Analysis of the allergenic proteins in black tiger prawn (Penaeus monodon) and characterization of the major allergen tropomyosin using mass spectrometry

Crustaceans are the third most prevalent cause of food‐induced anaphylaxis after peanuts and tree nuts. The severity of the allergenic proteins depends mainly on the amino acid sequence that induces production of IgE antibodies. In black tiger prawn (Penaeus monodon), the crude protein extract was profiled and its allergenic potency was examined against patient's sera. Proteins having strong immunoreactivity with patient's IgE were characterized using peptide mass fingerprinting (PMF). Tropomyosin (TM) (33 kDa), myosin light chain (20 kDa), and arginine kinase (40 kDa) were identified as allergenic proteins. Tropomyosin, the most abundant and potent allergen, was purified using ion‐exchange chromatography for de novo sequencing experiments. Using bottom up tandem mass spectrometry, the full amino acid sequence was achieved by a combination of matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) tandem mass spectrometry (QqToF). Myosin light chain and arginine kinase were also characterized, and their related peptides were de novo sequenced using the same approach. The immunological reactivity of the crude prawn extracts and purified TM samples were analyzed using a large number of patients' sera. A signature peptide was assigned for the TM protein for future quantification work of black tiger prawn TM levels in different matrices (i.e. water, air, food) in the seafood industry. Copyright © 2010 John Wiley & Sons, Ltd.     

Absolute quantification method and validation of airborne snow crab allergen tropomyosin using tandem mass spectrometry

In this work, the determination of rare earth elements (REEs), i.e. Y, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb and Lu in marine biological tissues by inductively coupled-mass spectrometry (ICP-MS) after a sample preparation method based on ultrasound-assisted extraction (UAE) is described. The suitability of the extracts for ICP-MS measurements was evaluated. For that, studies were focused on the following issues: (i) use of clean up of extracts with a C18 cartridge for non-polar solid phase extraction; (ii) use of different internal standards; (iii) signal drift caused by changes in the nebulization efficiency and salt deposition on the cones during the analysis. The signal drift produced by direct introduction of biological extracts in the instrument was evaluated using a calibration verification standard for bracketing (standard-sample bracketing, SSB) and cumulative sum (CUSUM) control charts. Parameters influencing extraction such as extractant composition, mass-to-volume ratio, particle size, sonication time and sonication amplitude were optimized. Diluted single acids (HNO(3) and HCl) and mixtures (HNO(3)+HCl) were evaluated for improving the extraction efficiency. Quantitative recoveries for REEs were achieved using 5 mL of 3% (v/v) HNO(3)+2% (v/v) HCl, particle size <200 μm, 3 min of sonication time and 50% of sonication amplitude. Precision, expressed as relative standard deviation from three independent extractions, ranged from 0.1 to 8%. In general, LODs were improved by a factor of 5 in comparison with those obtained after microwave-assisted digestion (MAD). The accuracy of the method was evaluated using the CRM BCR-668 (mussel tissue). Different seafood samples of common consumption were analyzed by ICP-MS after UAE and MAD.     

Top-down characterization of proteins and drug-protein complexes using nanoelectrospray tandem mass spectrometry

We report a 'top-down' approach for characterization of proteins, and identification of binding sites in protein-drug complexes using nanoelectrospray ionization hybrid quadrupole time-of-flight tandem mass spectrometry (nanoESI-MS/MS). The efficiency of direct fragmentation of intact protein ions and the feasibility of this method were initially demonstrated using several well-characterized proteins with different molecular weights including metallothionein (6126 Da), cytochrome c (horse, 12360 Da), myoglobin (horse, 16592 Da), and hemoglobin (human, 64453 Da). Simply varying collision energy without enzyme digestion and gel or LC separation generated a range of peptide fragments of these proteins. Over 80% of these peptide ions matched those in the SWISS-PROT database with mass accuracy of 8 to 32 ppm with external calibration. This technique was further applied to fragment a cisplatin-metallothionein complex to identify the binding sites, demonstrating a potential application in the study of drug-protein binding.     

