发现锌液中含有硅、铝、钙、钛和镁等元素的夹杂物,为消除干扰提出了用稀盐酸除去锌层的方法。采用定性法与定量法鉴别时分别对缺陷区域和无缺陷区域进行单火花分析,并将单火花数据按规则进行计算,进而鉴别缺陷属于划伤还是夹杂物,鉴别结论与能谱法吻合。方法灵敏度高,简便快速,分析1个试样时间少于10 min。     

Transforming growth factor-beta enhances the ultraviolet-mediated stress response in p53-/- keratinocytes

This review focuses on the contributions of modern mass spectrometry to neuropeptide research. An introduction to newer mass spectrometric techniques is provided. Also, the use of mass spectrometry in combination with high-resolution separation techniques for neuropeptide identification in biological samples is illustrated. The amino acid sequence information that is important for the identification and analysis of known, novel, or chemically modified neuropeptides may be obtained using mass spectrometric techniques. Because mass spectrometry techniques can be used to reflect the dynamic properties associated with neuropeptide processing in biological systems, they may be used in the future to monitor peptide profiles within organisms in response to environmental challenges such as disease and stress.     

Odorant identification and quality perception following methyl bromide-induced lesions of the olfactory epithelium.

Using a 5-odorant identification confusion matrix task, the authors assessed the consequence of olfactory epithelial damage on odorant quality perception in the rat. After establishing prelesion identification performance, each rat's epithelium was subjected to 330 ppm methyl bromide gas for 6 hr. Comparison of prelesion and 3-day postlesion performance demonstrated a significant decrease in identification as a consequence of 95%-98% epithelial destruction. Further, there was a differential effect of lesion on the ability of different animals to identify the different individual odorants. Evaluation of the anatomical state of the epithelium relative to performance on the identification task demonstrated a significant relationship between the extent and location of anatomical sparing and changes in individual odorant identifications. Assessment of pre- and postlesion quality perception for the individual rats demonstrated a highly significant shift in quality perception that was independent of any decrease in performance. These results provide strong support for the proposition that the regional variations in mucosal sensitivities within and across olfactory receptor gene expression zones are fundamentally important for the encoding of odorant quality. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Determination of hepatotoxic pyrrolizidine alkaloids by on-line high performance liquid chromatography mass spectrometry with an electrospray interface

The present study describes the determination of two different types of hepatotoxic pyrrolizidine alkaloids (PAs) and also distinguishing the hepatotoxic PAs from non-toxic ones by both in-source collision-induced dissociation high performance liquid chromatography mass spectrometry (CID-HPLC/MS) and HPLC/MS/MS (CID in the collision cell), using electrospray ionization. The mass spectra provided molecular ions and characteristic fragment ions, which could be used readily for a rapid identification of different types of PAs. Applications of both in-source CID-HPLC/MS and HPLC/MS/MS analytical methods were successful for the determination of PAs in blood samples obtained from rats dosed with PAs and in the PA-containing plant. The results demonstrated that the developed HPLC/MS methods with two different CID techniques provided a very simple and rapid analysis for an unequivocal diagnosis of PA poisoning and for definitive identification of PAs in plants or herbal medicines.     

Identification of the active site serine of penicillin-binding protein 2a from methicillin-resistant Staphylococcus aureus by electrospray mass spectrometry

Penicillin-binding protein 2a (PBP2a), a high molecular mass PBP, is the primary enzyme responsible for the beta-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA). Inhibition of a PBP such as PBP2a by beta-lactams is due to covalent modification of an active site serine residue. Based on the sequence alignment with well studied beta-lactamases, DD-carboxypeptidases and other high molecular mass PBPs, the serine of a tetrad S403XXK in PBP2a was tentatively identified as the penicillin-binding site. However, direct evidence for the involvement of serine403 has not been reported. In this study, a method which combines liquid chromatography/electrospray mass spectrometry (LC/MS) and nano-electrospray MS for the identification of the active site serine in PBP2a is described. The covalent binding of the beta-lactams was carried out in vitro with the recombinant PBP2a. Peptide mapping of the cyanogen bromide fragments from penicilloyl-PBP2a, using microbore LC/MS, provided a rapid identification of the modified peptide with a 334 Da mass increase. The acylated peptide was isolated and further digested with trypsin. Nano-electrospray MS/MS sequencing of the acylated peptide in the tryptic digest showed that the penicillin was indeed attached to serine403.     

