Showing papers on "Electrospray ionization published in 1997"

Studying noncovalent protein complexes by electrospray ionization mass spectrometry

Abstract: Electrospray ionization mass spectrometry has been used to study protein interactions driven by noncovalent forces The gentleness of the electrospray ionization process allows intact protein complexes to be directly detected by mass spectrometry Evidence from the growing body of literature suggests that the ESI-MS observations for these weakly bound systems reflect, to some extent, the nature of the interaction found in the condensed phase Stoichiometry of the complex can be easily obtained from the resulting mass spectrum because the molecular weight of the complex is directly measured For the study of protein interactions, ESI-MS is complementary to other biophysical methods, such as NMR and analytical ultracentrifugation However, mass spectrometry offers advantages in speed and sensitivity The experimental variables that play a role in the outcome of ESI-MS studies of noncovalently bound complexes are reviewed Several applications of ESI-MS are discussed, including protein interactions with metal ions and nucleic acids and subunit protein structures (quaternary structure)

Quantitative analysis of biological membrane lipids at the low picomole level by nano-electrospray ionization tandem mass spectrometry.

Abstract: Nano-electrospray tandem mass spectrometry allows qualitative and quantitative analysis of complex membrane lipid mixtures at the subpicomole level. We have exploited this technique to selectively detect individual classes of phospholipids from unprocessed total cellular lipid extracts by either precursor ion or neutral loss scanning. This way phosphatidylcholine, sphingomyelin, phosphatidylinositol and -phosphates, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidic acid, and their plasmalogen analogues can be detected. The optimized ionization and fragmentation conditions described together with the principle of internal standardization by nonnatural analogues allow the rapid and quantitative determination of membrane lipid compositions down to sample amounts of 1000 cells.

Electrospray ionization mass spectrometry : fundamentals, instrumentation, and applications

Abstract: Partial table of contents: FUNDAMENTAL ASPECTS OF ELECTROSPRAY IONIZATION (ESI). On the Mechanism of Electrospray Mass Spectrometry (P. Kebarle & Y. Ho). The Electrolytic Nature of Electrospray (G. Van Berkel). ELECTROSPRAY COUPLING TO MASS ANALYZERS. Electrospray Ionization Time--of--Flight Mass Spectrometry (I. Chernushevich, et al.). Electrospray Ionization Quadrupole Ion--Trap Mass Spectrometry (M. Bier & J. Schwartz). INTERFACING OF SOLUTION--BASED SEPARATION TECHNIQUES TO ELECTROSPRAY. Combining Liquid Chromatography with Electrospray Mass Spectrometry (R. Voyksner). APPLICATIONS OF ELECTROSPRAY IONIZATION. Electrospray Ionization Mass Spectrometry of Peptides and Proteins (J. Loo & R. Loo). Index.

A Predictive Model for Matrix and Analyte Effects in Electrospray Ionization of Singly-Charged Ionic Analytes

Abstract: In electrospray ionization (ESI), droplets with a surface excess charge are created. The rate of production of surface excess charge is a constant and is equal to the rate of ion production. The ions appearing in the mass spectrum are postulated to be those that formed the surface excess charge at the time of droplet formation (or their collision products). An equilibrium model based on competition among the ions in the solution for the limited number of excess charge sites has been developed. This model accurately predicts the response curves of singly-charged ionic analytes as a function of the concentration of electrolyte and other analytes and provides an explanation for the selective effectiveness of ESI. At low concentrations of total analyte (micromolar and less), the response curves are linear, indifferent to the presence of other low concentration analytes, and suppressed by electrolyte concentrations in excess of the minimum required. At higher analyte concentrations, the response becomes independent of analyte concentration but highly affected by the presence of other analytes.

Multichannel microchip electrospray mass spectrometry.