基于高分辨质谱技术的婴幼儿食品中过敏原蛋白质的高灵敏检测

基于高效液相色谱-串联质谱(LC-MS/MS)技术,选择稳定性好、灵敏度高的特征肽段,利用平行反应监测(PRM)技术,实现了多类过敏原蛋白质的高灵敏度同时检测,并成功应用于婴幼儿食品中过敏原成分的分析。对于婴幼儿食品中蛋白质的提取,与传统的丙酮沉淀法比,采用膜上原位样品预处理方法(i-FASP)可实现更高的蛋白质提取效率和抗干扰能力。所检测的过敏原蛋白质的定量限(LOQ)最小可达到0.028 mg/L,其线性范围最宽可跨越4个数量级,且线性关系良好(相关系数R~2≥0.99)。该方法为食品中过敏原蛋白质组学快速分析提供了一种可靠的分析方法。     

Poly(amic acid) and polyimide characterization using gas chromatography/mass spectrometry and particle beam liquid chromatography/mass spectrometry

A number of polymers were hydrolyzed in NH₄OH and studied using gas chromatography/ mass spectrometry (GC/MS) and particle beam liquid chromatography/mass spectrometry (particle beam LC/MS) techniques. The polymers studied in this report were as follows: BPDA-PDA, BPDA-PDA-ODA, BPDA-PDA-GFDA, PMDA-ODA, and BTDA-APB. Some of the polymer samples were hydrolyzed in both their acid and imide forms to see if any mass spectrometric differences could be detected. ln all cases, the acid and imide spectra looked the same. GC/MS was unable to determine either the amine or acid portion of these polymers via a direct injection of the sample, but when the samples were first extracted with diethyl ether and this ether extract was injected into the chromatograph, the amine portion of the polymers was readily detected. The acid portion was, again, not detected in either the sample or the ether extract. The particle beam was able to detect both the amine and acid monomeric units in the nonextracted sample.     

Analysis of metal-binding proteins separated by non-denaturating gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins - protein FAM44B and cathepsin B precursor - were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins.     

Determination of allergenic hydroperoxides in essential oils using gas chromatography with electron ionization mass spectrometry

Fragrance monoterpenes are widely used commercially due to their pleasant scent. In previous studies, we have shown that air‐exposed monoterpenes form hydroperoxides that are strong skin sensitizers. Methods for detection and quantification of the hydroperoxides in essential oils and scented products are thus desirable. Due to thermolability and low UV absorbance, this is a complicated task. We have recently developed a sensitive LC–ESI‐MS method, but with limited structural information and separation efficiency for positional isomers and stereoisomers. In the present study, we investigated derivatization with a trimethyl silyl reagent and subsequent GC with electron ionization MS for the determination of monoterpene hydroperoxides. All investigated monoterpene hydroperoxides could be chromatographed as thermostable trimethyl silyl derivatives and yielded the fragment m/z 89 ([OSi(CH₃)₃]⁺) at a higher extent compared to corresponding alcohols. Limonene‐2‐hydroperoxide and four other hydroperoxide isomers of limonene were separated and detected in sweet orange oil autoxidized for two months. The concentration of limonene‐2‐hydroperoxide isomers was found to be 19 μg/mg in total. Also isomers of linalyl acetate hydroperoxide and linalool hydroperoxide were detected in autoxidized petitgrain oil (two months). The presented GC–MS method showed concentrations in the same order as previous LC–MS/MS analysis of the same type of oils.     

Top down characterization of larger proteins (45 kDa) by electron capture dissociation mass spectrometry.

The structural characterization of proteins expressed from the genome is a major problem in proteomics. The solution to this problem requires the separation of the protein of interest from a complex mixture, the identification of its DNA-predicted sequence, and the characterization of sequencing errors and posttranslational modifications. For this, the "top down" mass spectrometry (MS) approach, extended by the greatly increased protein fragmentation from electron capture dissociation (ECD), has been applied to characterize proteins involved in the biosynthesis of thiamin, Coenzyme A, and the hydroxylation of proline residues in proteins. With Fourier transform (FT) MS, electrospray ionization (ESI) of a complex mixture from an E. coli cell extract gave 102 accurate molecular weight values (2-30 kDa), but none corresponding to the predicted masses of the four desired enzymes for thiamin biosynthesis (GoxB, ThiS, ThiG, and ThiF). MS/MS of one ion species (representing approximately 1% of the mixture) identified it with the DNA-predicted sequence of ThiS, although the predicted and measured molecular weights were different. Further purification yielded a 2-component mixture whose ECD spectrum characterized both proteins simultaneously as ThiS and ThiG, showing an additional N-terminal Met on the 8 kDa ThiS and removal of an N-terminal Met and Ser from the 27 kDa ThiG. For a second system, the molecular weight of the 45 kDa phosphopantothenoylcysteine synthetase/decarboxylase (CoaBC), an enzyme involved in Coenzyme A biosynthesis, was 131 Da lower than that of the DNA prediction; the ECD spectrum showed that this is due to the removal of the N-terminal Met. For a third system, viral prolyl 4-hydroxylase (26 kDa), ECD showed that multiple molecular ions (+98, +178, etc.) are due to phosphate noncovalent adducts, and MS/MS pinpointed the overall mass discrepancy of 135 Da to removal of the initiation Met (131 Da) and to formation of disulfide bonds (2 x 2 Da) at C32-C49 and C143-C147, although 10 S-S positions were possible. In contrast, "bottom up" proteolysis characterization of the CoaBC and the P4H proteins was relatively unsuccessful. The addition of ECD substantially increases the capabilities of top down FTMS for the detailed structural characterization of large proteins.     