Novel assay for identification of semicarbazide-sensitive amine oxidase by a priority-based strategy in mass spectrometry

A novel assay for the identification of semicarbazide-sensitive amine oxidase in human umbilical artery tissue by a priority-based strategy in the mass spectrometry was developed.The protein extract was separated by SDS-PAGE,and then an entire band at 96 KDa was excised and digested by trypsin.The digested peptides were separated by capillary C18 analytical column and detected by ESI-MS-MS.In the direct data-dependent method(also called traditional method),the semicarbazide-sensitive amine oxidase(SSAO)cannot be identified by LC-ESI-MS-MS.Compared with the traditional method,our assay by a priority-based strategy in the mass spectrometry can successfully identify the target protein-SSAO in the complex biological sample.As 60μg,120μg,240μg of total protein extract were loaded on the SDS-PAGE,the Mascot result showed that SSAO score was 46,86 and 137,the sequence coverage was 2%,5%and 10%,and the peptide count was 2,6 and 10,respectively.The MS/MS spectra of two unique peptides of SSAO were confirmed by manual identification.The band at 96 KDa included SSAO was validated by the Western blot.The assay significantly improved the score and coverage of target protein and enhanced the identification of reliability and the confidence.     

电感耦合等离子体原子发射光谱法测定高纯铁中痕量磷

在0.57～1.43 mol/L硝酸介质中, 用甲基异丁基酮(MIBK)萃取钼酸铵与磷生成的磷钼杂多酸, 使磷与基体铁分离后, 选择波长213.618 nm的谱线作为分析线, 采用电感耦合等离子体原子发射光谱法(ICP-AES)测定了高纯铁中磷的含量。校准曲线的线性回归方程为I= 15.04ρ+0.012 1, 相关系数为0.999 6, 方法的检出限为0.020 mg/L。通过萃取分离和选择合适的谱线作为磷的分析线, 基体和共存元素对测定没有干扰。方法用于多个高纯铁标准物质中磷的测定, 测定值与认定值一致, 测定结果的相对标准偏差(n=10)在0.54%～2.9%之间。方法适用于高纯铁中0.000 10%～0.010%磷的测定。     

Chromatography and mass spectrometry of chemical warfare agents, toxins and related compounds: state of the art and future prospects

Methods for the identification of chemical warfare agents, toxins, bioregulators and related products are frequently reported in literature. These methods are often based on instrumental analysis using chromatography (gas and liquid) and mass spectrometry. Here, these instrumental techniques are discussed in several applications, new developments and trends based on a review of the literature published since 1990. Apart from new instrumental developments, it is shown that modern analytical chemistry can be successfully applied to perform identification in the broad field of analytes ranging from chemical to biological warfare agents.     

Plasma Nitriding of 722M24 Steel with Pure Lanthanum, Cerium and Neodymium as Sputter Sources

Ｓｉｎｃｅｔｈｅｉｎｉｔｉａｌｒｅｐｏｒｔｉｎｔｈｅｅａｒｌｙ 1 980ｓｔｈａｔｒａｒｅｅａｒｔｈａｄｄｉｔｉｏｎｈａｓａｃａｔａｌｙｔｉｃｅｆｆｅｃｔｏｎｔｈｅｇａｓｃａｒｂｏｎｉｔｒｉｄｉｎｇｐｒｏｃｅｓｓｏｆｓｔｅｅｌｓ[1] ,ａｌａｒｇｅａｍｏｕｎｔｏｆｗｏｒｋｈａｓｂｅｅｎｃｏｎｄｕｃｔｅｄｔｏｉｎ ｖｅｓｔｉｇａｔｅｔｈｅｉｎｆｌｕｅｎｃｅｏｆｒａｒｅｅａｒｔｈａｄｄｉｔｉｏｎｓｏｎｖａｒｉｏｕｓｔｈｅｒｍｏｃｈｅｍｉｃａｌｐｒｏｃｅｓｓｅｓ[2～ 8] .Ｉｔｈａｓｂｅｅｎｅｓｔａｂｌｉｓｈｅ…     

