Detection and identification of gibberellins in Sitka spruce (Picea sitchensis) of different ages and coning ability by bioassay, radioimmunoassay and gas chromatography – mass spectrometry

Gibberellins Al (GA₁), GA₃, GA₄, GA₉, and after enzymatic hydrolysis of GA-conjugate-like fractions, GA₉ and GA₁₅, were identified in shoots of Sitka spruce [ Picea sitchensis (Bong.) Carr.] of different ages by combined gas chromatography-mass spectrometry (GC-MS). The purification and separation of the GAs involved the use of reverse phase and normal phase high performance liquid chromatography (HPLC). The Tan-ginbozu dwarf rice bioassay and binding to antibodies raised against GA₁, GA₄ and GA₉ were used for detection of GA-like substances. The qualitative differences between the three ages of plant material were the presence of GA₃ and GA₁ in the 48-year-old material and the absence of detectable amounts of GA₄ in the same material. This indicates a difference in GA metabolism which may reflect the difference in ability to form reproductive buds.     

Quantitation of gibberellins and indoleacetic acid in Begonia leaves: Relationship with environment, regeneration and flowering

Indole-3-acetic acid (IAA) was purified by high performance liquid chromatography (HPLC) and identified by gas chromatography - mass spectrometry (GC-MS) in leaf extracts of Begonia × cheimantha Everett cv. Nova. The content of IAA and of gibberellins A4, A9, A19 and A20 (GAs) previously identified in this material, were quantified by GC-MS in leaves of Begonia plants grown under different temperature and daylength conditions, using deuterated compounds as internal standards. GA1, which was also identified, was present in too low quantities for reliable quantitation. Rapid and significant decreases (within 2–4 days) occurred in the content of both IAA and GAs when the plants were transferred from conditions which are non-inductive for adventitious bud formation and flowering (24°C/long day) to inductive conditions (24°C/short day, 15°C/long day or 15°C/short day). GA4 and GA9 were affected by photoperiod only, whereas IAA, GA19 and GA20 were affected by both photoperiod and temperature. The data suggest that biosynthesis of GA9 and GA4 are blocked in short days at a step located before GA9. Conversion of GA19 to GA20 seemed to be blocked by both short days and low temperature, while an additional block located before GA19 seemed to be imposed in 15°C/short day. The results confirm earlier results and support the hypothesis that photoperiod and temperature effects in Begonia are mediated by endogenous hormones.     

Estimation of N-amino-N,N-dimethylaminoethanol, choline and their acetate esters by gas chromatography mass spectrometry

A method is described for measurement of choline, N-aminodeanol, and their acetyl esters by gas chromatography mass spectrometry. The preparation of N-aminodeanol and its isotopic variants is also described. This method allows a thorough quantitative analysis of the replacement of true with false neurotransmitter in biological preparations.     

Detection and identification of gibberellins in needles of silver fir (Abies alba Mill.) by combined gas chromatography-mass spectrometry

Extracts of mature silver fir (Abies alba Mill.) needles, current-year, and one-year old (A) and seven to nine-year old (B), were purified by reversed and normal phase HPLC. Gibberellin (GA)-like compounds were detected by the Tan-ginbozu dwarf rice micro-drop bioassay and corresponding fractions were analyzed by GC-MS. GA₉ was present in small amounts, while a major component was a cellulase-hydrolysable GA₉ conjugate which was assumed to be GA₉ glucosyl ester. It is proposed that GA₉ glucosyl ester plays a key role in the regulation of endogenous GA levels in silver fir needles.     

Determination of 4-chloroindole-3-acetic acid methyl ester in Lathyrus, Vicia and Pisum by gas chromatography – mass spectrometry

4-Chloroindole-3-acetic acid methyl ester was identified unequivocally in Lathyrus latifolius L., Vicia faba L. and Pisum sativum L. by thin layer chromatography, gas chromatography and mass spectrometry. The gas chromatographic system was able to separate underivatized chloroindole-3-acetic acid methyl ester isomers. The quantitative determination of 4-chloroindole-3-acetic acid methyl ester in immature seeds of these three species was performed by gas chromatography – mass spectrometry using deuterium labelled 4-chloro-indole-3-acetic acid methyl ester as an internal standard. P. sativum contained approximately 25 mg kg^-1, V. faba 1–2 mg kg^-1 and L. latifolius 2 mg kg^-1 dry weight.     

