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.     

A capillary mixer with adjustable reaction chamber volume for millisecond time-resolved studies by electrospray mass spectrometry

A novel continuous-flow apparatus for on-line kinetic studies of (bio)chemical solution-phase processes by electrospray ionization mass spectrometry (ESI-MS) is described. The device is based on two concentric capillaries. Fluid is released from the inner capillary into the intercapillary space, where it mixes with solution flowing through the outer capillary, thus initiating the reaction of interest. Gas-phase analyte ions are formed near the tip of the outer capillary by pneumatically assisted ESI. This setup allows the mixer to be placed directly within the ion source, thus providing a minimal dead volume of ~8 nL. Time-resolved data can be recorded in both "spectral" and "kinetic" modes. In the former case, the position of the inner capillary is fixed at various points, such that entire mass spectra can be recorded for selected reaction times. For experiments in kinetic mode, the mass spectrometer monitors the signal intensity at selected m/z values, while the inner capillary is continuously pulled back, thus providing intensity-time profiles for specific reactive species. A theoretical framework is developed that allows the measured kinetics to be analyzed by taking into account the effects of laminar flow within the reaction capillary. Failure to take these effects into account results in erroneous rate constants. Studies on the demetalation kinetics of chlorophyll reveal that the apparatus can reliably measure rate constants up to at least 100 s-1. This represents a substantial improvement over previous ESI-MS-based kinetic methods. Spectral mode experiments on the refolding of ubiquitin show the changing proportions of denatured and tightly folded protein subpopulations in solution. When monitored in kinetic mode, the refolding process was found to proceed with a rate constant of 5.2 s-1.     

Signal response of coexisting protein conformers in electrospray mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is a commonly used tool for characterizing conformational changes of proteins in solution. Different conformations can be distinguished on the basis of their ESI charge state distributions. ESI-MS studies carried out under semidenaturing conditions result in bi- or multimodal distributions that reflect the presence of coexisting conformers. This study explores whether the concentration ratios of these species in solution are reflected in the measured ion intensities. Experiments on two model proteins, lysozyme and myoglobin, reveal that non-native polypeptide chains tend to result in a much stronger signal response than natively folded species. The measured ion intensity ratios can differ from the actual concentration ratios by as much as 2 orders of magnitude. It is proposed that the higher ionization efficiency of unfolded proteins is due to their partially hydrophobic character, which results in a larger surface activity and facilitates protein transfer into ion-producing progeny droplets. Conversely, natively folded proteins have a lower affinity for the air/liquid interface, such that ionization of these conformers is suppressed. The extent of ion suppression is strongly dependent on the experimental conditions such as flow rate and protein concentration, which determine if ESI occurs in a charge deficient or a charge surplus regime. These aspects should be taken into account for the design of ESI-MS-based protein folding experiments and for studies that use ion intensity ratios for the determination of protein-ligand binding affinities.     

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.     

Neutral loss analysis in MALDI-MS using an ion-gate scanning mode.

Matrix-assisted laser desorption/ionization reflector time-of-flight (MALDI-reTOF) and electrospray ionization (ESI) mass spectrometry (MS) have become essential tools for the characterization of peptides and proteins. Whereas ESI in combination with a triple quadrupole analyzer allows product ion, precursor ion, and neutral loss analyses, MALDI-reTOF instruments can only be used to record product ion spectra based on the in-source or postsource decay (PSD). We describe a new method to perform neutral loss analyses in MALDI-reTOF instruments in a manner that identifies posttranslationally modified peptides and furthermore retrieves sequence information from peptides. The method is based on the selection of ions in a small time interval to record only signals within the corresponding mass interval. By stepping the time interval through the complete mass range, we obtained a spectrum of stable ions by combining the signals of all individually recorded time intervals. This method furthermore permits PSD fragment ions to be identified, since they reach the detector earlier than the stable ions transmitted in the chosen time interval. The neutral loss analysis were calculated by correlating the PSD fragment ions to the corresponding parent ion detected in this time interval. Moreover, this MALDI-MS mode increased the number of detectable signals in complex peptide mixtures and the signal-to-noise ratio.     

Analytical characterization of the electrospray ion source in the nanoflow regime

A detailed characterization of a conventional low-flow electrospray ionization (ESI) source for mass spectrometry (MS) using solution compositions typical of reversed-phase liquid chromatography is reported. Contrary to conventional wisdom, the pulsating regime consistently provided better ESI-MS performance than the cone-jet regime for the interface and experimental conditions studied. This observation is supported by additional measurements showing that a conventional heated capillary interface affords more efficient sampling and transmission for the charged aerosol generated by a pulsating electrospray. The pulsating electrospray provided relatively constant MS signal intensities over a wide range of voltages, while the signal decreased slightly with increasing voltage for the cone-jet electrospray. The MS signal also decreased with increasing emitter-interface distance for both pulsating and cone-jet electrosprays due to the expansion of the charged aerosol plume. At flow rates below 100 nL/min, the MS signal increased with increasing flow rate due to increased number of gas-phase ions produced. At flow rates greater than 100 nL/min, the signal reached a plateau due to decreasing ionization efficiency at larger flow rates. These results suggest approaches for improving MS interface performance for low-flow (nano- to micro-) electrosprays.     

