Real-time measurement of myosin-nucleotide noncovalent complexes by electrospray ionization mass spectrometry

Nanoelectrospray ionization mass spectrometry has been used to measure the binding of ATP and ADP to the active site of rabbit skeletal myosin-S1. Increases in the molecular mass of myosin-S1 of 425 +/- 10 Da were obtained with the binding of ADP to the active site and by 530 +/- 10 Da with either ATP or hydrolysis products ADP and phosphate. Active site titrations of myosin-S1 with ADP gave a stoichiometry of approximately 1 ADP/S1 with an affinity in the micromolar range. The binding of ATP to myosin-S1 could be observed in the presence of up to 60 muM of excess MgATP without nonspecific binding of MgATP to the myosin. Conversion of the nucleotide complex containing an equilibrium mixture of ATP and ADP-Pi bound to myosin-S1 to one containing only bound ADP occurs at a rate consistent with that of the known steady-state rate of ATP hydrolysis. We expect this method to be of considerable use in the analysis of ligand binding and hydrolysis by the active sites of expressed myosin and myosin subfragments, which are not available in sufficient quantities for conventional methods of measurement of ligand binding.     

Macromolecular assembly of the transition state regulator AbrB in its unbound and complexed states probed by microelectrospray ionization mass spectrometry

The Bacillus subtilis global transition-state regulator AbrB specifically recognizes over 60 different DNA regulatory regions of genes expressed during cellular response to suboptimal environments. Most interestingly the DNA regions recognized by AbrB share no obvious consensus base sequence. To more clearly understand the functional aspects of AbrB activity, microelectrospray ionization mass spectrometry has been employed to resolve the macromolecular assembly of unbound and DNA-bound AbrB. Analysis of the N-terminal DNA binding domain of AbrB (AbrBN53, residues 1-53) demonstrates that AbrBN53 is a stable dimer, showing no apparent exchange with a monomeric form as a function of pH, ionic strength, solvent, or protein concentration. AbrBN53 demonstrates a capacity for DNA binding, underscoring the role of the N-terminal domain in both DNA recognition and dimerization. Full-length AbrB is shown to exist as a homotetramer. An investigation of the binding of AbrBN53 and AbrB to the natural DNA target element sinIR shows that AbrBN53 binds as a dimer and AbrB binds as a tetramer. This study represents the first detailed characterization of the stoichiometry of a transition-state regulator binding to one of its target promoters.     

Calcium and zinc complexes of pyrroglutamate analogs detected by electrospray ionization mass spectrometry

The complexation of calcium and zinc cations by pyrroglutamate analogs has been studied in the gas phase by means of electrospray ionization mass spectrometry (ESI–MS). Complexes were obtained from the solutions of calcium perchlorate and zinc perchlorate in acetonitrile. The complexes with calcium are singly and doubly charged with various stoichiometries while zinc complexes are singly charged except for one ligand. Solvation with acetonitrile and presence of perchlorate counter-ions are observed when the complexes are in the gas phase. The complexes formed with both metals are mainly L₂M and LM species. All tested compounds are better complexing agents for calcium than for zinc.     

Analysis of chirality by femtosecond laser ionization mass spectrometry

Recent progress in the field of chirality analysis employing laser ionization mass spectrometry is reviewed. Emphasis is given to femtosecond (fs) laser ionization work from the author's group. We begin by reviewing fundamental aspects of determining circular dichroism (CD) in fs-laser ionization mass spectrometry (fs-LIMS) discussing an example from the literature (resonant fs-LIMS of 3-methylcyclopentanone). Second, we present new data indicating CD in non-resonant fs-LIMS of propylene oxide. Chirality 24:684-690, 2012. ? 2012 Wiley Periodicals, Inc.     

Determination of branched beta-cyclodextrin-prostaglandin complexes using electrospray ionization mass spectrometry

Mass spectral measurements by electrospray ionization mass spectrometry (ESI-MS) detected the ions of beta-cyclodextrin (betaCD) or branched betaCDs (glucosyl-, galactosyl-, mannosyl- and maltosyl-betaCD)-prostaglandins (PGs: PGA(2), PGD(2), PGE(1), PGE(2), PGF(2alpha) and PGJ(2)) complexes, i.e., betaCD-PG complexes, with a host:guest ratio of 1:1 in the negative ion mode. This is the first study to report the ions of branched betaCD-PG complexes using ESI-MS. The inclusion complexes were determined by a flow injection analysis using acetonitrile/water. We could confirm by this method the presence of a betaCD-PGE(2) complex with a host:guest ratio of 1:1 in a solution-dissolved pharmaceutical formulation consisting of betaCD-PGE(2) (Prostarmon E tablet).     