Accurate and efficient method for quantification of urinary histamine by gas chromatography negative ion chemical ionization mass spectrometry

Because of the recognized inaccuracy and unreliability of currently available methods for the quantification of histamine in biological fluids, a method for quantification of urinary histamine by stable isotope dilution assay with negative ion chemical ionization mass spectrometry has been developed. Following the addition of [²H₄]histamine to 1 ml of urine, histamine is extracted into butanol, back-extracted into HCl, derivatized to the pentafluorobenzyl derivative (CH₂C₆F₅)₃-histamine, extracted into methylene chloride, and then quantified with negative ion chemical ionization mass spectrometry by selected ion monitoring of the ratio of ions $\text{m}\text{z}$$\text{430}\text{434}$. Twenty samples can be assayed in 2 days. Precision of the assay is ±2.7% and the accuracy is 97.6%. Lower limits of sensitivity are approximately 100–500 fg injected on-column. This assay provides a very sensitive, accurate, and efficient method for the quantification of histamine in human urine.     

Gas chromatography and mass spectrometry of disaccharides from glycoproteins.

Partial acid hydrolysis and methanolysis released disaccharides and disaccharide methylglycosides from the glycoproteins, ovomucoid and porcine gastric mucin in amounts of 0.5--7 microgram disaccharide per mg of glycoprotein. These disaccharides were fractionated by gas chromatography as the trimethylsilyl (Me3Si) derivatives. The composition of recovered disaccharides has been determined by hydrolysis and rechromatography of the Me3Si monosaccharides. The intersaccharide linkages of the disaccharides have been determined by electron impact mass spectrometry. This simple and rapid method can give structural information on small glycoprotein samples.     

Large-scale identification of proteins in human salivary proteome by liquid chromatography/mass spectrometry and two-dimensional gel electrophoresis-mass spectrometry

Human saliva contains a large number of proteins and peptides (salivary proteome) that help maintain homeostasis in the oral cavity. Global analysis of human salivary proteome is important for understanding oral health and disease pathogenesis. In this study, large-scale identification of salivary proteins was demonstrated by using shotgun proteomics and two-dimensinal gel electrophoresis-mass spectrometry (2-DE-MS). For the shotgun approach, whole saliva proteins were prefractionated according to molecular weight. The smallest fraction, presumably containing salivary peptides, was directly separated by capillary liquid chromatography (LC). However, the large protein fractions were digested into peptides for subsequent LC separation. Separated peptides were analyzed by on-line electrospray tandem mass spectrometry (MS/MS) using a quadrupole-time of flight mass spectrometer, and the obtained spectra were automatically processed to search human protein sequence database for protein identification. Additionally, 2-DE was used to map out the proteins in whole saliva. Protein spots 105 in number were excised and in-gel digested; and the resulting peptide fragments were measured by matrix-assisted laser desorption/ionization-mass spectrometry and sequenced by LC-MS/MS for protein identification. In total, we cataloged 309 proteins from human whole saliva by using these two proteomic approaches.     

Rapid and comprehensive evaluation of microalgal fatty acids via untargeted gas chromatography and time‐of‐flight mass spectrometry

Due to their high triacylglyceride content, microalgae are intensively investigated for bio‐economy and food applications. However, lipid analysis is a laborious task incorporating extraction, transesterification and typically gas chromatographic measurement. Co‐elution induces a significant risk of fatty acid misidentification and thus, additional purification steps like thin layer chromatography are needed. Contrary to database matching approaches, solely targeted analysis is facilitated as compound identification is driven by matching retention times or indices with standard substances. In this context, a rapid workflow for the analysis of algal fatty acids is presented. In‐situ transesterification was used to simplify sample preparation and conditions were optimized towards fast processing. If results are needed at the very day of sampling, direct processing without a preceding drying step is feasible to obtain a rough estimate about the occurrence of the major compounds. Coupling gas chromatography and time‐of‐flight mass spectrometry enables untargeted analysis. Unknown compounds may be identified by structural reconstruction of their respective fragmentation patterns and by database matching to routinely avoid mismatches by co‐elution of disturbing agents. The developed workflow was successfully applied to derive the exact stereochemistry of all fatty acids from Chlorella vulgaris and a systematic shift depending on physiological state of the cells was confirmed.     

Detection and identification of gibberellins in Douglas fir (Pseudotsuga menziesii) shoots

Extracts of Douglas fir ( Pseudotsuga menziesii [Mirb.] Franco) shoots were purified by reversed and normal phase HPLC; gibberellin (GA)-like compounds detected by radioimmunoassay with antibodies against GA₄ and the Tan-ginbozu dwarf rice micro-drop biossay were analyzed by GC-MS. Three major components were identified as GA₄, GA₇, and GA₉ while smaller amounts of GA¹, GA₃ and putative GA⁹-glucosyl ester were also present.     

