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.     

Interfacing capillary-based separations to mass spectrometry using desorption electrospray ionization

The powerful hybrid analysis method of capillary-based separations followed by mass spectrometric analysis gives substantial chemical identity and structural information. It is usually carried out using electrospray ionization. However, the salts and detergents used in the mobile phase for electrokinetic separations suppress ionization efficiencies and contaminate the inlet of the mass spectrometer. This report describes a new method that uses desorption electrospray ionization (DESI) to overcome these limitations. Effluent from capillary columns is deposited on a rotating Teflon disk that is covered with paper. As the surface rotates, the temporal separation of the eluting analytes (i.e., the electropherogram) is spatially encoded on the surface. Then, using DESI, surface-deposited analytes are preferentially ionized, reducing the effects of ion suppression and inlet contamination on signal. With the use of this novel approach, two capillary-based separations were performed: a mixture of the rhodamine dyes at milligram/milliliter levels in a 10 mM sodium borate solution was separated by capillary electrophoresis, and a mixture of three cardiac drugs at milligram/milliliter levels in a 12.5 mM sodium borate and 12.5 mM sodium dodecyl sulfate solution was separated by micellar electrokinetic chromatography. In both experiments, the negative effects of detergents and salts on the MS analyses were minimized.     

Differentiation of maturity and quality of fruit using noninvasive extractive electrospray ionization quadrupole time-of-flight mass spectrometry

Maturity is an essential factor that determines storage-life and final quality of most fruits and vegetables. Maturity monitoring is thus of paramount importance for postharvest handling and fruit quality regulation. Ideal analytical procedures for maturity investigation require high sensitivity, specificity, and high throughput and should be noninvasive. For the purpose of maturity differentiation, extractive electrospray ionization quadrupole time-of-flight mass spectrometry (EESI-QTOF-MS) is developed for rapid fingerprinting of compounds released from various fruits. Ripening stages of bananas, grapes, and strawberries are successfully differentiated by performing principal component analysis (PCA) of the mass spectral fingerprints of the fruits. Methodological reproducibility was also evaluated experimentally and in terms of PCA clusters. The data indicate that EESI-QTOF-MS is a useful noninvasive tool for rapid investigation and differentiation of maturity and quality of fruits without sample preparation.     

Identifying specific small-molecule interactions using electrospray ionization mass spectrometry

A simple method for establishing whether complexes composed of small molecules detected by electrospray ionization mass spectrometry (ES-MS) originate from specific interactions in solution or nonspecific binding during the ES process is described. The technique, referred to as the nonspecific probe method, exploits the tendency of small molecules to bind nonspecifically to macromolecules during the ES process to establish the presence of specific noncovalent interactions. To implement the method, a macromolecule probe (P(NS)), which does not bind specifically to any of the components present in solution, is added prior to ES-MS analysis. The existence of specific small-molecule complexes is determined from an analysis of the measured distributions of the small molecules bound nonspecifically to P(NS). The principal assumption on which this methodology is based is that nonspecific binding of small molecules and their complexes to P(NS) during ES is a statistical (random) process. A mathematical framework for establishing the presence of specific heterocomplexes is presented. The reliability of the method for distinguishing specific from nonspecific small-molecule interactions is illustrated for peptide-antibiotic and metal ion-ligand interactions in water.     

Tissue imaging using nanospray desorption electrospray ionization mass spectrometry

Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 μm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.     

Selective detection of proteins in mixtures using electrospray ionization mass spectrometry: influence of instrumental settings and implications for proteomics

We studied the effects of electrospray mass spectrometric instrumental settings on the relative and absolute detection of individual proteins in a five-component mixture. Conditions that were effective for a given protein could be very poor for the others, and vice versa, such that to a good approximation it was possible to find conditions for selective detection of individual proteins in a complex mixture without prior analytical separation. Some of these could be rationalized on the basis of the known biophysical properties of the individual proteins. The ability to vary the conditions of a mass spectrometric detection method on-line provides an important degree of freedom for the selective detection, and hence discrimination, of individual proteins and peptides in complex mixtures and has implications in proteomics, in particular with respect to top-down strategies for proteomic characterizations.     