Analysis of phenylethylamines by gas chromatography-chemical ionization mass spectrometry.

A method is described for the simultaneous analysis of several biologically important phenylethylamines using gas chromatography/mass spectrometry. The amines are converted to N-dinitrophenyl, O-trimethylsilyl derivatives prior to their separation by gas chromatography. By using selected ion monitoring in the chemical ionization mode, we have been able to quantitate the endogenous concentrations of phenylethylamine and phenylethanolamine in rat brain.     

Analysis of heparan sulfate oligosaccharides by nano-electrospray ionization mass spectrometry.

A highly sensitive method to identify and quantify heparan sulfate (HS) oligosaccharides by using nano-electrospray ionization mass spectrometry (nESI-MS) is described. The new approach allows us to detect approximately 50 nM of a chemically synthesized pentasaccharide with a structure of GlcNS6S-GlcA-GlcNS6S-IdoA2S-GlcNS6SOMe (3-OH pentasaccharide). Typically, solutions were infused for a total of 5 min, at an average flow rate of 30 nl/min, and the remaining sample was recovered from the nanovial. The spectra shown were obtained by summing scans for 1--3 min. Hence, our data indicated that as little as 3 x 10(-15) mole of the pentasaccharide was consumed to obtain a reasonable spectrum at the concentration as low as 50 nM. In addition, we found a linear relationship between the relative response of the molecular ion and the concentration of the analyzed 3-OH pentasaccharide, demonstrating that this approach can be used to determine the amount of HS oligosaccharides. To this end, a 3-O-sulfated pentasaccharide was prepared by incubating the 3-OH pentasaccharide with purified HS 3-O-sulfotransferase-1 and 3'-phosphoadenosine-5'-phospho[(35)S]sulfate. The resulting 3-O-sulfated pentasaccharide was purified and analyzed by nESI-MS. Based on the standard curve constructed with the 3-OH pentasaccharide, we calculated the concentration of the 3-O-sulfated pentasaccharide by the relative response. The result indicates that this value is very close to the value measured by [(35)S]sulfate radioactivity. In conclusion, nESI-MS provides both high sensitivity and the capacity to quantify HSs. This approach is likely to become a very important tool for structural analysis and sequencing of HS and heparin oligosaccharides.     

Protein analysis by electrospray ionization mass spectrometry

The applicability of electrospray ionization (ESI) mass spectrometry to protein analyses has been studied. The molecular weight of hen egg lysozyme (HEL) was determined with an accuracy of +/- 2 u. The choice of solvents and additives in sample preparations was important to achieve high sensitivity as well as high precision of molecular weight measurements.     

Surfactant effects on protein structure examined by electrospray ionization mass spectrometry.

Electrospray ionization mass spectrometry (ESI-MS) has proven to be a useful tool for examining noncovalent complexes between proteins and a variety of ligands. It has also been used to distinguish between denatured and refolded forms of proteins. Surfactants are frequently employed to enhance solubilization or to modify the tertiary or quaternary structure of proteins, but are usually considered incompatible with mass spectrometry. A broad range of ionic, nonionic, and zwitterionic surfactants was examined to characterize their effects on ESI-MS and on protein structure under ESI-MS conditions. Solution conditions studied include 4% acetic acid/50% acetonitrile/46% H2O and 100% aqueous. Of the surfactants examined, the nonionic saccharides, such as n-dodecyl-beta-D-glucopyranoside, at 0.1% to 0.01% (w/v) concentrations, performed best, with limited interference from chemical background and adduct formation. Under the experimental conditions used, ESI-MS performance in the presence of surfactants was found to be unrelated to critical micelle concentration. It is demonstrated that surfactants can affect both the tertiary and quaternary structures of proteins under conditions used for ESI-MS. However, several of the surfactants caused significant shifts in the charge-state distributions, which appeared to be independent of conformational effects. These observations suggest that surfactants, used in conjunction with ESI-MS, can be useful for protein structure studies, if care is used in the interpretation of the results.     

