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1.
In this paper, we describe a novel technique—ultrasonication-assisted spray ionization (UASI)—for the generation of singly charged and multiply charged gas-phase ions of biomolecules (e.g., amino acids, peptides, and proteins) from solution; this method employs a low-frequency ultrasonicator (ca. 40 kHz) in place of the high electric field required for electrospray ionization. When a capillary inlet is immersed into a sample solution within a vial subjected to ultrasonication, the solution is continually directed to the capillary outlet as a result of ultrasonication-assisted capillary action; an ultrasonic spray of the sample solution is emitted at the outlet of the tapered capillary, leading to the ready generation of gas-phase ions. Using an ion trap mass spectrometer, we found that singly charged amino acid and multiply charged peptides/proteins ions were generated through this single-step operation, which is both straightforward and extremely simple to perform. The setup is uncomplicated: only a low-frequency ultrasonicator and a tapered capillary are required to perform UASI. The mass spectra of the multiply charged peptides and proteins obtained from sample solutions subjected to UASI resemble those observed in ESI mass spectra.  相似文献   

2.
Summary Chlorination step (C-step), spent bleach liquor from a kraft pulp mill and the product from the chlorination of guaiacol in aqueous solution have been investigated for their content of chlorinated catechols. After separation the samples were derivatized with diazoethane and analysed by glass capillary gas chromatography with an electron capture detector. The external standard mixture of all ethylated chlorocatechols was used in the measurements. The structures of the compounds identified were confirmed by gas chromatography/mass spectrometry. Eight chlorinated catechols were identified from a sample prepared by chlorination of guaiacol with Cl2 in aqueous solution. However, only four of these compounds could be detected from a bleach liquor sample.  相似文献   

3.
We have constructed an electrospray-assisted laser desorption/ionization (ELDI) source which utilizes a nitrogen laser pulse to desorb intact molecules from matrix-containing sample solution droplets, followed by electrospray ionization (ESI) post-ionization. The ELDI source is coupled to a quadrupole ion trap mass spectrometer and allows sampling under ambient conditions. Preliminary data showed that ELDI produces ESI-like multiply charged peptides and proteins up to 29 kDa carbonic anhydrase and 66 kDa bovine albumin from single-protein solutions, as well as from complex digest mixtures. The generated multiply charged polypeptides enable efficient tandem mass spectrometric (MS/MS)-based peptide sequencing. ELDI-MS/MS of protein digests and small intact proteins was performed both by collisionally activated dissociation (CAD) and by nozzle-skimmer dissociation (NSD). ELDI-MS/MS may be a useful tool for protein sequencing analysis and top-down proteomics study, and may complement matrix-assisted laser desorption/ionization (MALDI)-based measurements.  相似文献   

4.
The C‐terminal domain of lung surfactant protein C (CTC) precursor (proSP‐C) is involved in folding of the transmembrane segment of proSP‐C. CTC includes a Brichos domain with homologs in cancer‐ and dementia‐associated proteins. Mutations in the Brichos domain cause misfolding of proSP‐C and hence amyloid fibril formation in interstitial lung disease. Electrospray ionization mass spectrometry (ESI‐MS) with collision‐induced dissociation (CID) experiments was applied to study non‐covalent interactions between human recombinant CTC or its Brichos domain, and SP‐C analogs, homotripeptides and peptides designed to model amyloid fibril formation. The results show that the Brichos domain contains the peptide‐binding function of CTC. In titration experiments, apparent dissociation constants (KD) were in the micromolar range where triple‐valine showed the lowest KD and triple‐tyrosine the highest. Non‐hydrophobic peptides failed to form complexes with Brichos. CID revealed that complexes with aromatic peptide ligands are more stable in the gas phase than complexes with non‐aromatic ligands. The Brichos domain was also shown to bind fibril‐forming peptides containing aromatic/hydrophobic residues. Copyright © 2009 John Wiley & Sons, Ltd.  相似文献   

