Determination of dissociation constants for protein-ligand complexes by electrospray ionization mass spectrometry

A fully automated biophysical assay based on electrospray ionization mass spectrometry (ESI-MS) for the determination of the dissociation constants (KD) between soluble proteins and low molecular mass ligands is presented. The method can be applied to systems where the relative MS response of the protein and the protein-ligand complexes do not reflect relative concentrations. Thus, the employed approach enables the use of both electrostatically and nonpolar bound complexes. The dynamic range is wider than for most biological assays, which facilitates the process of establishing a structure-activity relationship. This fully automated ESI-MS assay is now routinely used for ligand screening. The entire procedure is described in detail using hGHbp, a 25-kDa extracellular soluble domain of the human growth hormone receptor, as a model protein.     

On-line dynamic titration: determination of dissociation constants for noncovalent complexes using Gaussian concentration profiles by electrospray ionization mass spectrometry

A new method for determination of dissociation constants (Kd) using on-line titration by electrospray ionization mass spectrometry is presented. Unlike in common titration experiments, where a set of discrete solutions with a fixed concentration of host and increasing concentration of guest is measured, here a continuous Gaussian concentration profile of guest, formed by band-broadening dispersion during passage through a long tubing, is utilized. An equation allowing access to dissociation constant values from experimental data fit to a 1:1 binding model was derived and incorporated into an in-house-written computer program for automated data processing. The new method is demonstrated for noncovalent complexes of cinchona alkaloid carbamate chiral selectors with N-dinitrobenzoylleucine enantiomers and a series of cyclodextrins with sulfonated azo dyes.     

Thermal dissociation of multimeric protein complexes by using nanoelectrospray mass spectrometry

The behavior of macromolecular systems at different temperatures is often crucial to their biological activity and function. While heat-induced changes of individual proteins are readily monitored by a number of spectroscopic methods, changes in noncovalent complexes of biomolecules are more challenging to interpret. Nanoelectrospray mass spectrometry is becoming increasingly powerful in the study of large noncovalent complexes, and here we describe the design, characterization, and application of a novel probe that allows the thermocontrol of the solution in the electrospray capillary. The transition temperature for the unfolding of the protein lysozyme is readily obtained and correlates closely with that measured by fluorescence spectroscopy, thereby demonstrating the validity of this approach. We apply this technique to the study of the 200-kDa complex of the small heat shock protein TaHSP16.9, revealing both its dissociation into suboligomeric species and an increase in its size and polydispersity at elevated temperatures. In contrast, gas-phase activation of this complex is also carried out and yields a dissociation pathway fundamentally different from that observed for thermal activation in solution. As such, this probe allows the study of the reversible heat-induced changes of noncovalent complexes in a biologically relevant manner.     

Development of multi-ESI-sprayer, multi-atmospheric-pressure-inlet mass spectrometry and its application to accurate mass measurement using time-of-flight mass spectrometry

The atmospheric pressure sampling nozzle (orifice, heated capillary, or inlet) of a high mass accuracy time-of-flight mass spectrometer (TOF-MS) was modified by replacing its single nozzle with multiple atmospheric pressure nozzles. This allowed multiple streams of liquids to be introduced into the MS in parallel (an electrosprayer for each nozzle), with minimum analyte interactions between the streams. The chemical contents of all liquid streams were analyzed concurrently using a single mass spectrometer. To obtain a higher mass accuracy by providing internal reference on each scan (acquisition) and to evaluate the suitability of TOF-MS for molecular-formula confirmation, a dual-ESI-sprayer, dual-nozzle version of this design was used. The accurate masses of tens of organic compounds in the mass range of 200-3000 Da were measured, and the results were compared with those obtained using dual-sprayer, single-nozzle TOF-MS. A significant improvement in mass accuracy was observed when the former technique was used. Comparison between the mass accuracy using dual-ESI-sprayer, dual-nozzle TOF-MS and that obtained using a double-focusing mass spectrometer operating under chemical ionization (CI) and fast atom bombardment (FAB) shows the suitability of the technique for elemental-composition confirmation. Approximately 85% of samples analyzed had mass errors of less than 5 ppm, and the other 15% had mass errors less than 8 ppm. Using a high-performance liquid chromatography (HPLC) as a device for introduction of one liquid stream (sample) and a syringe pump as a device for introduction of the second liquid stream (reference standard), the accurate mass of a tryptic digest of cytochrome c was measured. The range of mass errors was from -6.1 ppm to +3.6 ppm, a significant improvement over our previously reported mass accuracy for this digest using single-nozzle TOF-MS. The interactions between analytes in the liquid streams also were investigated using a variety of sample-introduction and nozzle-design combinations, including single-ESI-sprayer, single-nozzle; dual-ESI-sprayer, single-nozzle; dual-ESI-sprayer, Y-shaped inlet; and dual-ESI-sprayer, dual-inlet. The results demonstrated that the dual-ESI-sprayer, dual-inlet design provides reference peaks on every acquisition with minimum analyte-reference interaction and, therefore, higher consistent mass accuracy.     