Abstract: Microfabricated multiple-channel glass chips were successfully interfaced to an electrospray ionization mass spectrometer (ESI-MS). The microchip device was fabricated by standard photolithographic, wet chemical etching, and thermal bonding procedures. A high voltage was applied individually from each buffer reservoir for spraying sample sequentially from each channel. With the sampling orifice of the MS grounded, it was found that a liquid flow of 100−200 nL/min was necessary to maintain a stable electrospray. The detection limit of the microchip MS experiment for myoglobin was found to be lower than 6 × 10-8 M. Samples in 75% methanol were successfully analyzed with good sensitivity, as were aqueous samples. The parallel multiple-channel microchip system allowed ESI-MS analysis of different samples of standard peptides and proteins in one chip.

Analysis of Oligonucleotides by HPLC-Electrospray Ionization Mass Spectrometry.

Abstract: A new interface procedure has been developed that allows, for the first time, the high-efficiency analysis of synthetic oligonucleotides up to 75 bases by reversed-phase HPLC and on-line electrospray ionization mass spectrometry. For oligonucleotides up to 30 bases in length, single-base resolution can be obtained with low levels of cation adduct formation in the negative ion electrospray mass spectra. A key part of the method uses 1,1,1,3,3,3-hexafluoro-2-propanol as an additive to the HPLC mobile phase, adjusted to pH 7.0 with triethylamine. This novel additive results in both good HPLC separation and efficient electrospray ionization. The broad potential of this new method is demonstrated for synthetic homopolymers of thymidine (PolyT), fragments based on the pBR322 plasmid sequence, and phosphorothioate ester antisense oligonucleotides. This approach will be of particular utility for the characterization of DNA probes and PCR primers and quality control of antisense compounds such as phosphorothioates...

A microfabricated device for rapid protein identification by microelectrospray ion trap mass spectrometry.

Abstract: Nanoelectrospray mass spectrometry, the infusion at low flow rates of unseparated peptide mixtures representing protein proteolytic digests into an electrospray ionization mass spectrometer (MS), has been shown to be a suitable method for the analysis of small amounts of proteins. However, the current technique is time consuming, tedious, and difficult to automate. We used microfabrication technologies to construct a device for the sequential infusion of different peptide samples into an electrospray ionization MS without the need for sample manipulation. In this device, etched sample and buffer reservoirs are connected via etched channels to a microelectrospray ion source. Peptide samples, typically unseparated tryptic digests of proteins, are applied to different reservoirs. A flow of liquid originating from a specific reservoir is generated and selectively directed toward the microsprayer and the MS by electroosmotic pumping. The analyte proteins are identified by searching sequence databases with the ...

Acid-Induced Unfolding of Cytochrome c at Different Methanol Concentrations: Electrospray Ionization Mass Spectrometry Specifically Monitors Changes in the Tertiary Structure†

Abstract: The acid-induced denaturation of ferricytochrome c (cyt c) was examined in aqueous solutions containing different concentrations of methanol by electrospray ionization mass spectrometry (ESI MS) and optical spectroscopy. Circular dichroism, fluorescence, and absorption spectroscopy show that at a low concentration of methanol (3%) a decrease in pH induces a cooperative unfolding transition at around pH 2.6 that is accompanied by a breakdown of the native secondary and tertiary structure of the protein. In 50% methanol the breakdown of the tertiary structure occurs at around pH 4.0, whereas the α-helical content remains largely intact over the whole pH range studied. In ESI MS different protein conformations in solution are monitored by the different charge state distributions they generate during ESI. The ESI mass spectra recorded at near-neutral pH for both methanol concentrations are very similar and show a maximum at (cyt c + 8H+)8+. Despite the different conformations of the protein in solution, the a...

Disulfide-Intact and -Reduced Lysozyme in the Gas Phase: Conformations and Pathways of Folding and Unfolding

Abstract: The conformations of gaseous lysozyme ions (+5 through +18) produced by electrospray ionization have been studied in the gas phase using ion mobility mass spectrometry techniques. When solutions containing the disulfide-intact and disulfide-reduced lysozyme are electrosprayed, the gas-phase ions that are produced have distinctly different collision cross sections. Disulfide-intact ions favor two conformer types: a highly folded conformer with a cross section near that calculated for the crystal structure and a partially unfolded conformer that is formed when the ions are injected into the drift tube at high injection voltages. Ions formed from the disulfide-reduced solution have collision cross sections that are much larger than any observed for the disulfide-intact protein, showing that these ions are largely unfolded. Gas-phase proton-transfer reactions in the ion source can be used to favor lower charge states for both solutions. When protons are removed from disulfide-intact lysozyme ions, highly fol...