Quantification of isoflavones and lignans in urine using gas chromatography/mass spectrometry

Phytoestrogens (isoflavones and lignans) are of increasing interest due to their potential to prevent certain types of complex diseases. However, epidemiological evidence is needed on the levels of phytoestrogens and their metabolites in foods and biological fluids in relation to risk of these diseases. We report an assay for phytoestrogens which is sensitive, accurate, and uses low volumes of sample. Suitable for epidemiological studies, the assay consists of a simple sample preparation procedure and has been developed for the analysis of five isoflavones (daidzein, O-desmethylangolensin, equol, genistein, and glycitein) and two lignans (enterodiol and enterolactone), which requires only 200 microl of urine and utilizes one solid-phase extraction stage for sample preparation prior to derivatization for GC/MS analysis. Limits of detection were in the region 1.2 ng/ml (enterodiol) to 5.3ng/ml (enterolactone) and the method performed well in the UK Government's Food Standards Agency-sponsored quality assurance scheme for phytoestrogens. For the first time, average levels of all the above phytoestrogens were measured in samples of urine collected from a free living population sample of women. Results show a large range in both the amount and the type of phytoestrogens excreted.     

Detection, quantification and identification of fungal extracellular laccases using polyclonal antibody and mass spectrometry

This study presents a combined method to analyze extracellular fungal laccases using a new anti-laccase antibody together with the identification of tryptic laccase peptides by mass spectrometry (nanoLC–ESI–MS/MS). The polyclonal anti-laccase antibody LccCbr2 was raised against peptides designed from the copper binding region II of fungal laccases using in silico data obtained from GenBank database. As a consequence, detection requires denaturation of the enzymes due to the stable conformation of the copper binding region II. The specificity of the antibody was shown with denatured laccase Lcc1 of Coprinopsis cinerea and laccase of Hypholoma fasciculare. LccCbr2 detected amounts as low as 5 ng of highly purified laccase, indicating a possible use of the antibody for quantification of laccase proteins. Denatured extracellular laccases from culture supernatants of the basidiomycetes C. cinerea, H. fasciculare, Lentinula edodes, Mycena sp., Piriformospora indica, Pleurotus cornucopiae, Pleurotus ostreatus, Pycnoporus cinnabarinus, Trametes versicolor and furthermore the ascomycete Verpa conica were detected with apparent molecular masses between 60 and 70 kDa by LccCbr2. The identity of extracellular laccases from C. cinerea, H. fasciculare, P. ostreatus, P. cinnabarinus and T. versicolor were verified by tryptic peptides using nanoLC–ESI–MS/MS.     

Detection and characterization of N-alpha-chloramines by electrospray tandem mass spectrometry

Hypochlorous acid (HOCl) is a major product of activated neutrophils and may be important in antimicrobial activities of cells by oxidation or chlorination of susceptible amino acids. Three major peaks separated using C18 reverse phase-high-performance liquid chromatography RP-HPLC after incubation of leucine enkephalin (LeuEnk) with HOCl. Electrospray mass spectrometry showed masses of m/z 556.2, 590.2, and 624.4 corresponding to unmodified LeuEnk and peptides altered by addition of one or two chlorines (Cl). Formation of stable N-alpha-chloramines was indicated because the chlorinated peptides were readily reduced with the physiological reductants glutathione and ascorbic acid to LeuEnk (m/z 556.2) within 10 min. Sequence-specific ions observed in product ion spectra of single-charged monochlorinated and dichlorinated peptides were consistent with modification of the N-terminal amine. There was no evidence for chlorination of the Tyr aromatic ring in any spectra. Similar RP-HPLC profiles were obtained after oxidation of des-Tyr1-LeuEnk (GGFL) with the masses of the major products being m/z 393.3, 427.2, and 461.1. These were identified as unmodified GGFL, N-alpha-Cl-GGFL, and N-alpha-Cl2-GGFL based on comparison of tandem mass spectra. Oxidation of Met and formation of disulfide dimers was observed after incubation of either N-alpha-Cl-LeuEnk or N-alpha-Cl2-LeuEnk with a protein, indicating that both peptide N-alpha-chloramines were able to readily modify sulfur-containing amino acids within proteins. These data indicate initial formation of stable N-alpha-chorinated peptides after incubation with HOCl and suggest that N-alpha-chlorinated peptides may exist for some hours in the absence of physiological reducing agents or sulfur-containing amino acids.     