5.
Improving the sensitivity of detection and fragmentation of peptides to provide reliable sequencing of peptides is an important goal of mass spectrometric analysis. Peptides derivatized by bicyclic quaternary ammonium ionization tags: 1‐azabicyclo[2.2.2]octane (ABCO) or 1,4‐diazabicyclo[2.2.2]octane (DABCO), are characterized by an increased detection sensitivity in electrospray ionization mass spectrometry (ESI‐MS) and longer retention times on the reverse‐phase (RP) chromatography columns. The improvement of the detection limit was observed even for peptides dissolved in 10 mM NaCl. Collision‐induced dissociation tandem mass spectrometry of quaternary ammonium salts derivatives of peptides showed dominant a‐ and b‐type ions, allowing facile sequencing of peptides. The bicyclic ionization tags are stable in collision‐induced dissociation experiments, and the resulted fragmentation pattern is not significantly influenced by either acidic or basic amino acid residues in the peptide sequence. Obtained results indicate the general usefulness of the bicyclic quaternary ammonium ionization tags for ESI‐MS/MS sequencing of peptides. Copyright © 2014 John Wiley & Sons, Ltd.  相似文献   

6.
β,β-Dideuterated poly(viny1 chloride) (β,β -d2-PVC) was chlorinated under conditions of photochemical suspension chlorination in concentrated hydrochloric acid with addition of chloroform as a swelling agent, and also under conditions of thermal solution chlorination in tetrachloroethane. The 1H-NMR spectra of chlorinated β,β -d2PVC (β,β -d2CPVC) were measured. The deuterium concentration was determined in combustion products of β,β -d2CPVC as the D2O:H2 O ratio by means of mass spectroscopy. It was found that the concentration of CHC1- groups decreases in both the photochemical suspension chlorination and in the thermal solution chlorination. The structural nonidentity of β,β -d2PVC chlorinated in suspension and in solution was confirmed.  相似文献   

7.
In this study, a polarization‐induced electrospray ionization mass spectrometry (ESI‐MS) was developed. A micro‐sized sample droplet was deposited on a naturally available dielectric substrate such as a fruit or a stone, and then placed close to (~2 mm) the orifice of a mass spectrometer applied with a high voltage. Taylor cone was observed from the sample droplet, and a spray emitted from the cone apex was generated. The analyte ion signals derived from the droplet were obtained by the mass spectrometer. The ionization process is similar to that in ESI although no direct electric contact was applied on the sample site. The sample droplet polarized by the high electric field provided by the mass spectrometer initiated the ionization process. The dielectric sample loading substrate facilitated further the polarization process, resulting in the formation of Taylor cone. The mass spectral profiles obtained via this approach resembled those obtained using ESI‐MS. Multiply charged ions dominated the mass spectra of peptides and proteins, whereas singly charged ions dominated the mass spectra of small molecules such as amino acids and small organic molecules. In addition to liquid samples, this approach can be used for the analysis of solid and viscous samples. A small droplet containing suitable solvent (5–10 µl) was directly deposited on the surface of the solid (or viscous) sample, placed close the orifice of mass spectrometer applied with a high voltage. Taylor cone derived from the droplet was immediately formed followed by electrospray processes to generate gas‐phase ions for MS analysis. Analyte ions derived from the main ingredients of pharmaceutical tablets and viscous ointment can be extracted into the solvent droplet in situ and observed using a mass spectrometer. Copyright © 2015 John Wiley & Sons, Ltd.  相似文献   

8.
In this study, we developed a novel electrospray ionization (ESI) technique based on household aluminum foil (Al foil) and demonstated the desirable features and applications of this technique. Al foil can be readily cut and folded into desired configuration for effective ionization and for holding sample solution in bulk to allowing acquisition of durable ion signals. The present technique was demonstrated to be applicable in analysis of a wide variety of samples, ranging from pure chemical and biological compounds, e.g., organic compounds and proteins, to complex samples in liquid, semi-solid, and solid states, e.g., beverages, skincare cream, and herbal medicines. The inert, hydrophobic and impermeable surface of Al foil allows convenient and effective on-target extraction of solid samples and on-target sample clean-up, i.e., removal of salts and detergents from proteins and peptides, extending ESI device from usually only for sample loading and ionization to including sample processing. Moreover, Al foil is an excellent heat-conductor and highly heat-tolerant, permitting direct monitoring of thermal reactions, e.g., thermal denaturation of proteins. Overall, the present study showed that Al-foil ESI could be an economical and versatile method that allows a wide range of applications.  相似文献   