A method for the determination of binding constants by electrospray ionization mass spectrometry

A new method for the determination of binding constants using electrospray ionization mass spectrometry is presented. The intensity of a reference complex with a known log K value is monitored before and after addition of a second host or guest. On the basis of the change in intensity of the reference complex and extrapolation from a calibration curve, the log K value is then derived for the complex of interest using a set of simultaneous equilibrium equations. Binding constants of several crown ether-alkali metal cation complexes that were previously studied were determined to validate this strategy. Log K values for complexes involving dibenzo-16-crown-5 and its sym-oxyacetate derivative with Na+ or K+ were also determined.     

Interrogation of N-Linked oligosaccharides using infrared multiphoton dissociation in FT-ICR mass spectrometry

The structural elucidation of oligosaccharides remains a major challenge. Mass spectrometry provides a rapid and convenient method for structural elucidation based on tandem mass spectrometry. Ions commonly are selected and subjected to collision-induced dissociation (CID) to obtain structural information. Unfortunately, N-linked oligosaccharides are relatively large compounds and are not readily fragmented using CID. In this report, we illustrate the use of infrared multiphoton dissociation (IRMPD) to obtain structural information for large N-linked oligosaccharides. The IRMPD and CID behavior of oligosaccharides were compared for high-mannose-type oligosaccharides. Fragmentation that could not be obtained through conventional CID in Fourier transform ion cyclotron resonance mass spectrometry was observed with N-linked oligosaccharides. O-Linked and N-linked glycans of similarly large sizes were compared. It was found that internal cross-ring cleavages were observed only for N-linked oligosaccharides. The mannose branch points of N-linked oligosaccharides are apparently more susceptible to cross-ring cleavages.     

Investigation of enzyme kinetics using quench-flow techniques with MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry is combined off-line with rapid chemical quench-flow methods to investigate the pre-steady-state kinetics of a protein-tyrosine phosphatase (PTPase). PTPase kinetics are generally interrogated spectrophotometrically by the employment of an artificial, chromophoric substrate. However, that methodology places a constraint on the experiment, hampering studies of natural, biochemically relevant substrates that do not incorporate a chromophore. The mass spectrometric assay reported herein is based on the formation of a covalent phosphoenzyme intermediate during substrate turnover. This species is generated in the reaction regardless of the substrate studied and has a molecular weight 80 Da greater than that of the native enzyme. By following the appearance of this intermediate in a time-resolved manner, we can successfully measure pre-steady-state kinetics, regardless of the incorporation of a chromophore. The strengths of the mass-spectrometric assay are its uniform response to all substrates, simple and direct detection of covalent enzyme-substrate intermediates, and facile identification of enzyme heterogeneities that may affect enzymatic activity.     

Comparison of fast atom bombardment mass spectrometry and thermal ionization quadrupole mass spectrometry for the measurement of zinc absorption in human nutrition studies

A method is described for the measurement of apparent zinc absorption in human nutrition studies. An enriched source of the stable isotope 67Zn was given to adult subjects together with a wheat cereal and the unabsorbed 67Zn measured in the feces. After drying, subsamples of the homogenized fecal material were ashed at 480 degrees C, purified for analysis by ion exchange chromatography, and the 64Zn/67Zn ratios determined by both fast atom bombardment mass spectrometry and thermal ionization quadrupole mass spectrometry. Good agreement was found between the two sets of results with mean precisions of approximately 0.5% for both techniques.     