The determination of 2,3,7,8-tetrachlorodibenzo-p-dioxin in biological extracts by gas chromatography mass spectrometry

Using both gas chromatography low resolution mass spectrometry and gas chromatography high resolution mass spectrometry, 2,3,7,8-tetrachlorodibenzo-p-dioxin at the part per trillion level may be determined in pre-concentrated extracts of bovine fat, liver and milk; human milk; rats; rice; grass; soil and water. Criteria are set forth for the objective determination of detection limit, signal and noise as applied to these determinations.     

Biologically active gibberellins in immature seeds of Pyrus serotin

Biologically active gibberellins were isolated from immature seeds of Japanese pear (Pyrus serotina). The major one was tentatively identified as GA₄ by selected ion monitoring. Evidence for the possible presence of GA₇ in the seeds was inconclusive but GA₃ was not detected by either the rice seedling or barley half-seed bioassay.     

气相色谱-质谱定量检测水产品腐败菌群体感应DKPs信号分子

【目的】为了分析水产品腐败菌群体感应的新型信号分子二酮哌嗪(DKPs)化合物,建立一种简便、灵敏的气相色谱-质谱(GC-MS)定量检测方法。【方法】通过优化气相色谱和质谱条件、培养基和提取溶剂建立定量检测方法,确定Cyclo-(L-Pro-L-Gly)、Cyclo-(L-Pro-L-Leu)、Cyclo-(L-Leu-L-Leu)和Cyclo-(L-Pro-L-Phe)4种DKPs标准品的特征离子,并检测水产品腐败菌荧光假单胞菌(Pseudomonas fluorescens)和波罗的海希瓦氏菌(Shewanella baltica)中的DKPs。【结果】4种DKPs化合物在1-200 mg/L范围内线性良好,检测限为0.06、0.10、0.06和0.04 mg/L,定量限为0.16、0.18、0.14和0.12 mg/L,回收率为51.8%-88.5%,标准偏差为1.4%-8.3%。以LB培养基为细菌培养基,氯仿作为萃取溶剂,检测的DKPs含量较高。两种水产品腐败菌都检测到DKPs活性,主要种类为Cyclo-(L-Pro-L-Leu)和Cyclo-(L-Pro-L-Phe)。随着两种细菌的生长,培养上清中DKPs含量显著增加,在12 h达到最高。【结论】建立了检测细菌DKPs的GC-MS定量方法,具有较高精密度、准确度,能够准确定量分析4种DKPs的含量。为探究水产品特定腐败菌DKPs的调控机制奠定基础。     

Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry

During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.     

Rapid identification of an antibody DNA construct rearrangement sequence variant by mass spectrometry

During cell line development for an IgG1 antibody candidate (mAb1), a C-terminal extension was identified in 2 product candidate clones expressed in CHO-K1 cell line. The extension was initially observed as the presence of anomalous new peaks in these clones after analysis by cation exchange chromatography (CEX-HPLC) and reduced capillary electrophoresis (rCE-SDS). Reduced mass analysis of these CHO-K1 clones revealed that a larger than expected mass was present on a sub-population of the heavy chain species, which could not be explained by any known chemical or post-translational modifications. It was suspected that this additional mass on the heavy chain was due to the presence of an additional amino acid sequence. To identify the suspected additional sequence, de novo sequencing in combination with proteomic searching was performed against translated DNA vectors for the heavy chain and light chain. Peptides unique to the clones containing the extension were identified matching short sequences (corresponding to 9 and 35 amino acids, respectively) from 2 non-coding sections of the light chain vector construct. After investigation, this extension was observed to be due to the re-arrangement of the DNA construct, with the addition of amino acids derived from the light chain vector non-translated sequence to the C-terminus of the heavy chain. This observation showed the power of proteomic mass spectrometric techniques to identify an unexpected antibody sequence variant using de novo sequencing combined with database searching, and allowed for rapid identification of the root cause for new peaks in the cation exchange and rCE-SDS assays.     

Protein identification in cerebrospinal fluid using packed capillary liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry

The identification and characterization of proteins in complex biological samples such as body fluids, require powerful and reliable tools. Mass spectrometry is today one of the most important methods in such research. This paper reports on the results from the first experiment where a tryptic digest of cerebrospinal fluid was analyzed applying reversed phase liquid chromatography coupled on-line to a 9.4 T Fourier transform ion cyclotron resonance mass spectrometer. In total, 70 204 peaks were detected, which originated from 16 296 isotopic clusters corresponding to 6551 unique peptide masses. From these masses, 39 proteins were identified in the sample. The amount of sample required for one experiment corresponds to 32 microL of cerebrospinal fluid.