Quantifying ligand binding to large protein complexes using electrospray ionization mass spectrometry

An electrospray ionization mass spectrometry (ESI-MS) method for quantifying protein-ligand complexes that cannot be directly detected by ESI-MS is described. The proxy protein ESI-MS method combines direct ESI-MS binding measurements with competitive protein-ligand binding. To implement the method, a proxy protein (P(proxy)), which interacts specifically with the ligand of interest with known affinity and can be detected directly by ESI-MS, is used to quantitatively monitor the extent of ligand binding to the protein of interest. A mathematical framework for establishing the association constant (K(a)) for protein-ligand binding by the proxy protein ESI-MS method, implemented with a P(proxy) containing a single ligand binding site, is given. A modified form of the proxy protein ESI-MS method, which accounts for real-time changes in ligand concentration, is also described. The reliability of these methods is demonstrated for the interactions between the 180 kDa wildtype homotrimeric tailspike protein of the bacteriophage P22 and its endorhamnosidase point mutant (D392N) with its ligands comprising two and three O-antigen repeats from Salmonella enterica serovar Typhimurium: octasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](2)) and dodecasaccharide ([α-Gal-(1→2)-[α-Abe-(1→3)]-α-Man-(1→4)-α-Rha](3)). A 27 kDa single chain antibody, which binds to both ligands, served as P(proxy). The results of binding measurements performed at 10 and 25 °C are in excellent agreement with K(a) values measured previously using a fluorescence quenching assay.     

Development of submillisecond time-resolved mass spectrometry using desorption electrospray ionization

Reaction kinetics studied by mass spectrometry (MS) has previously been limited to millisecond time resolution. This paper presents the development of a submillisecond time-resolved mass spectrometric method for fast reaction kinetic study, based on the capability of desorption electrospray ionization (DESI) for direct and fast ionization of a high-speed liquid jet stream. The principle underlying this methodology is that two reactant solutions undergo rapid mixing to produce a free liquid jet which is ionized by DESI at different positions corresponding to different reaction times. Due to the high velocity of the liquid jet, high time resolution can be achieved. In this study, the fast reduction reaction of 2, 6-dichlorophenolindophenol (DCIP) and L-ascorbic acid (L-AA) was chosen as an example to demonstrate this concept, and the reaction rate constant was successfully measured with an unprecedented time resolution of 300 μs. The good agreement of the measured value of (116 ± 3) s(-1) with that measured by the stopped-flow optical method (105 ± 2) s(-1) validates the feasibility of such a DESI-MS approach. Unlike classical spectroscopic techniques that require either chromophoric substrates or labeling, MS is a general detector with high chemical specificity. Therefore, this time-resolved DESI-MS method should find wide applications in fast (bio)chemical reaction investigations.     

Sequence confirmation of modified oligonucleotides using chemical degradation, electrospray ionization, time-of-flight, and tandem mass spectrometry

We report the sequencing of highly modified oligonucleotides containing a mixture of 2'-deoxy, 2'-fluoro, 2'-O-methyl, abasic, and ribonucleotides. The passenger and guide strands each containing 48% and 86% of modified nucleotides, respectively, are representative sequences of synthetic short interfering RNAs (siRNAs). We describe herein the sequence confirmation of both strands using a series of robust chemical reactions, followed by analysis via ESI-TOF and ion trap mass spectrometry (ITMS). The following method enables the rapid sequence confirmation of highly modified oligonucleotides.     

Thin-layer chromatography and mass spectrometry coupled using desorption electrospray ionization

Desorption electrospray ionization (DESI) was demonstrated as a means to couple thin-layer chromatography (TLC) with mass spectrometry. The experimental setup and its optimization are described. Development lanes were scanned by moving the TLC plate under computer control while directing the stationary DESI emitter charged droplet plume at the TLC plate surface. Mass spectral data were recorded in either selected reaction monitoring mode or in full scan ion trap mode using a hybrid triple quadrupole linear ion trap mass spectrometer. Fundamentals and practical applications of the technique were demonstrated in positive ion mode using selected reaction monitoring detection of rhodamine dyes separated on hydrophobic reversed-phase C8 plates and reversed-phase C2 plates, in negative ion full scan mode using a selection of FD&C dyes separated on a wettable reversed-phase C18 plate, and in positive ion full scan mode using a mixture of aspirin, acetaminophen, and caffeine from an over-the-counter pain medication separated on a normal-phase silica gel plate.     

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.     