Subunit diversity and tissue distribution of human glutathione S-transferases: interpretations based on electrospray ionization-MS and peptide sequence-specific antisera

Abstract: Uncertainties about the composition and identities of glutathione S-transferases (GSTs) in human tissue have impeded studies on their biological functions. A rigorous protocol has therefore been developed to characterize the human proteins. Cytosolic GST subunits were resolved by reverse-phase HPLC methods, individual components were assigned to Alpha, Mu and Pi classes on the basis of their immunoreactivities, and peptide-sequence-specific antisera were used to distinguish among five different Mu-class subunits (GSTM1-GSTM5). Each subunit type was characterized and identified unambiguously by electrospray ionization-MS. Acetylation of N-terminal residues in the GSTA1, GSTA2, GSTM3 and GSTM4 subunits were the only natural post-translational modifications detected. The unique structure of GSTM3, with N- and C-terminal peptide extensions predicted from cDNA sequences, was confirmed. Only testis and brain were rich sources of GSTM3 subunits. Subunit profiles were distinct and characteristic of the particular tissue type, and this tissue specificity in GST expression was evident even in organs from different individuals. For instance, livers had relatively simple GST compositions, consisting of a preponderance of Alpha-class subunits and GSTM1 (when present). By contrast, representation of most subunit types was a characteristic feature of testis, which had the highest levels of GSTs. GSTM4 and GSTM5 subunits, here identified for the first time in human tissue extracts, were minor components, with GSTM5 found only in brain, lung and testis. Specimens devoid of GSTM1 subunits, particularly those from null-genotype individuals, were readily discerned at the protein level. Liver was the only rich source of the GSTM1 subunit (although it also constituted a major fraction of adrenal GSTs), and so the functional consequences of the GSTM1 gene deletion are likely to vary in extrahepatic tissues.

Study of 4-Quinolone Antibiotics in Biological Samples by Short-Column Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry

Abstract: Simultaneous detection and confirmation of 15 quinolone antibiotics was accomplished by fast short-column liquid chromatography coupled with electrospray ionization tandem mass spectrometry (LC/MS/MS). Several physicochemical parameters such as hydrophobicity and aqueous dissociation constants were calculated from the structural formulas of the quinolone drugs, and their impact on both chromatographic and mass spectrometric behavior was studied. Additionally, a possible influence of bulk solution pH on electrospray detection sensitivity of 4-quinolones was investigated and compared to predictions based on solution-phase equilibria. A signal intensity comparison of the MH+ ions at different pH values for all 15 compounds did not reveal any pH effect, despite variations by several orders of magnitude in equilibrium concentrations in bulk solution. To demonstrate the potential of the LC/MS/MS method, its application to trace analysis in several biological matrices such as milk, salmon, and human urine was in...