熔融制样-X射线荧光光谱法测定稀土铝中间合金中稀土元素

稀土铝中间合金中稀土含量(质量分数,下同)一般约在0.5%~20%之间,文献中鲜见稀土铝中间合金标样和测定稀土含量大于10%的方法。实验通过选择钐的Lβ1线,镧、铈的Lα线,钇的Kα线,采用纯物质法配制标准溶液解决无标样问题,采用特散比法校正基体效应,对熔片条件以及仪器参数进行优化,建立了一套熔融制样-X射线荧光光谱法(XRF)测定稀土铝中间合金中镧、铈、钐、钇的方法。实验表明,称样0.2g,用5mL盐酸(1+1)熔样,四硼酸锂-偏硼酸锂混合熔剂熔融,稀释比选择1∶30,以4mL溴化铵溶液为脱模剂,控制熔样温度为1050℃,熔样时间为15min,熔样效果较好。实验方法应用于镧铝、铈铝、镧铈铝、钐铝、钇铝5类稀土铝中间合金中稀土元素的测定,测定结果与电感耦合等离子体原子发射光谱法(ICP-AES)结果基本一致,相对标准偏差(RSD)均在2%以下。方法可用于测定镧铝、铈铝、镧铈铝、钐铝、钇铝5类稀土铝中间合金中含量范围为0.5%~20%的镧、铈、钐、钇。     

Evidence that loss of chromosome 18q is associated with tumor progression

Toxicological investigation of suspected cocaine-related deaths routinely involves the identification of cocaine (COC) and its metabolites including benzoylecgonine (BE) and ecgonine methyl ester (EME) in postmortem specimens. We utilized solid-phase extraction followed by gas chromatography/mass spectrometry for the qualitative and quantitative analysis of cocaine and eight cocaine-related analytes. These analytes included anhydroecgonine methyl ester (AEME), a unique product formed during cocaine smoking, and cocaethylene (CE), formed by transesterification of cocaine in the presence of ethanol. Thirteen pairs of postmortem heart blood and urine specimens were analyzed from cases of death due to acute cocaine intoxication, multiple drug intoxication, or other non-drug related causes. COC, EME, and BE were detected in all specimens. The range of concentrations in blood were: COC, 23-2088 ng/mL; BE, 215-9195 ng/mL; and EME, 220-7275 ng/ mL. AEME was identified in 2 blood and 10 urine specimens, and CE was identified in 1 blood specimen and 4 urine specimens. The identification of AEME in the specimens indicated that "crack" cocaine had been smoked, and the presence of CE indicated co-administration of cocaine and ethanol. The presence of these unique cocaine analysis in postmortem specimens provides valuable information regarding the cause and manner of death.     

火花放电原子发射光谱法测定奥氏体易切削不锈钢中高含量硫

奥氏体易切削不锈钢中S含量(质量分数,下同)大于0.2％,常规的测量方法有红外吸收法和电感耦合等离子体原子发射光谱法(ICP-AES),而上述分析方法存在制样慢、分析元素种类少等缺点,不能满足炉前炼钢的快速分析要求.实验采用火花放电原子发射光谱法研究了奥氏体易切削不锈钢中高含量S的分析技术.首先通过分析奥氏体易切削不锈...     