Affinity capture and elution/electrospray ionization mass spectrometry assay of phosphomannomutase and phosphomannose isomerase for the multiplex analysis of congenital disorders of glycosylation types Ia and Ib

We report a new application of affinity capture-elution electrospray mass spectrometry (ACESI-MS) to assay the enzymes phosphomannomutase (PMM) and phosphomannose isomerase (PMI), which when deficient cause congenital disorders of glycosylation CDG-type Ia and type Ib, respectively. The novel feature of this mass-spectrometry-based assay is that it allows one to distinguish and quantify enzymatic products that are isomeric with their substrates that are present simultaneously in complex mixtures, such as cultured human cell homogenates. This is achieved by coupled assays in which the PMM and PMI primary products are in vitro subjected to another enzymatic reaction with yeast transketolase that changes the mass of the products to be detected by mass spectrometry. The affinity purification procedure is fully automated, and the mass spectrometric analysis is multiplexed in a fashion that is suitable for high-throughput applications.     

Quantitative determination of noncovalent binding interactions using automated nanoelectrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) has proven to be an extremely powerful tool for studying biomolecular structures and noncovalent interactions. Here we report a method using a fully automated, chip-based nanoESI-MS system to determine the dissociation constants (Kd) for the complexes of two different proteins with their ligands. The automated nanoelectrospray system, consisting of the NanoMate and ESI chip, serves functionally as a combination of autosampler and nanoelectrospray ionization source. This system provides all the advantages of conventional nanoelectrospray plus automated, high-throughput analyses without carryover. The automated nanoESI system was used to investigate quantitative noncovalent interactions between ribonuclease A (RNase A) and cytidylic acid ligands (2'-CMP, CTP), a well-characterized model protein-ligand complex, and between an inactive endocellulase mutant (Thermobifida fusca Cel6A D117Acd) and four oligosaccharide ligands (cellotriose, cellotetraose, cellopentaose, cellohexaose). Both titration and competitive binding approaches were performed prior to automated nanoESI-MS analysis with a Q-TOF mass spectrometer. Dissociation constants for each complex were calculated from the sum of ion peak areas of free and complexed proteins during the titration and competition experiments. The measured Kd values for the RNase A-CMP and Cel6A D117Acd-G3 complexes were found to be in excellent agreement with the available published values obtained by standard spectroscopic titration techniques. To our knowledge, this is the first report of using an ESI-MS approach to study the interactions between a cellulase and oligosaccharides. The results provide new insights for understanding the nature of cellulase-cellulose interactions.     

Absolute isotopic composition and atomic weight of commercial zinc using inductively coupled plasma mass spectrometry

Inductively coupled plasma mass spectrometry is introduced as a method for determining the absolute isotopic composition of zinc. The high ionization efficiency and time-independent characteristics of the mass spectrometry permit the absolute isotopic composition of high ionization potential elements. The mass discrimination of the instrument is calibrated by synthetic isotope mixtures prepared from highly enriched isotopes of zinc. The resulting isotope ratios yield atomic percents of 64Zn, 49.188 +/- 0.030; 66Zn, 27.792 +/- 0.041; 67Zn, 4.041 +/- 0.009; 68Zn, 18.378 +/- 0.050; and 70Zn, 0.600 +/- 0.003. This isotopic composition is different from those of conventional mass spectrometric measurements. Their differences depend on the mass differences about 0.8-1.2%/amu with enhancement of heavier isotopes. The atomic weight calculated from our isotopic composition is 65.3756 +/- 0.0040. The obtained atomic weight is fully consistent with that of a precise coulometric measurement in contrast to the previous mass spectrometric measurements. An isotopic variation of commercial zinc reagents has been investigated. A mass-dependent fractionation of 0.12%/amu is observed in a high-purity metal zinc, NIST-SRM 682, among five reagents. This mass dependence is probably inherited through their purification process.     