‘Wrong‐way‐round’ Electrospray Ionization of Amino Acids

Abstract: ‘Wrong-way-round’ electrospray ionization, in the present context, refers to observation of intense [M + H]+ ions electrosprayed from strongly basic solutions and of [M − H]− ions from strongly acidic solutions. Most previous investigations of this phenomenon have been directed at variations in charge-state distributions for polyfunctional peptides and proteins as a function of bulk solution pH. The present work extends that of Hiraoka et al. (J. Mass Spectrom. Soc. Japan43, 127 (1995)) on the pH dependence of absolute mass spectral intensities of ions in electrospray mass spectra of amino acids. This choice of test analyte permits investigation of both positive- and negative-ion mass spectra without potential complications from changes in secondary and tertiary structures as the pH is varied. The intensities of [M + H]+ and [M − H]− ions, over the pH range 3 to 11, varied by factors of 3–5 despite calculated variations of several orders of magnitude in equilibrium concentrations in the bulk solution. The same behaviour was observed for derivatized amino acids such as amides and methyl esters. Measurements of pH of collected spray, and of the total current carried by the charged droplets, confirmed that these observations can not be accounted for in terms of wholesale pH switching from acidic to basic or vice versa by electrochemical reactions at the electrospray needle. Precursors of the ‘wrong-way-round’ ions were sought by conventional precursor-ion scanning experiments but with minimal declustering conditions in the atmospheric-pressure ion-source interface. In the case of added electrolytes, such as ammonia and acetic acid, which are both volatile and capable of Bronsted acid–base behaviour, the observations were consistent with earlier interpretations involving e.g. [M + NH4+] precursors for [M + H]+ ions. Such explanations were not applicable to similar observations made for solutions with added tetramethylammonium hydroxide or hydrochloric acid. © 1997 Crown Copyright, Canada

High-Sensitivity Analysis of Neutral Underivatized Oligosaccharides by Nanoelectrospray Mass Spectrometry

Abstract: Nanoscale electrospray ionization (nano-ESI) overcomes the sensitivity problems found and reported for neutral oligosaccharides in conventional microscale forced-flow ESI. For a series of compounds ranging from trisaccharides to larger polymers with molecular masses up to 6 kDa, sample concentrations of 10-5 M, i.e., 10 pmol total sample load, yielded very intense singly or multiply cationized molecule ions in an ion-trap mass spectrometer. In a dilution series, it is exemplified that, at the 10-8 M level, molecule ion signals can be clearly registered with a S/N ratio of about 7. Only 100 amol of sample has been consumed in this experiment. Investigation of an oligosaccharide−peptide mixture revealed that the oligosaccharide is suppressed in conventional ESI, whereas in nano-ESI both analytes are detected at comparable and high intensities. Mechanistic implications are discussed, emphasizing the influence of surface activity for the two ESI techniques. The very low flow rates inherent to nano-ESI of abou...

Analytical applications of ion-molecule reactions

Abstract: This review covers applications of ion-molecule reactions for solving increasingly complex analytical problems. Because gas-phase reactions are frequently fast and efficient, the use of ion-molecule reactions provides a diverse frontier for extending the boundaries of mass spectrometry. Product distributions from ion-molecule reactions may provide key diagnostic information for structure identification, and particular product ions may afford more structurally informative fragmentation patterns than those patterns of ions that are generated by conventional methods. Applications range from those areas involving the development of novel chemical ionization reagents that show structural specificity upon reactions with analytes, to those areas in which ion-molecule reactions are combined with collisionally activated dissociation in unusual sequences, to those areas involving ion-molecule reactions of species formed by laser desorption or electrospray ionization. In electrospray ionization applications, the use of ion-molecule reactions allows the concentration of ion current into fewer multicharged ions, permits the counting of acidic or basic sites, and provides indirect information about protein structures and thermochemical data about individual sites in large molecules. © 1997 John Wiley & Sons, Inc., Mass Spectrom Rev 16 (2), 91–110, 1997

Identification of Catechin Oligomers from Apple (Malus pumila cv. Fuji) in Matrix‐assisted Laser Desorption/Ionization Time‐of‐flight Mass Spectrometry and Fast‐atom Bombardment Mass Spectrometry

Abstract: Molecular size information for polymerized catechin larger than the decamer in unripe apple was obtained by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and by fast-atom bombardment mass spectrometry. Matrix-assisted laser desorption/ionization time of flight mass spectrometry provided evidence for the pentadecamer using trans-3-indoleacrylic acid as the matrix in the presence of silver ion. Even in the absence of silver ion, the dodecamer and undecamer were observed in the positive- and negative-ion modes, respectively. Fast-atom bombardment mass spectrometry also afforded evidence for the undecamer in both positive- and negative-ion modes.