New developments in biochemical mass spectrometry: electrospray ionization

The principles, development, and recent application of electrospray ionization-mass spectrometry (ESI-MS) to biological compounds are reviewed. ESI-MS methods now allow determination of accurate molecular weights for proteins extending to over 50,000, and in some cases well over 100,000. Similar capabilities are being developed for oligonucleotides. The instrumentation used for ESI-MS is briefly described and it is shown that, although ionization efficiency appears to be uniformly high, detector sensitivity may be directly correlated with molecular weight. The use of tandem mass spectrometry (e.g., MS/MS) for extending collision-induced dissociation (CID) methods to the structural studies of large molecules is described. For example, effective CID of various albumin species (molecular weight approximately 66,000) can be obtained, far larger than obtainable for singly charged molecular ions. The combination of capillary electrophoresis, in both free solution zone electrophoresis and isotachophoresis formats, as well as microcolumn liquid chromatography with ESI-MS, provides the capability for on-line separation and analysis of subpicomole quantities of proteins. These and other new developments related to ESI-MS are illustrated by a range of examples. Fundamental considerations suggest even more impressive developments may be anticipated related to detection sensitivity and methods for obtaining structural information.     

Quantification of diacylglycerol species from cellular extracts by electrospray ionization mass spectrometry using a linear regression algorithm

Diacylglycerols (DAGs) play significant roles in both intermediate metabolism and signal transduction. These lipid species are second messengers involved in modulating a plethora of cellular processes. Evaluation of DAG species concentrations has been hampered by the lack of a reliable method for molecular species analysis within a complex mixture of cellular lipids. We describe a new method for quantitative analysis of DAG species from complex biological extracts based on positive mode electrospray ionization mass spectrometry without prior derivatization. Quantification is achieved using internal standards and calibration curves constructed by spiking cell extracts with different concentrations of DAG species containing various acyl chain lengths and degrees of unsaturation. The new mass spectral data processing algorithm incorporates a multiple linear regression model including a factor accountable for possible interactions between experimental preparations and the slope of the curve for the standards, allowing the examinations of the effects of sample origin conditions (such as cell types, phenotypes, etc.) and instrument variability on this slope. Internal standards provide a basis for quantification of 28 DAG molecular species detected in RAW 264.7 cells after stimulation of a G-protein coupled receptor with platelet activating factor. This method displays excellent reproducibility over the established range of concentrations with variations of < or =10% and is highly sensitive with a detection limit of 0.1-0.4 pmol/microL depending upon acyl chain composition. We have shown differential effects on various DAGs in response to a ligand which illustrates the importance of examining lipids at the molecular species level rather than as a single homogeneous entity.     

Study of electrochemical reactions using nanospray desorption electrospray ionization mass spectrometry

The combination of electrochemistry (EC) and mass spectrometry (MS) is a powerful analytical tool for studying mechanisms of redox reactions, identification of products and intermediates, and online derivatization/recognition of analytes. This work reports a new coupling interface for EC/MS by employing nanospray desorption electrospray ionization, a recently developed ambient ionization method. We demonstrate online coupling of nanospray desorption electrospray ionization MS with a traditional electrochemical flow cell, in which the electrolyzed solution emanating from the cell is ionized by nanospray desorption electrospray ionization for MS analysis. Furthermore, we show first coupling of nanospray desorption electrospray ionization MS with an interdigitated array (IDA) electrode enabling chemical analysis of electrolyzed samples directly from electrode surfaces. Because of its inherent sensitivity, nanospray desorption electrospray ionization enables chemical analysis of small volumes and concentrations of sample solution. Specifically, good-quality signal of dopamine and its oxidized form, dopamine o-quinone, was obtained using 10 μL of 1 μM solution of dopamine on the IDA. Oxidation of dopamine, reduction of benzodiazepines, and electrochemical derivatization of thiol groups were used to demonstrate the performance of the technique. Our results show the potential of nanospray desorption electrospray ionization as a novel interface for electrochemical mass spectrometry research.     

Quantifying protein-protein interactions within noncovalent complexes using electrospray ionization mass spectrometry

Several electrospray-mass spectrometry (ESI-MS)-based methods are available for determining the constant of association (K(a)) between a protein and a small ligand, but current MS-based strategies are not fully adequate for measuring K(a) of protein-protein interactions accurately. We expanded the application of ESI-MS-based titration to determine the strength of noncovalent interactions between proteins, forming a complex. Taking into account relative response factors (probability of being ionized, transmitted, and detected), we determined K(a) values of an equilibrium between dimers and tetramers at three different pH values (6.8, 3.4, and 8.4). We investigated the association of the lectin concanavalin A, whose dimer-tetramer ratio in the gas phase is affected by solution concentration and by pH. To calculate the constants of association in solution, we also utilized isothermal titration calorimetry (ITC) for a comparison with MS-based titration. At pH 6.8 and pH 8.4, the K(a) values measured by MS and by ITC were in agreement. ITC results allowed us to restrain the response factor to a value close to 4. At pH 3.4, we were able to measure the K(a) only by MS, but not by ITC because of limited sensitivity of calorimetry. Our investigation illustrates the great potential MS for calculating the binding strength of protein-protein interactions within noncovalent complexes. The main advantages of MS over ITC are its sensitivity (i.e., the required amount of sample is >100 times less than the one necessary for ITC), and the possibility to obtain precise information on composition of protein complexes, their stoichiometry, their subunit interactions, and their assembly pathway. Compared to previous investigations, our study shows the strong influence of response factors on determining accurate protein-protein association constants by MS.     