Infrared multiphoton dissociation in quadrupole time-of-flight mass spectrometry: top-down characterization of proteins

The first implementation of infrared multiphoton dissociation (IRMPD) for a hybrid quadrupole time-of-flight (QqTOF) mass spectrometer is reported. Ions were trapped in the radio frequency-only quadrupole (q2), which normally serves as a collision cell, and irradiated by a continuous CO2 IR laser. The laser beam was introduced coaxially with the quadrupoles in order to maximize overlap with the ion path. The resolution of the TOF mass analyzer allowed direct charge state determination for fragments smaller than 7 kDa. For larger fragments, the charge state could be assigned using the multiple losses of water, characteristic for IRMPD of proteins. The analytical performance is demonstrated by top-down sequencing of several representative proteins (equine myoglobin, bovine casein, and human insulin and chaperonin 10). Various post-translational modifications such as phosphorylation, acetylation, formation of disulfide bridges, and removal of N-terminal methionine followed by acetylation are detected and characterized. The utility of IRMPD for the analysis of biological samples is demonstrated in a study of a recently identified potential marker for endometrial cancer, chaperonin 10.     

High mass measurement accuracy determination for proteomics using multivariate regression fitting: application to electrospray ionization time-of-flight mass spectrometry

Important factors that limit the mass measurement accuracy from a mass spectrometer are related to (1) the type of mass analyzer used and (2) the data processing/calibration methods used to obtain mass values from the raw data. Here, two data processing methods are presented that correct for systematic deviations when the mass of ions is measured using a time-of-flight (TOF) mass spectrometer. The first fitting method is one where m/z values are obtained from fitting peak distributions using double Gaussian functions. A second calibration method takes into account the slight nonlinear response of the TOF analyzer in addition to the drift in the calibration over time. Using multivariate regression, both of these two effects can be corrected for using a single calibration formula. Achievable performance was evaluated with a trypsin digestion of serum albumin and proteins from the organism D. radiodurans that was analyzed using gradient reversed-phase liquid chromatography combined with an electrospray ionization orthogonal TOF mass spectrometer. The root-mean-square deviation between the theoretical and experimental m/z values for serum albumin tryptic peptides was found to be 8 ppm using the double Gaussian-multivariate method compared to 29 ppm determined using linear calibration and normal peak centroiding. An advantage of the methods presented here is that no calibrant compounds need to be added to the mobile phase, thereby avoiding interference effects and signal suppression of analytes.     

Determination of bond dissociation energies using electrospray tandem mass spectrometry and a derived effective reaction path length approach

A new approach for calculating bond dissociation energies (BDEs) from ES-MS/MS measurements has been developed. The new method features a "derived effective reaction path length" that has been applied to measure BDEs of alkali metal (Li+) adducts and halide (Cl-) adducts of monoacylglycerol, 1,2-diacylglycerol, and 1,3-diacylglycerol lipids. Also studied were lithium-bound dimers of monoacylglycerols, 1,2-diacylglycerols, and 1,3-diacylglycerols. BDEs for the adducts and dimers of the lipids were derived from collision-induced dissociation experiments using a triple quadrupole mass spectrometer with electrospray as the ionization source. Mass spectral data were used to empirically derive a single-exponential growth equation that relates product cross section to collision energy. From these single-exponential equations, a general second-order polynomial was derived using a multivariate growth curve model that enables prediction of BDEs of unknown complexes. Mass spectral results were compared to computer-generated bond dissociation energies using Becke-style three-parameter density functional theory (B3LYP, employing the Lee-Yang-Parr correlation functional), with excellent agreement between experimental and theoretical energy values. The newly developed method is general in nature and can be used for the measurement of metal or halide ionic adduct bond dissociation energies and for the measurement of bond energies of noncovalent interactions such as dimer dissociation energies. The validity of the method has been rigorously established using a triple quadrupole, but it may also be applied to other mass spectrometers that allow user control of the collision cell potential.     