Ultra high-performance liquid chromatography-tandem mass spectrometry for the simultaneous analysis of asparagine, sugars, and acrylamide in Maillard reactions

We developed an automated microwave digestion labstation (MDL) combined with ultra high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method under the control of positive-negative ion switching as a robust kinetic study tool for rapid and simultaneous quantification of asparagine, glucose, fructose, and acrylamide in Maillard reaction products. Maillard reactions were conducted in a potato model via MDL. The two-step simple pretreatment procedures included the addition of isotope internal standards (15)N(2)-asparagine, (13)C(6)-glucose, and D(3)-acrylamide, followed by appropriate dilution with the mobile phase and filtration. Analytes were separated on a Hypercarb column and monitored by MS/MS. Study of matrix effects indicated Maillard reaction products induce an ionization suppression of both positive and negative precursor ions, but quantitative results are corrected through the use of isotopically labeled internal standards. Using this method, the limit of detection (LOD) and limit of quantification (LOQ) ranges of all analytes were calculated as 0.04-0.6 and 0.1-1.1 μM, respectively. Excellent repeatability (RSD < 9.6%) and acceptable within-laboratory reproducibility (RSD < 9.2%) substantially supported the use of this method for sample analysis. The present kinetic tools, with 10-50 min mimic of Maillard reactions and short instrumental run time (5.5 min per sample), were successfully validated and applied to simultaneous determination of acrylamide and its precursors and intermediates during Maillard reactions and kinetic elucidation. Furthermore, current tools of MDL combined with simple sample treatment procedures and UHPLC-MS/MS analysis reduce sample analysis time and labor in the kinetic study.     

A digital microfluidic system for the investigation of pre-steady-state enzyme kinetics using rapid quenching with MALDI-TOF mass spectrometry

A digital microfluidic system based on electrowetting has been developed to facilitate the investigation of pre-steady-state reaction kinetics using rapid quenching and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The device consists of individually addressable electrodes arranged to allow the combination of liquid droplets at well-defined time intervals and an integrated, electrohydrodynamically driven mixer. The device combines two droplets to initiate a reaction, then, with precise timing, combines a third droplet to quench the reaction, and finally combines a fourth droplet to form a matrix. Improvements to throughput when compared to traditional laboratory-scale methods, and previous MALDI-TOF MS digital microfluidic systems, were made. The device was tested against a model protein tyrosine phosphatase system, and results agreed well with published data. The system therefore allows for the analysis of reaction kinetics that were previously too rapid to analyze using MALDI-TOF MS.     

Monitoring of real-time streptavidin-biotin binding kinetics using droplet microfluidics

Rapid kinetic measurements are important in understanding chemical interactions especially for biological molecules. Herein, we present a droplet-based microfluidic platform to study streptavidin-biotin binding kinetics with millisecond time resolution. With integration of a confocal fluorescence detection system, individual droplets can be monitored and characterized online to extract kinetic information. Using this approach, binding kinetics between streptavidin and biotin were observed via fluorescence resonance energy transfer. The binding rate constant of streptavidin and biotin was found to be in a range of 3.0 x 10 (6)-4.5 x 10 (7) M (-1) s (-1).     

Ratiometric pulse-chase amidination mass spectrometry as a probe of biomolecular complex formation

Selective chemical modification of protein side chains coupled with mass spectrometry is often most informative when used to compare residue-specific reactivities in a number of functional states or macromolecular complexes. Herein, we develop ratiometric pulse-chase amidination mass spectrometry (rPAm-MS) as a site-specific probe of lysine reactivities at equilibrium using the Cu(I)-sensing repressor CsoR from Bacillus subtilis as a model system. CsoR in various allosteric states was reacted with S-methyl thioacetimidate (SMTA) for pulse time, t, and chased with excess of S-methyl thiopropionimidate (SMTP) (Δ = 14 amu), quenched and digested with chymotrypsin or Glu-C protease, and peptides were quantified by high-resolution matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry and/or liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). We show that the reactivities of individual lysines from peptides containing up to three Lys residues are readily quantified using this method. New insights into operator DNA binding and the Cu(I)-mediated structural transition in the tetrameric copper sensor CsoR are also obtained.     