Length and Base Composition of PCR-Amplified Nucleic Acids Using Mass Measurements from Electrospray Ionization Mass Spectrometry

Abstract: A generally applicable algorithm has been developed to allow base composition of polymerase chain reaction (PCR) products to be determined from mass spectrometrically measured molecular weights and the complementary nature of DNA. Mass measurements of arbitrary precision for single-stranded DNA species are compatible with an increasingly large number of possible base compositions as molecular weight increases. For example, the number of base compositions that are consistent with a molecular weight of 35 000 is ∼6000, based on a mass measurement precision of 0.01%. However, given the low misincorporation rate of standard DNA polymerases, mass measurement of both of the complementary single strands produced in the PCR reduces the number of possibilities to less than 100 at 0.01% mass precision, and base composition is uniquely defined at 0.001% mass precision. Taking into account the low misincorporation rate of standard DNA polymerases and the fact that the final PCR product also contains primers of known ...

High-resolution Fourier transform ion cyclotron resonance mass spectrometry of humic and fulvic acids by laser desorption/ionization and electrospray ionization

Abstract: High-field (9.4 T) Fourier transform ion cyclotron resonance (FT-ICR) mass spectra of standard Suwannee River humic and fulvic acids have been obtained by use of laser desorption (LDI) and electrospray (ESI) ionization. The LDI FT-ICR mass spectrum was similar to those observed previously, with ions at essentially every nominal value, 200 ≤ m/z ≤ 800. In contrast, the ESI FT-ICR mass spectrum, although still containing ions at most values in the 200 ≤ m/z ≤ 800 range, was dominated by a relatively few prominent species. ESI FT-ICR mass spectra of standard humic and fulvic acid isolates were similar. Although many ionic species appeared in both fulvic acid and humic acid ESI FT-ICR mass spectra, the fulvic acid mass spectrum contained more highly charged species. Subfractions of the fulvic acid mixture isolated by an HPLC procedure yielded similar mass spectra. The stability of high-mass ions produced by ESI combined with the high-mass resolution capability of FT-ICR MS allow for precise determination of m...

Effects of charge state on fragmentation pathways, dynamics, and activation energies of ubiquitin ions measured by blackbody infrared radiative dissociation.

Abstract: Blackbody infrared radiative dissociation spectra of the (M + 5H)5+ through (M + 11H)11+ ions of the protein ubiquitin (8.6 kDa) formed by electrospray ionization were measured in a Fourier-transform mass spectrometer. The 5+ ion dissociates exclusively by loss of water and/or ammonia, whereas the 11+ charge state dissociates only by formation of complementary y and b ions. These two processes are competitive for intermediate charge state ions, with the formation of y and b ions increasingly favored for the higher charge states. The y and b ions are formed by cleavage of the backbone amide bond on the C-terminal side of acidic residues exclusively, with cleavage adjacent to aspartic acid favored. Thermal unimolecular dissociation rate constants for the dissociation of each of these charge states were measured. From the temperature dependence of these rates, Arrhenius activation parameters in the rapid energy exchange limit are obtained. The activation energies (Ea) and preexponential factors (A) for the 5...

Characterizing Oligosaccharides Using Injected-Ion Mobility/Mass Spectrometry

Abstract: Injected-ion mobility/mass spectrometry techniques have been used to measure the reduced ion mobilities for negatively charged raffinose, melezitose and α-, β-, and γ-cyclodextrins formed by electrospray ionization. At low injection energies, the mass spectra are dominated by negatively charged (deprotonated) parent ions. At high injection energies, the mass spectra recorded for the cyclodextrins and raffinose display peaks that result from cross-ring cleavage of individual sugar units. Melezitose dissociates by cleavage of the glycosidic bonds. The ion mobility distributions can be used to distinguish between different isomeric forms of parent and fragment ions having the same mass-to-charge ratios.

Cytochrome c folding kinetics studied by time-resolved electrospray ionization mass spectrometry.