Electrospray ionization and collision induced dissociation mass spectrometry of primary fatty acid amides

Primary fatty acid amides are a group of bioactive lipids that have been linked with a variety of biological processes such as sleep regulation and modulation of monoaminergic systems. As novel forms of these molecules continue to be discovered, more emphasis will be placed on selective, trace detection. Currently, there is no published experimental determination of collision induced dissociation of PFAMs. A select group of PFAM standards, 12 to 22 length carbon chains, were directly infused into an electrospray ionization source Quadrupole Time of Flight Mass Spectrometer. All standards were monitored in positive mode using the [M + H](+) peak. Mass Hunter Qualitative Analysis software was used to calculate empirical formulas of the product ions. All PFAMs showed losses of 14 m/z indicative of an acyl chain, while the monounsaturated group displayed neutral losses corresponding to H(2)O and NH(3). The resulting spectra were used to propose fragmentation mechanisms. Isotopically labeled PFAMs were used to validate the proposed mechanisms. Patterns of saturated versus unsaturated standards were distinctive, allowing for simple differentiation. This determination will allow for fast, qualitative identification of PFAMs. Additionally, it will provide a method development tool for selection of unique product ions when analyzed in multiple reaction monitoring mode.     

Characterization of pyridine-octanethiolated gold nanoparticles: desorption kinetics by thermal analysis mass spectrometry

We describe a synthetic pathway to the formation of stable pyridine-functionalized octanethiolate mixed monolayer-protected Au clusters (MPCs). The spectroscopic characterization data of MPCs using NMR, UV-Vis, TEM, XPS, and thermal-analysis-mass techniques are discussed. TEM analysis showed that spherical nanoclusters of 3-5 nm were produced. Furthermore, the particle sizes are uniform with a narrow size distribution. The pyridine-functionalized MPCs formed 2D superlattices with hexagonal packing covering on the carbon-coated copper grids during the toluene evaporation. For all samples, the S 2p(3/2) and 2p(1/2) components that appeared at approximately 162 and approximately 163 eV, respectively, in the XPS spectra compare very well with the typical value of chemisorbed S species. Thermal analysis mass spectrometer was used to analyze desorption behavior of octanethiolated MPCs or pyridine-functionalized mixed MPCs. The TA-mass spectra have revealed that MCPs exist monomer and dimer desorption behavior from monomeric thiolate adsorbed on the surface.     

Enhanced screening of glutathione-trapped reactive metabolites by in-source collision-induced dissociation and extraction of product ion using UHPLC-high resolution mass spectrometry

A selective and sensitive approach, called extraction of product ion (XoPI) method, was developed for the detection of l-glutathione (GSH)-trapped reactive metabolites employing an Orbitrap high resolution mass spectrometer. Fragmentation of GSH conjugates in the negative ion mode leads to a product ion, deprotonated γ-glutamyl-dehydroalanyl-glycine (m/z 272.0888). As a means of utilizing this property, negative ion high resolution MS data were collected from in vitro incubations by monitoring ions from m/z 269.5 to 274.5 under in-source collision-induced dissociation. Extraction of product ions at m/z 272.0888 ± 5 ppm from this data resulted in a chromatogram exhibiting deprotonated γ-glutamyl-dehydroalanyl-glycine as the major peaks with no or very few interferences. Therefore, peaks in this extracted product ion chromatogram potentially came from GSH-trapped reactive metabolites. The GSH conjugate parent ions were then confirmed in the corresponding full scan MS data, and their structures were identified from their MS(2) fragmentation patterns. The effectiveness of the approach was assessed with four model compounds, amodiaquine, clozapine, diclofenac, and fipexide, all well-known to form GSH-trapped reactive metabolites, following incubation in human liver microsomes supplemented with β-nicotinamide adenine dinucleotide 2'-phosphate reduced tetrasodium salt (NADPH) and GSH. The results from XoPI method were compared to two other commonly employed liquid chromatography-mass spectrometry (LC-MS) methods: precursor ion scan method and mass defect filter method. Overall, the XoPI method was more selective and sensitive in detecting the GSH conjugates. Many GSH conjugates previously not reported were detected and characterized in this study.     