Structural characterization of poly(N-alkyl-4-vinylpyridinium) triflates using pyrolysis/tandem mass spectrometry

Desorption chemical ionization (DCI) and desorption electron ionization (DEI) of homo- and co-polymers of N-alkyl-4-vinylpyridinium triflates having ethyl, n-hexyl and n-dodecyl groups as N-alkyl substituents, produce mass spectra that display oligomeric ions. These positively charged ions are singly-charged and result from cleavage of the polymer into neutral oligomers and the loss of a single triflate anion per oligomer. Analogous negatively charged ions, in which each neutral oligomer carries an extra triflate anion, are observed in the desorption chemical ionization mass spectra. Each oligomer within the available mass range is represented in the mass spectra. The formation of cluster ions in which a single, multiply-charged cation is associated with a number of singly-charged anions, as observed for these ammonium polysalts, is unusual. Five major and three minor series of positively charged ions are observed in DCI and DEI methods of ionization. Ions in the different series correspond either to cleavage at different bonds between the constituent monomers or to hydrogen transfer in different directions. Unique and structurally diagnostic fragmentation processes are observed in tandem mass spectrometry (MS/MS) experiments performed using collision activated dissociation of mass-selected oligomeric ions.     

Operational variables in high-performance liquid chromatography-electrospray ionization mass spectrometry of peptides and proteins using poly(styrene-divinylbenzene) monoliths

Capillary reversed-phase high-performance liquid chromatography (RP-HPLC) utilizing monolithic poly(styrene-divinylbenzene) columns was optimized for the coupling to electrospray ionization mass spectrometry (ESI-MS) by the application of various temperatures and mobile phase additives during peptide and protein analysis. Peak widths at half height improved significantly upon increasing the temperature and ranged from 2.0 to 5.4 s for peptide and protein separations at 70 degrees. Selectivity of peptide elution was significantly modulated by temperature, whereas the effect on proteins was only minor. A comparison of 0.10% formic acid (FA), 0.050% trifluoroacetic acid (TFA), and 0.050% heptafluorobutyric acid (HFBA) as mobile phase additives revealed that highest chromatographic efficiency but poorest mass spectrometric detectabilities were achieved with HFBA. Clusters of HFBA, water, and acetonitrile were observed in the mass spectra at m/z values >500. Although the signal-to-noise ratios for the individual peptides diverged considerably both in the selected ion chromatograms and extracted mass spectra, the average mass spectrometric detectabilities varied only by a factor of less than 1.7 measured with the different additives. Limits of detection for peptides with 500 nl sample volumes injected onto a 60 mm x 0.20 mm monolithic column were in the 0.2-13 fmol range. In the analysis of hydrophobic membrane proteins, HFBA enabled highest separation selectivity at the cost of lower mass spectral quality. The use of 0.050% TFA as mobile phase additive turned out to be the best compromise between chromatographic and mass spectrometric performance in the analysis of peptides and proteins by RP-HPLC-ESI-MS using monolithic separation columns.     

Fast characterization of intact proteins using a high-throughput eight-channel parallel liquid chromatography/mass spectrometry system

The preparation of protein substrates requires that a large number of chromatographic fractions be analyzed for the presence of reactants, products and by-products. Analyses using linear matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) or single column liquid chromatography/mass spectrometry (LC/MS) have been inadequate because of mass resolution or throughput. Therefore, a high-throughput method employing an eight-channel parallel reverse-phase LC/MS system was developed. This system is capable of screening fractions from preparative ion-exchange chromatography with the required mass accuracy and throughput so that the protein purification process can be monitored in a relatively short period of time. As an example, the purification and analysis of an acylated protein with a molecular weight of 8.9 kDa is described and the detection of a contaminating by-product that differs in size by less than 20 Da is demonstrated. Using the current instrumentation and approach, it is practical to analyze 50 protein-containing fractions from column chromatography in less than 1 hour using parallel LC/MS.     