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.     

Alkylating tryptic peptides to enhance electrospray ionization mass spectrometry analysis

A major limitation of mass spectrometry-based proteomics is inefficient and differential ionization during electrospray ionization (ESI). This leads to problems such as increased limits of detection and incomplete sequence coverage of proteins. Incomplete sequence coverage is especially problematic for analyses that require the detection and identification of specific peptides from a protein, such as the analysis of post-translational modifications. We describe here the development and use of aldehyde-based chemistry for the alkylation of peptide primary amines to increase peptide hydrophobicity, providing increased ionization efficiency and concomitant signal enhancement. When employed to modify the peptide products of protein tryptic digests, increased sequence coverage is obtained from combined modified and unmodified digests. To evaluate the utility of alkylation of peptides for selected reaction monitoring (SRM) assays, we alkylated a peptide from the protein Oct4, known to play a role in regulating stem cell differentiation. Increased chromatographic retention and ionization efficiency is observed for the alkylated Oct4 peptide compared to its unmodified form.     

Simple coupling of gas chromatography to electrospray ionization mass spectrometry

A simple method for direct coupling of gas chromatography (GC) with electrospray ionization mass spectrometry (ESI/MS) has been developed. The outlet of the GC capillary column was placed between the ESI needle and the atmospheric pressure ionization (API) source of a mass spectrometer. The ionization occurs via dissolution of neutral compounds into the charged ESI droplet followed by ion evaporation or via a gas-phase proton transfer reaction between a protonated solvent molecule and an analyte. The mass spectra of organic volatile compounds showed abundant protonated molecules with little fragmentation, being very similar to those produced by normal liquid ESI. The quantitative performance of the system was evaluated by determining the limit of detection (LOD), linearity ( r (2)), and repeatability (RSD). The GC-ESI/MS method was shown to be stable, providing high sensitivity and good quantitative performance.     

Interfaces to connect thin-layer chromatography with electrospray ionization mass spectrometry

This study has developed two interfaces to connect small-size thin-layer chromatography (TLC) with electrospray ionization mass spectrometry (ES-MS) for the continuous analysis of organic mixtures. The interfaces are (1) two bound optical fibers inserted into the C18-bonded particles at the exit of a small TLC channel and (2) a small commercial TLC strip with a sharpened tip. A reservoir continuously supplied a makeup solution to the tip of the TLC channel. The high voltage required for electrospray ionization was introduced into the makeup solution or mobile phase through a Pt wire, and electrospray was generated at the tip of the bonded optical fibers and at the sharp end of the TLC strip. Since small-size TLC channels were used, the elution time was short and less than 0.2 microL of the sample solution and 200 microL of the eluting solvent were required. Organic mixtures were separated successfully and detected on-line using the TLC/ES-MS techniques.     

On-chip proteolytic digestion and analysis using "wrong-way-round" electrospray time-of-flight mass spectrometry

Rapid protein digestion and analysis using a hybrid microchip nanoelectrospray device and time-of-flight mass spectrometry detection are reported. The device consists of a planar glass chip with microfabricated channels coupled to a disposable nanospray emitter. Reactions between substrate and enzyme (trypsin), mixed off-chip and then immediately loaded into a sample reservoir on the device, are monitored in real time following the onset of electrospray. Protein cleavage products are determined at the optimum pH for generating tryptic fragments, directly from the digestion buffer using "wrong-way-round" electrospray, i.e., monitoring (MH)+ ions from basic solutions. Intense tryptic peptide ions are observed within a few minutes following sample loading on the microchip. Proteins were identified from low femtomole or even attomole quantities of analyte/spectrum using peptide mass fingerprinting, loading 0.1-2 pmol/microL of sample on the chip. The sequence coverage for analyzed proteins ranged from 70 to 95%. The rapid analysis of human hemoglobin is demonstrated using the technique.