Enzyme kinetics by directly imaging a porous silicon microfluidic reactor using desorption/ionization on silicon mass spectrometry

Enzyme kinetics were obtained in a porous silicon microfluidic channel by combining an enzyme and substrate droplet, allowing them to react and deposit a small amount of residue on the channel walls, and then analyzing this residue by directly ionizing the channel walls using a matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) laser source. The porous silicon of the channel walls functions in a manner analogous to the matrix in MALDI-MS, and is referred to as a desorption/ionization on silicon mass spectrometry (DIOS-MS) target when used in this configuration. Mass spectrometry signal intensity of substrate residue correlates with relative concentration, and position in the microchannel correlates with time, thus allowing determination of kinetic parameters. The system is especially suitable for initial reaction velocity determination. This microreactor is broadly applicable to time-resolved kinetic assays as long as at least one substrate or product of the reaction is ionizable by DIOS-MS.     

Acrylamide polymerization kinetics in gel electrophoresis capillaries. A Raman microprobe study.

The formation of 3.5% T, 3.3% C cross-linked polyacrylamide is monitored in 75-microns-i.d. electrophoresis capillaries by Raman microprobe spectroscopy. The disappearance of the acrylamide 1292-cm-1 band is followed with 60-s time resolution for 30 min, and 2-4 min resolution for up to 10 h. Polymerization is 98% complete in 1.5 h and greater than 99% complete after 2 h. In the 900-1700-cm-1 region no bands attributable to cross-linking are observable. Reaction in the capillary follows second-order kinetics. The reaction is faster in the bulk system because heat dissipation is not sufficient to maintain a constant temperature.     

Direct interfacing of high-speed countercurrent chromatography to frit electron ionization, chemical ionization, and fast atom bombardment mass spectrometry.

Direct interfacing of analytical high-speed countercurrent chromatography (HSCCC) to mass spectrometry (MS) was demonstrated for the first time, and its performance was evaluated in terms of chromatography and mass spectrometry. HSCCC/MS interface was based upon Frit electron ionization (EI), chemical ionization (CI), and fast atom bombardment (FAB). Separations were conducted by newly developed HSCCC-4000 with a 2.5-cm revolutional radius and 0.3 mm or 0.55 mm i.d. multilayer coiled column which is capable of operating at a maximum speed of 4000 rpm. To demonstrate the potential capability of HSCCC/frit MS, three indole auxin mixtures, two mycinamicin (macrolide antibiotics) mixtures, and a colistin complex (peptide antibiotics) were analyzed under HSCCC/frit EI, CI, and FABMS conditions, respectively. The data obtained indicated that interfacing to frit/MS does not adversely affect the chromatographic resolution and mass spectra provide structural information. The HSCCC system interfaced with a frit-equipped mass spectrometer will offer a new dimension in the separation of biologically important substances.     

Single hair cocaine consumption monitoring by mass spectrometric imaging

Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) was used to image the distribution of cocaine and its metabolites in intact single hair samples from chronic users down to a concentration of 5 ng/mg. Acquisitions were performed in rastering mode, at a speed of 1 mm/s and in the selected reaction monitoring (SRM) mode on a MALDI triple quadrupole linear ion trap fitted with a high repetition rate laser (1 kHz). Compared to traditional methods based on LC-MS/MS or GC-MS(/MS) which require to segment the hair to obtain spatial resolution, MALDI-MSI, with a straightforward sample preparation beforehand, allowed obtaining a spatial resolution of 1 mm and thus the chronological information about cocaine consumption contained in a single intact hair over several months could be monitored. The analysis time of an intact single hair sample of 6 cm is approximately of 6 min. Cocaine and its metabolites benzoylecgonine, ethylcocaine, and norcocaine were investigated in nine sets of hair samples for forensic purposes. The analyses were accomplished by spraying α-cyano-4-hydroxycinnamic acid (CHCA), 4-chloro-α-cyano-cinnamic acid (Cl-CCA), or (E)-2-cyano-3-(naphthalen-2-yl)acrylic acid (NpCCA) as MALDI matrices. We also propose a rapid strategy for sensitive confirmatory analyses with both MS/MS and MS(3) experiments performed directly on intact hair samples. Since only part of the hair strand is analyzed, additional analyses are possible at any time on the remaining hair from the strand.     

Colloidal graphite-assisted laser desorption/ionization MS and MS(n) of small molecules. 2. Direct profiling and MS imaging of small metabolites from fruits

Due to a high background in the low-mass region, conventional MALDI is not as useful for detecting small molecules (molecular masses <500 Da) as it is for large ones. Also, spatial inhomogeneity that is inherent to crystalline matrixes can degrade resolution in imaging mass spectrometry (IMS). In this study, colloidal graphite was investigated as an alternative matrix for laser desorption/ionization (GALDI) in IMS. We demonstrate its advantages over conventional MALDI in the detection of small molecules such as organic acids, flavonoids, and oligosaccharides. GALDI provides good sensitivity for such small molecules. The detection limit of fatty acids and flavonoids in the negative-ion mode are in the low-femtomole range. Molecules were detected directly and identified by comparing the MS and MS/MS spectra with those of standards. Various fruits were chosen to evaluate the practical utility of GALDI since many types of small molecules are present in them. Distribution of these small molecules in the fruit was investigated by using IMS and IMS/MS.     