Effectiveness of continuous intravenous prostaglandin E 1 against pulmonary hypertension]

Teratogenicity studies of methyl bromide, a widely used fumigant, were conducted in rats and rabbits. Methyl bromide was dissolved in corn oil and administered orally to groups of 24 copulated female Crj:CD (SD) rats at dose levels of 0 (corn oil), 3, 10 or 30 mg/kg/day on days 6-15 of gestation and to groups of 18 artificially inseminated female Kbl:JW rabbits at 0, 1, 3 or 10 mg/kg/day on days 6-18 of gestation. Maternal rats and rabbits were euthanized on respective days 20 and 27 of gestation. Foetuses were examined for survival, growth and teratological alterations. Maternal toxicity was evident in the high-dose groups for both species. In these groups, maternal body weight gains and food consumption were significantly decreased during the dosing and post-dosing periods. Necropsy of maternal rats also revealed erosive lesions in the stomach and the surrounding organs. However, no treatment-related adverse effects were found in foetuses of the treated groups for both rat and rabbit studies. These results led to the conclusion that methyl bromide was not foetotoxic or teratogenic to rat and rabbit foetuses up to dose levels of 30 and 10 mg/kg/day, respectively, at which maternal toxicity was evident for both species.     

Hemoglobin solutions: volume replacement or oxygen therapy?

The development of haemoglobin solutions has progressed significantly in the last 15 years because of a perceived short fall in allogeneic blood within the next decades and increased concern about transmitted infectious diseases. Animal studies have shown that modern highly purified and chemically modified haemoglobin preparations are free of toxic side effects, provide adequate volume replacement and have vasoconstrictive effects that enhance systemic vascular resistance and mean arterial pressures after haemorrhage and in models of nearly complete blood replacement. Microcirculatory effects of haemoglobin-based oxygen carriers are dependent on the respective organ and species in which they are applied and on their degree of purification and chemical modification. Because of different physico-chemical properties in comparison with red cells, haemoglobin solutions provide sufficient tissue oxygenation in areas with critically restricted perfusion even when applied in small doses. First studies in volunteers and patients showed efficacy and tolerability of different newly developed haemoglobin solutions during acute normovolaemic haemodilution and in perioperative blood replacement. However, only little information exists to date in terms of metabolism of haemoglobin preparations and their potential immunogenicity and immunosuppressive side effects. Technical problems with the clinical use of haemoglobin solutions arise because of interference of plasma haemoglobin with routine laboratory tests and oximetry. Future indications for haemoglobin solutions as an oxygen therapeutic allow for application of small doses of such preparations and may help to avoid major technical problems. More clinical studies have to be undertaken to confirm the effectivity and safety of the different haemoglobin solutions and to find out the optimal indications beyond acute preclinical and perioperative blood replacement.     

Rapid identification of comigrating gel-isolated proteins by ion trap-mass spectrometry

In the search for novel nuclear binding proteins, two bands from a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gel were analyzed and each was found to contain a number of proteins that subsequently were identified by tandem mass spectrometry (MS/MS) on a quadrupole ion trap instrument. The bands were digested with trypsin in situ on a polyvinylidene difluoride (PVDF) membrane following electroblot transfer. Analysis of a 2.5% aliquot of each peptide mixture by matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) followed by an initial database search with the peptide masses failed to identify the proteins. The peptides were separated by reversed-phase capillary high performance liquid chromatography (HPLC) in anticipation of subsequent Edman degradation, but mass analysis of the chromatographic fractions by MALDI-MS revealed multiple, coeluting peptides that precluded this approach. Selected fractions were analyzed by capillary HPLC-electrospray ionization-ion trap mass spectrometry. Tandem mass spectrometry provided significant fragmentation from which full or partial sequence was deduced for a number of peptides. Two stages of fragmentation (MS3) were used in one case to determine additional sequence. Database searches, each using a single peptide mass plus partial sequence, identified four proteins from a single electrophoretic band at 45 kDa, and four proteins from a second band at 60 kDa. Many of these proteins were derived from human keratin. The protein identifications were corroborated by the presence of multiple matching peptide masses in the MALDI-MS spectra. In addition, a novel sequence, not found in protein or DNA databases, was determined by interpretation of the MS/MS data. These results demonstrate the power of the quadrupole ion trap for the identification of multiple proteins in a mixture, and for de novo determination of peptide sequence. Reanalysis of the fragmentation data with a modified database searching algorithm showed that the same sets of proteins were identified from a limited number of fragment ion masses, in the absence of mass spectral interpretation or amino acid sequence. The implications for protein identification solely from fragment ion masses are discussed, including advantages for low signal levels, for a reduction of the necessary interpretation expertise, and for increased speed.