Analysis of protein complexes using mass spectrometry

The versatile combination of affinity purification and mass spectrometry (AP-MS) has recently been applied to the detailed characterization of many protein complexes and large protein-interaction networks. The combination of AP-MS with other techniques, such as biochemical fractionation, intact mass measurement and chemical crosslinking, can help to decipher the supramolecular organization of protein complexes. AP-MS can also be combined with quantitative proteomics approaches to better understand the dynamics of protein-complex assembly.     

The observation of chaperone-ligand noncovalent complexes with electrospray ionization mass spectrometry.

Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) was applied for the study of noncovalent chaperone SecB-ligand complexes produced in solution and examined in the gas phase with the aid of electrospray ionization (ESI). Since chaperone proteins are believed to recognize and bind only with ligands with nonnative tertiary structure, this work required careful unfolding of the ligand and subsequent reaction with the intact chaperone (the noncovalent tetrameric protein, SecB). A high denaturant concentration was employed to produce nonnative structures of the OppA, and microdialysis of the resulting solutions containing the chaperone-ligand complexes was carried out to rapidly remove the denaturant prior to analysis. Multistage mass spectrometry was essential to the successful study of these complexes since the initial mass spectra indicated extensive adduction that precluded mass measurements, even after microdialysis. However, low energy collisional activation of the ions in the FTICR trap proved useful for adduct removal, and careful control of excitation level preserved the intact complexes of interest, revealing a 1:1 SecB:OppA stoichiometry. To our knowledge, these results present the first direct observation of chaperone-ligand noncovalent complexes and the highest molecular weight heterogeneous noncovalent complex observed to date by mass spectrometry. Furthermore, these results highlight the capabilities of FTICR for the study of such complex systems, and the development of a greater understanding of chaperone interactions in protein export.     

Analysis of fluorescently labeled oligosaccharides by capillary electrophoresis and electrospray ionization mass spectrometry

Neutral oligosaccharides were fluorescently conjugated with 7-amino-1, 3-naphthalenedisulfonic acid. A mixture of fluorescently labeled chitobiose, chitotriose, and chitotetrose were successfully separated by preparative capillary electrophoresis (CE) and the individual components characterized by electrospray ionization-mass spectrometry (ESI-MS). By combining fluorescent labeling with CE, the use of highly specific exoglycosidases and ESI-MS, a more structurally complex N-linked glycan was analyzed.     

Negative and positive ion chemical ionization mass spectrometry of dioximes and their nickel complexes

The negative ion mass spectra of Ni(LH)₂ (where LH₂ is glyoxime, methylglyoxime, dimethylglyoxime and diphenylglyoxime), in the presence of ammonia or methane at 0.5 torr, are reported and compared with the spectra of the free ligands. In each case, the base peak of the complex is the molecular negative ion and the extent of fragmentation was found to decrease gradually going from the glyoximato to the diphenylglyoximato derivative. In the chemical ionization mass spectra of the free ligands, the [M]⁻ ion is absent in all cases. The base peak is [M  H]⁻ for methylglyoximine, dimethylglyoxime and diphenylglyoxime and [M  H  H₂]⁻ for glyoxime. The fragmentation occurs largely by loss of H, OH, H₂O and NO species. The positive ion chemical ionization mass spectra of the same complexes show very abundant [M + H]⁺ and [M]⁺ ions and weak fragments, whilst a rather high fragmentation is observed for the corresponding free ligands.     

Mammalian electron transferring flavoprotein.flavoprotein dehydrogenase complexes observed by microelectrospray ionization-mass spectrometry and surface plasmon resonance