Direct analysis in real time for reaction monitoring in drug discovery

Direct analysis in real time (DART) is a novel ionization technique that provides for the rapid ionization of small molecules under ambient conditions. In this study, several commercially available drugs as well as actual compounds from drug discovery research were examined by LC/UV/ESI-MS and DART interfaced to a quadrupole mass spectrometer. For most compounds, the molecular ions observed by ESI-MS were observed by DART/MS. DART/MS was also studied as a means to quickly monitor synthetic organic reactions and to obtain nearly instantaneous molecular weight confirmations of final products in drug discovery. For simple, synthetic organic transformations, the trends in the intensities of the mass spectral signals for the reactant and product obtained by DART/MS scaled closely with those of the diode array or the total ion chromatogram obtained by LC/UV/ESI-MS. In summary, DART is a new tool that complements electrospray ionization for the rapid ionization and subsequent mass spectral analysis of compounds in drug discovery.     

Real-time, on-line characterization of diesel generator air toxic emissions by resonance-enhanced multiphoton ionization time-of-flight mass spectrometry

The laser-based resonance-enhanced multiphoton ionization time-of-flight mass spectrometry (REMPI-TOFMS) technique has been applied to the exhaust gas stream of a diesel generator to measure, in real time, concentration levels of aromatic air toxics. Volatile organic compounds, as well as several polycyclic aromatic hydrocarbons were detected in the concentration range of 10-200 ppb in the steady-state diesel generator exhaust. The results were verified and compared with conventional extractive sampling and analytical techniques using gas chromatography/mass spectrometry (GC/MS). The high isomer selectivity of the REMPI-TOFMS instrument provided data for individual xylene isomers that are otherwise (partially) coeluting in standard GC/MS analyses. Good agreement was observed between results for volatile and semivolatile organic compounds obtained with REMPI-TOFMS and conventional extractive sampling. Transient events, such as cold start-ups of the diesel generator, resulted in sharp (less than 15 s) peak emissions that were, for benzene, up to a factor of 90 higher than the predominately constant concentrations observed during steady-state operation; warm restarts resulted in lower peak concentrations by a factor of 2.5. These fast transient emissions are only detectable using a real-time approach (1-s resolution) as demonstrated here using REMPI-TOFMS.     

Multidimensional analysis of single algal cells by integrating microspectroscopy with mass spectrometry

We demonstrate a facile label-free approach for performing multidimensional chemical analysis on individual single-cell organisms by combining optical, fluorescence, and Raman microspectroscopy with matrix-free laser desorption/ionization mass spectrometry (MS). Single unicellular algae are seeded on a bare stainless steel plate and analyzed microspectroscopically. This provides information on the content and distribution of photoactive species, such as β-carotene, as well as chlorophyll and other components of the photosynthetic apparatus. Exactly the same cells are then analyzed by mass spectrometry in the negative ion mode. Phospholipid species are readily ionized by laser desorption/ionization of intact cells, without the need for an auxiliary matrix. This not only facilitates sample preparation but also preserves high spatial resolution and high sensitivity. Using this method, we were able to study the content and arrangement of proplastids and photosystem components, as well as the amounts of various phospholipid species in individual algal cells. The methodology can be used in the fundamental biological studies on these unicellular organisms, which require information on the internal structure as well as the chemical composition of individual cells.     

Solid-state stability studies of crystal form of tebipenem

The aim of this study was to determine the kinetic and thermodynamic parameters of tebipenem degradation in the solid state. The process was analyzed based on the results obtained by a high performance liquid chromatography (HPLC) method using ultraviolet diode-array detector (DAD)/electrospray ionization tandem mass spectrometry (Q-TOF-MS/MS), Fourier transform infrared spectroscopy (FT-IR) and Raman spectroscopic (RS) studies. In dry air, the degradation of tebipenem was a first-order reaction depending on the substrate concentration while at an increased relative air humidity tebipenem was degraded according to the kinetic model of autocatalysis. The thermodynamic parameters: energy of activation (E_a), enthalpy (ΔH^≠a) and entropy (ΔS^≠a) of tebipenem degradation were calculated. Following a spectroscopic analysis of degraded samples of tebipenem, a cleavage of the β-lactam bond was proposed as the main degradation pathway, next confirmation using HPLC-Q-TOF-MS/MS method.