9.
At high pH and in the presence of dissolved CO2, the N-terminus and ε-amino groups of amino acids, peptides, and proteins can form carbamino adducts with CO2, R-NH2 + CO2 ↔ R-NHCOO + H+. We report the first study of carbamino group formation by electrospray ionization (ESI) mass spectrometry (MS). Angiotensin II, bradykinin, substance P, and insulin have been studied. A careful optimization of the instrumental parameters was necessary to allow the transfer of the fragile adducts into vacuum for mass analysis. Particularly, dissociation of the adducts in the ion sampling process and pH changes in ESI must be minimized. With these precautions, levels of carbamino group formation of angiotensin II and bradykinin determined from mass spectra agree with those expected to be in solution, calculated from literature equilibrium constants. Thus, ESI MS can quantitatively measure ratios of carbamino adduct to total peptide concentration in solution. Values of equilibrium constants for carbamino group formation with substance P (pKc = 4.77 ± 0.18) and insulin (pKc = 4.99 ± 0.05) are reported for the first time.  相似文献   

10.
Recently, two ionization sources, electrospray (ESI) and matrix-assisted laser desorption (MALDI) have been used in parallel to exploit their complementary nature and to increase proteome coverage. In this study, a method using bidimensional (2D) nanoLC coupled online with ESI quadrupole time-of-flight (Q-TOF) with the simultaneous collection of fractions for analyses by LC–MALDI Q-TOF–MS/MS was developed. A total of 39 bovine proteins were identified to a high degree of confidence. To help in differentiating peptide detection following ESI and MALDI with the same mass spectrometer, we compared physico–chemical characteristics of the peptides (molecular mass, charge and size) by principal component analysis (PCA) and analysis of variance on the results of PCA. More hydrophobic peptides with a wider mass coverage were identified when ESI was used, whereas more basic and smaller peptides were identified when MALDI was used. However, the generally accepted differentiation between ESI and MALDI according to the presence of basic amino acids residues Lys and Arg and the ratio Lys/Arg was not shown as significant in this study. Moreover, we pointed out the importance of the type of mass spectrometer used in complement to both ionization sources for achieving a global increase of proteome coverage.  相似文献   

11.
江玮  喻钢 《分析测试学报》2012,31(4):430-435
自建了简易的电喷雾解吸电离源(DESI),优化了DESI源喷口的位置和角度,并将其用于常见多肽和蛋白质的分析。多肽和小质量蛋白质(<20 kDa)可以容易地从表面解吸电离,生成清晰的质谱。而牛血清白蛋白(66.4 kDa)不能产生清晰的多电荷分布的质谱,说明当前DESI源的设计可能存在一个电离的分子量上限。通过比较不同的实验条件并对比ESI-MS,发现溶剂分子的挥发过程对电荷分布以及峰宽均有显著影响,可能是由于ESI更软引起。载样表面的性质对DESI-MS的信号强度有较大影响。金表面的自组装单分子膜(SAM)相对于纯金表面有较好的绝缘性,并有助于产生较强信号,说明来自表面的电子转移(电中和)是电喷雾解吸电离过程中的一个重要因素。该文的研究有助于对DESI-MS的实验条件和载样表面的选择,同时增进了对电喷雾解吸电离机理的了解。  相似文献   

12.
Mono‐ and poly‐adenosine diphosphate (ADP)‐ribosylation are common post‐translational modifications incorporated by sequence‐specific enzymes at, predominantly, arginine, asparagine, glutamic acid or aspartic acid residues, whereas non‐enzymatic ADP‐ribosylation (glycation) modifies lysine and cysteine residues. These glycated proteins and peptides (Amadori‐compounds) are commonly found in organisms, but have so far not been investigated to any great degree. In this study, we have analyzed their fragmentation characteristics using different mass spectrometry (MS) techniques. In matrix‐assisted laser desorption/ionization (MALDI)‐MS, the ADP‐ribosyl group was cleaved, almost completely, at the pyrophosphate bond by in‐source decay. In contrast, this cleavage was very weak in electrospray ionization (ESI)‐MS. The same fragmentation site also dominated the MALDI‐PSD (post‐source decay) and ESI‐CID (collision‐induced dissociation) mass spectra. The remaining phospho‐ribosyl group (formed by the loss of adenosine monophosphate) was stable, providing a direct and reliable identification of the modification site via the b‐ and y‐ion series. Cleavage of the ADP‐ribose pyrophosphate bond under CID conditions gives access to both neutral loss (347.10 u) and precursor‐ion scans (m/z 348.08), and thereby permits the identification of ADP‐ribosylated peptides in complex mixtures with high sensitivity and specificity. With electron transfer dissociation (ETD), the ADP‐ribosyl group was stable, providing ADP‐ribosylated c‐ and z‐ions, and thus allowing reliable sequence analyses. Copyright © 2010 John Wiley & Sons, Ltd.  相似文献   