Microelectrospray ionization-mass spectrometry was used to directly observe electron transferring flavoprotein.flavoprotein dehydrogenase interactions. When electron transferring flavoprotein and porcine dimethylglycine dehydrogenase or sarcosine dehydrogenase were incubated together in the absence of substrate, a relative molecular mass corresponding to the flavoprotein.electron transferring flavoprotein complex was observed, providing the first direct observation of these mammalian complexes. When an acyl-CoA dehydrogenase family member, human short chain acyl-CoA dehydrogenase, was incubated with dimethylglycine dehydrogenase and electron transferring flavoprotein, the microelectrospray ionization-mass spectrometry signal for the dimethylglycine dehydrogenase.electron transferring flavoprotein complex decreased, indicating that the acyl-CoA dehydrogenases have the ability to compete with the dimethylglycine dehydrogenase/sarcosine dehydrogenase family for access to electron transferring flavoprotein. Surface plasmon resonance solution competition experiments revealed affinity constants of 2.0 and 5.0 microm for the dimethylglycine dehydrogenase-electron transferring flavoprotein and short chain acyl-CoA dehydrogenase-electron transferring flavoprotein interactions, respectively, suggesting the same or closely overlapping binding motif(s) on electron transferring flavoprotein for dehydrogenase interaction.     

Formation and study of single metal ion-phospholipid complexes in biphasic electrospray ionization mass spectrometry

Single metal ion-phospholipid complexes are observed in biphasic electrospray ionization mass spectrometry (BESI-MS) using a dual-channel microsprayer. Such a microsprayer makes it possible to put into contact two immiscible liquids within the Taylor cone. Thus, L-α-dipalmitoyl phosphatidylcholine (DPPC) dissolved in 1,2-dichloroethane (DCE) reacts with aqueous metal cations (M = Na(+), K(+), Ca(2+), Cu(2+), La(3+)) yielding the formation of [M-DPPC(n)](z+) complexes. The number of phospholipid molecules ranges from 1 to 4 for monovalent ions, to 8 for divalent and to more than 10 for trivalent ions respectively. The large number of ligands observed involves the formation of solvent free single ion-phospholipid complexes.     

Application of electrospray ionization mass spectrometry for studying human immunodeficiency virus protein complexes

Mass spectrometry (MS) with electrospray ionization (ESI) has shown utility for studying noncovalent protein complexes, as it offers advantages in sensitivity, speed, and mass accuracy. The stoichiometry of the binding partners can be easily deduced from the molecular weight measurement. In many examples of protein complexes, the gas phase-based measurement is consistent with the expected solution phase binding characteristics. This quality suggests the utility of ESI-MS for investigating solution phase molecular interactions. Complexes composed of proteins from the human immunodeficiency virus (HIV) have been studied using ESI-MS. Multiply charged protein dimers from HIV integrase catalytic core (F185K) and HIV protease have been observed. Furthermore, the ternary complex between HIV protease dimer and inhibitor pepstatin A was studied as a function of solution pH. Zinc binding to zinc finger-containing nucleocapsid protein (NCp7) and the NCp7-psi RNA 1:1 stoichiometry complex was also studied by ESI-MS. No protein-RNA complex was observed in the absence of zinc, consistent with the role of the zinc finger motifs for RNA binding. Proteins Suppl. 2:28–37, 1998. © 1998 Wiley-Liss, Inc.     

Liquid ionization mass spectrometry of phospholipids

Several phosphatidylcholines (PC) and a phosphatidylethanolamine (PE) were subjected to liquid ionization (LI) mass spectrometry, in which a sample is ionized through energy transfer from metastable argon atoms under atmospheric pressure. Commercially available and synthesized, saturated or unsaturated fatty acid containing phospholipids and their mixtures were studied. A sample either as a concentrated chloroform-methanol solution or with glycerol (matrix) gave characteristic peaks such as MH+ and four fragment ions. One of the fragment ions (e.g., m/z 551 of PC 16:0, 16:0) containing both fatty acid residues has been commonly observed with other ionization methods such as CI, FD, and FAB, but the other fragment ions have not been observed in other mass spectra with one exception on desorption CI. Ions b and d (e.g., m/z 464 and 328, respectively, for PC 16:0, 16:0) contain one fatty acyl residue and the other ion containing the phosphorylcholine moiety appears at m/z 196 for PC. Thus the masses of the MH+ ion and these fragment ions provide useful structural information even in the case of a mixture. The ion b (e.g., m/z 488 of PC 18:0, 18:2) observed during an early period of heating was formed mainly by the loss of one acyl group at sn-1 of the glycerol backbone and thus may be used to differentiate the positional specificities of the constituent fatty acids. The temperature of the sample, however, should be controlled precisely, because it has a significant effect on the mass spectrum. The present method (LI) also provided useful information for a mixture of PC and PE.