Fast characterization of foodstuff by headspace mass spectrometry (HS-MS)

The field of the rapid characterization of products by HS-MS is reviewed. The general principle of HS-MS systems consists of introducing volatile components present in the HS of a sample without prior chromatographic separation into the ionization chamber of a mass spectrometer. The spectrum resulting from simultaneous ionization and fragmentation of the mixture of molecules introduced constitutes a “fingerprint” that is characteristic of the product being analyzed. Exploitation of this spectral information allows one determine the composition of the sample.     

多维离子交换色谱分离和串联质谱鉴定在小鼠肝脏膜蛋白质组分析中的应用

膜蛋白质在变性剂作用下能够较充分地溶解。根据这一特点,我们试图在变性剂溶液中采用串联离子交换色谱法分离小鼠肝脏膜蛋白质。将小鼠肝脏膜蛋白质溶解于含有4 mol/L尿素,20 mmol/L三羟甲基氨基甲烷(Tris)-盐酸缓冲液(pH 9.0)中,用Q-Sepharose FF和Sephacryl S-200HR树脂组成的色谱柱结合大部分溶解的膜蛋白质,然后采用氯化钠线性梯度洗脱蛋白质,分步收集后采用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)进一步分离洗脱组分的蛋白质。利用胶内胰蛋白酶消化技术将SDS-PAGE胶内分离的蛋白质降解为相应的肽段,然后以反相高效液相色谱分离和离子阱质谱仪鉴定肽段。根据文献报道和蛋白质的功能分类,在所鉴定的392个蛋白质中有306个可能为膜蛋白质或膜结合蛋白质。蛋白质的疏水性计算表明,GRAVY(grand average of hydropathicity)得分大于或等于0.00的蛋白质有83个。综上所述,我们有理由认为本实验方法基本符合小鼠肝脏膜蛋白质组学研究的要求。     

Analysis of food proteins and peptides by chromatography and mass spectrometry

The research topics and the analytical strategies dealing with food proteins and peptides are summarized. Methods for the separation and purification of macromolecules of food concern by both high-performance liquid chromatography (HPLC) on conventional packings and perfusion HPLC are examined. Special attention is paid to novel methodologies such those based on multi-dimensional systems that comprise liquid-phase based protein separation, protein digestion and mass spectrometry (MS) analysis of food peptide and proteins. Recent applications of chromatography and MS-based techniques for the analysis of proteins and peptides in food are discussed.     

Electrospray ionization mass spectrometry in the structural characterization of some diphenyllead(IV) thiosemicarbazonates

The behavior in electrospray ionization mass spectrometry (ESI-MS) conditions of some complexes formed by Pb(C6H5)2(OAc)2 with salicylaldehyde, 2-ketobutyric acid, pyridine-2-carbaldehyde, 2-acetylpyridine, and 2-benzoylpyridine thiosemicarbazones, was studied in detail with the aid of collisional experiments performed by ion trap. The homoleptic complexes of tridentate thiosemicarbazonate dianions (TSC2-) lose the phenyl groups first, destabilizing the high oxidation state of the metal and leading to Pb(II) complexes in which the TSC2- ligand or a part of it remains coordinated to the metal. The main difference among the complexes derives from the presence of a carboxylate group on the 2-ketobutyric acid thiosemicarbazonato ligand, which probably interacts with a Na+ cation leading to ESI-generated [M+Na]+ species. In the absence of the carboxylate group, the production of abundant protonated molecules is observed. These different behaviors have been rationalized from the structural point of view. The heteroleptic mononuclear complexes, including thiosemicarbazonate and AcO- monoanions in the coordination sphere, do not yield intact ionized molecular species, and the most abundant ESI-generated ion is due to AcO- loss. The spectrum of the binuclear heteroleptic complex formed by the salicylaldehyde thiosemicarbazonate monoanion is conditioned by the weak bonding interaction displayed by the acetate bridge. MSn experiments yield important information on the relative ligand-Pb bond strengths. This work forms part of the search for chelating agents that can be used for effective chemotherapy of organolead poisoning.