13.
A series of growth hormone-releasing factor analogs have been studied by both circular dichroism and electrospray ionization mass spectrometry (ESI/MS). The peptides are 32 residues long and are known to adopt a random-coil structure in aqueous solution but become increasingly helical as the proportion of organic solvent is increased. Deuterium exchange was observed as an increase in mass of the peptide, as measured by ESI/MS. Rates of exchange were measured and half-lives calculated for analogs containing amino acid substitutions designed to promote or discourage helix formation. Exchange was slower in peptides that are helical (as shown by circular dichroism) than in randomly coiled peptides. Solution conditions that favor helix formation also produced slower exchange rates. These studies suggest that ESI/MS can provide date about the extent and stability of helix formation.  相似文献   

14.
The analysis of acylated proteins by mass spectrometry (MS) has largely been overshadowed in proteomics by the analysis of glycosylated and phosphorylated proteins; however, lipid modifications on proteins are proving to be of increasing importance in biomedical research. In order to identify the marker ions and/or neutral loss fragments that are produced upon collision-induced dissociation, providing a means to identify the common lipid modifications on proteins, peptides containing an N-terminally myristoylated glycine, a palmitoylated cysteine and a farnesylated cysteine were chemically synthesized. Matrix-assisted laser desorption/ionization time-of-flight time-of-flight (MALDI-TOF-TOF), electrospray ionization quadrupole time-of-flight (ESI Q-TOF), and electrospray ionization hybrid triple-quadrupole/linear ion trap (ESI QqQ(LIT)) mass spectrometers were used for the analysis. The peptide containing the N-terminally myristoylated glycine, upon CID, produced the characteristic fragments a1 (240.4 Th) and b1 (268.4 Th) ions as well as a low-intensity neutral loss of 210 Da (C14H26O). The peptides containing a farnesylated cysteine residue fragmented to produce a marker ion at a m/z of 205 Th (C15H25) as well as other intense farnesyl fragment ions, and a neutral loss of 204 Da (C15H24). The peptides containing a palmitoylated cysteine moiety generated neutral losses of 238 Da (C16H30O) and 272 Da (C16H32OS); however, no marker ions were produced. The neutral losses were more prominent in the MALDI-TOF-TOF spectra, whereas the marker ions were more abundant in the ESI QqQ(LIT) and Q-TOF mass spectra.  相似文献   

15.
Electrospray laser desorption ionization mass spectrometry (ELDI/MS) allows the rapid desorption and ionization of proteins from solutions under ambient conditions. In this study, we have demonstrated the use of ELDI/MS to efficiently examine the integrity of the proteins stored in various solutions before they were further used for other biochemical tests. The protein standards were prepared in the solutions containing buffers, organic salts, inorganic salts, strong acid, strong base, and organic solvents, respectively, to simulate those collected from solvent extraction, filtration, dialysis, or chromatographic separation. Other than the deposit of a drop of the sample solution on the metallic sample plate in an ELDI source, no additional sample pretreatment is needed. The sample drop was then irradiated with a pulsed laser; this led to desorption of the analyte molecules, which subsequently entered the ESI plume to undergo post-ionization. Because adjustment of the composition of the sample solution is unnecessary, this technique appears to be useful for rapidly evaluating the integrity of proteins after storage or prior to further biochemical treatment. In addition, when using acid-free and low-organic-solvent ESI solutions for ELDI/MS analysis, the native conformations of the proteins in solution could be detected.
Figure
?  相似文献   

16.
Our goal was to compare two popular analytical techniques used nowadays in proteomic investigations for proteins/peptides sequencing and identification, a widely used nanoLC‐MS/MS approach applied in the bottom‐up proteomics and electron transfer dissociation/proton transfer reaction fragmentation preferably used when top‐down strategy is applied. Comparison was carried out with the aid of the ESI‐quadrupole ion‐trap instrument using the following criteria: total time of analysis including sample preparation, sequence coverage, Mascot scoring, capability to detect modifications, quality of the results as a function of protein molecular weight and sample consumption. Copyright © 2012 John Wiley & Sons, Ltd.  相似文献   