Abstract: A new method for studying the folding kinetics of proteins is described. The method combines a continuous flow mixing technique with an electrospray mass spectrometer. Different protein conformations in solution are detected by the different charge states they produce during electrospray ionization. Unfolded proteins generally have more accessible protonation sites and give higher charge states than native proteins. The method is applied to study the refolding of acid-denatured cytochrome c. Global data analysis is used to obtain the exponential lifetimes which are associated with the refolding process. The kinetics can be described by two lifetimes of 0.17 +/- 0.02 and 8.1 +/- 0.9 s which are in accordance with the results of stopped flow experiments previously described in the literature. These lifetimes are associated with roughly 90 and 10% of the total intensity changes in the mass spectrum, respectively, and most likely reflect fast and slow refolding subpopulations of cytochrome c in solution.

Mass spectrometry as a readout of protein structure and function

Abstract: Proteins have evolved to carry out very specific functions within the cell by interacting with a diverse set of biomolecules. Understanding how a protein's higher order structure relates to its function is important for defining the molecular basis of these interactions. In recent years, mass spectrometry has become an important tool for dissecting protein structure and function. Using electrospray ionization (ESI)- and matrix-assisted laser desorption/ionization (MALDI)-based approaches, it has been possible to monitor protein folding, characterize noncovalent protein complexes, and assess the contribution of individual amino acid residues to a protein's function. Here, it is our goal to summarize these approaches and highlight recent, biologically relevant applications where mass spectrometry has provided unique insight into the mysteries of protein structure and function.

Integrated multichannel microchip electrospray ionization mass spectrometry: analysis of peptides from on-chip tryptic digestion of melittin.

Abstract: In continuation of our work to develop an integrated multichannel microchip interfaced to electrospray mass spectrometry (ESI-MS), this paper demonstrates one of several applications of this approach in monitoring tryptic digestion products. The multichannel microchip allowed integration of sample preparation onto the microchip to facilitate the analysis process. Melittin was selected as a model oligopeptide because it possesses a cluster of four adjacent basic residues which enable probing the site specificity of trypsin as a function of digest times. Reactions were performed on-chip in different wells for specific time periods and then analyzed by infusion from the microchip by ESI-MS, using leucine enkephalin as internal standard. The rate of formation and disappearance of the molecular ion and individual fragments was followed for a melittin to trypsin concentration ratio of 300:1. The results indicate the potential of integrating enzymatic reactions with multichannel microchip ESI-MS for automated optimization of reaction condition while consuming only small amounts of sample.

New sialic acids from biological sources identified by a comprehensive and sensitive approach: liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) of SIA quinoxalinones

Abstract: Sialic acids are a family of 9-carbon carboxylated sugars, where different substitutions of the backbone define over 30 members. Biological roles of these substitutions have been missed until recently because of their low abundance and lability to conventional isolation/purification methods. This new approach characterizes sialic acids using electrospray ionization-mass spectrometry (ESI-MS) to monitor the HPLC separation of their DMB (1,2-diamino-4,5-methylenedioxy-benzene) derivatives (quinoxalinones). A combination of retention times and spectra characteristics allows definition of the type and position of the various substituents. This approach requires no previous purification, involving a simple derivatization reaction followed by direct injection on the microbore HPLC column. A complete spectrum, including molecular ions and CAD fragments of a sialic acid quinoxalinone, is obtained by injecting 10-20 pmol of the compound. Individual quinoxalinones can be purified by regular RP-HPLC and analyzed by direct-injection ESI-MS or LSIMS. Using this approach, we identified 28 different sialic acids, including the following new species: Neu5Gc9Lt (BSM), anhydro derivatives of Neu5Ac other than the 4,8-anhydro (horse serum hydrolyzates), KDN5(7)Ac and KDN5(7),9Ac2 (amphibian Pleurodeles waltl), four isomers of Neu5Gc8MexAc and three anhydro derivatives of Neu5Gc8Me (glycolipids of the starfish Pisaster brevispinus), and Neu5Ac8S (in addition to Neu5Gc8S, in the glycolipids of the sea urchin Lovenia cordiformis). Results show the usefulness of LC-ESI-MS to study sialic acid diversity, and identification of small amounts of unexpected sialic acids or new members of their family.