Dynamic titration: determination of dissociation constants for noncovalent complexes in multiplexed format using HPLC-ESI-MS

With recent growth in fields such as life sciences and supramolecular chemistry, there has been an ever increasing need for high-throughput methods that would permit determination of binding affinities for noncovalent complexes of various host-guest systems. These are traditionally measured by titration experiments where concentration-dependent signals of species participating in solution-based binding equilibria are monitored by methods such as UV-vis spectrophotometry, calorimetry, or nuclear magnetic resonance spectrometry. Here we present a new titration technique that unifies and allows chromatographic separation of guests with determination of dissociation constants by electrospray mass spectrometry in a multiplexed format. A theoretical model has been derived that describes the complex formation for the guests eluted from a chromatographic column when hosts are admixed postcolumn. The model takes possible competition equilibria into account; i.e., it can deal with unresolved peaks of guests with the possible addition of multiple hosts in one experiment. This on-line workflow makes determination of binding affinities for large libraries of compounds possible. The potential of the method is demonstrated on the determination of dissociation constants for complexes of beta- and gamma-cyclodextrins with nonsteroidal antiinflammatory drugs ibuprofen, naproxen, and flurbiprofen.     

Native electrospray and electron-capture dissociation FTICR mass spectrometry for top-down studies of protein assemblies

The high sensitivity, extended mass range, and fast data acquisition/processing of mass spectrometry and its coupling with native electrospray ionization (ESI) make the combination complementary to other biophysical methods of protein analysis. Protein assemblies with molecular masses up to MDa are now accessible by this approach. Most current approaches have used quadrupole/time-of-flight tandem mass spectrometry, sometimes coupled with ion mobility, to reveal stoichiometry, shape, and dissociation of protein assemblies. The amino-acid sequence of the subunits, however, still relies heavily on independent bottom-up proteomics. We describe here an approach to study protein assemblies that integrates electron-capture dissociation (ECD), native ESI, and FTICR mass spectrometry (12 T). Flexible regions of assembly subunits of yeast alcohol dehydrogenase (147 kDa), concanavalin A (103 kDa), and photosynthetic Fenna-Matthews-Olson antenna protein complex (140 kDa) can be sequenced by ECD or "activated-ion" ECD. Furthermore, noncovalent metal-binding sites can also be determined for the concanavalin A assembly. Most importantly, the regions that undergo fragmentation, either from one of the termini by ECD or from the middle of a protein, as initiated by CID, correlate well with the B-factor from X-ray crystallography of that protein. This factor is a measure of the extent an atom can move from its coordinated position as a function of temperature or crystal imperfections. The approach provides not only top-down proteomics information of the complex subunits but also structural insights complementary to those obtained by ion mobility.     

Probing ligand binding to duplex DNA using KMnO4 reactions and electrospray ionization tandem mass spectrometry

An electrospray ionization tandem mass spectrometry (ESI-MS/MS) strategy employing the thymine-selective KMnO4 oxidation reaction to detect conformational changes and ligand binding sites in noncovalent DNA/drug complexes is reported. ESI-MS/MS is used to detect specific mass shifts of the DNA ions that are associated with the oxidation of thymines. This KMnO4 oxidation/ESI-MS/MS approach is an alternative to conventional gel-based oxidation methods and affords excellent sensitivity while eliminating the reliance on radiolabeled DNA. Comparison of single-strand versus duplex DNA indicates that the duplexes exhibit a significant resistance to the reaction, thus confirming that the oxidation process is favored for unwound or single-strand regions of DNA. DNA complexes containing different drugs including echinomycin, actinomycin-D, ethidium bromide, Hoechst 33342, and cis-C1 were subjected to the oxidation reaction. Echinomycin, a ligand with a bisintercalative binding mode, was found to induce the greatest KMnO4 reactivity, while Hoechst 33342, a minor groove binder, caused no increase in the oxidation of DNA. The oxidation of echinomycin/DNA complexes containing duplexes with different sequences and lengths was also assessed. Duplexes with thymines closer to the terminal ends of the duplex demonstrated a greater increase in the degree of oxidation than those with thymines in the middle of the sequence. Collisional activated dissociation (CAD) and infrared multiphoton dissociation (IRMPD) experiments were used to determine the site of oxidation based on oligonucleotide fragmentation patterns.