17.
Comparative MS/MS studies of singly and doubly charged electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) precursor peptide ions are described. The spectra from these experiments have been evaluated with particular emphasis on the data quality for subsequent data processing and protein/amino acid sequence identification. It is shown that, once peptide ions are formed by ESI or MALDI, their charge state, as well as the collision energy, is the main parameter determining the quality of collision-induced dissociation (CID) MS/MS fragmentation spectra of a given peptide. CID-MS/MS spectra of singly charged peptides obtained on a hybrid quadrupole orthogonal time-of-flight mass spectrometer resemble very closely spectra obtained by matrix-assisted laser desorption/ionization post-source decay time-of-flight mass spectrometry (MALDI-PSD-TOFMS). On the other hand, comparison of CID-MS/MS spectra of either singly or doubly charged ion species shows no dependence on whether ions have been formed by ESI or MALDI. This observation confirms that, at the time of precursor ion selection, further mass analysis is effectively decoupled from the desorption/ionization event. Since MALDI ions are predominantly formed as singly charged species and ESI ions as doubly charged, the associated difference in the spectral quality of MS/MS spectra as described here imposes direct consequences on data processing, database searching using ion fragmentation data, and de novo sequencing when ionization techniques are changed.  相似文献   

18.
Protein identification is routinely accomplished by peptide sequencing using mass spectrometry (MS) after enzymatic digestion. Site-specific chemical modification may improve peptide ionization efficiency or sequence coverage in mass spectrometry. We report herein that amino group of lysine residue in peptides can be selectively modified by reaction with a peroxycarbonate and the resulting lysine peroxycarbamates undergo homolytic fragmentation under conditions of low-energy collision-induced dissociation (CID) in electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) MS. Selective modification of lysine residue in peptides by our strategy can induce specific peptide cleavage at or near the lysine site. Studies using deuterated analogues of modified lysine indicate that fragmentation of the modified peptides involves apparent free-radical processes that lead to peptide chain fragmentation and side-chain loss. The formation of a-, c-, or z-types of ions in MS is reminiscent of the proposed free-radical mechanisms in low-energy electron capture dissociation (ECD) processes that may have better sequence coverage than that of the conventional CID method. This site-specific cleavage of peptides by free radical- promoted processes is feasible and such strategies may aid the protein sequencing analysis and have potential applications in top-down proteomics.  相似文献   

19.
Department of Chemistry, University of New Orleans, New Orleans, Louisiana, USA The effect of solvent composition on negative ion electrospray ionization (ESI) mass spectrometry was examined. The onset potentials for ES1 of a series of chlorinated solvents and methanol were found to be within the range predicted by D. P. H. Smith, based on differences in the surface tension of the solvents used. The tendency toward electric discharge decreased with increasing percent weight of chlorine in the solvent. This effect has been attributed to an increasing propensity for electron capture for more highly chlorinated solvents. Addition of the electron scavenger gas SF, was even more effective at suppressing corona discharge phenomena. In a comparison of ultimate signal intensity obtainable for a test analyte in 10% methanol, the highest signal, which was stable over the widest range of temperatures, was exhibited by chloroform compared to dichloromethane, 1,2-dichloroethane, carbon tetrachloride, and methanol (100%). Chloroform, thus, is a recommended solvent for negative ion electrospray mass spectrometry (ES/MS) when solubility is not a limiting issue. Solvent polarity was shown to exhibit a profound influence on the distribution of charge states in negative ion ES/MS. For both chlorinated and nonchlorinated organic solvents, the higher the solution dielectric constant, the more the charge-state distribution is shifted toward higher charge states. These observations build on the “electrophoretic” mechanism of droplet charging. Solvents with high solution dielectric constants are considered to be most effective at stabilizing multiply charged ions (where charge separation is greatest), and they are likely to increase the level of droplet charging. Solvents with high basicities (gas phase and solution phase) and high proton affinities, yet low dielectric constants, favor lower charge states in ES mass spectra of lipid A and cardiolipin from Escherichia coli. This indicates that gas-phase processes and solvent basicity contribute much less toward ion formation than solution-phase solvation via preferred orientation of the solvent dipole.  相似文献   

20.
Chlorinated β,β-dideuterated poly(vinyl chloride) (β,β-d2-CPVC) was prepared by photochemical suspension chlorination in concentrated hydrochloric acid with chloroform as swelling agent, and thermally in tetrachloroethane solution. 1H-NMR (nuclear magnetic resonance) spectra of β,β-d2-CPVC were recorded. The content of deuterium was determined in combustion products of β,β-d2-CPVC as the D2O:(H1D)2O ratio by means of mass spectroscopy. Both in the photochemical suspension chlorination and in the thermal solution chlorination of β,β-d2-PVC, a decrease of the concentration of ? CHCl? groups was observed. The differences in structure of the suspension chlorinated and solution chlorinated β,β-d2-PVC were confirmed.  相似文献   

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