Quantification of Protein-Ligand Interactions by Laser Electrospray Mass Spectrometry.

Laser electrospray mass spectrometry (LEMS) measurement of the dissociation constant (Kd) for hen egg white lysozyme (HEWL) and N,N',N″-triacetylchitotriose (NAG3) revealed an apparent Kd value of 313.2 ± 25.9 µM for the ligand titration method. Similar measurements for N,N',N″,N″'-tetraacetylchitotetraose (NAG4) revealed an apparent Kd of 249.3 ± 13.6 µM. An electrospray ionization mass spectrometry (ESI-MS) experiment determined a Kd value of 9.8 ± 0.6 µM. In a second LEMS approach, a calibrated measurement was used to determine a Kd value of 6.8 ± 1.5 µM for NAG3. The capture efficiency of LEMS was measured to be 3.6 ± 1.8% and is defined as the fraction of LEMS sample detected after merging with the ESI plume. When the dilution is factored into the ligand titration measurement, the adjusted Kd value was 11.3 µM for NAG3 and 9.0 µM for NAG4. The calibration method for measuring Kd developed in this study can be applied to solutions containing unknown analyte concentrations. Graphical Abstract.

Direct Analysis of Proteins from Solutions with High Salt Concentration Using Laser Electrospray Mass Spectrometry.

The detection of lysozyme, or a mixture of lysozyme, cytochrome c, and myoglobin, from solutions with varying salt concentrations (0.1 to 250 mM NaCl) is compared using laser electrospray mass spectrometry (LEMS) and electrospray ionization-mass spectrometry (ESI-MS). Protonated protein peaks were observed up to a concentration of 250 mM NaCl in the case of LEMS. In the case of ESI-MS, a protein solution with salt concentration > 0.5 mM resulted in predominantly salt-adducted features, with suppression of the protonated protein ions. The constituents in the mixture of proteins were assignable up to 250 mM NaCl for LEMS and were not assignable above a NaCl concentration of 0.5 mM for ESI. The average sodium adducts (< n >) bound to the 7+ charge state of lysozyme for LEMS measurements from salt concentrations of 2.5, 25, 50, and 100 mM NaCl are 1.71, 5.23, 5.26, and 5.11, respectively. The conventional electrospray measurements for lysozyme solution containing salt concentrations of 0.1, 1, 2, and 5 mM NaCl resulted in < n > of 2.65, 6.44, 7.57, and 8.48, respectively. LEMS displays an approximately two orders of magnitude higher salt tolerance in comparison with conventional ESI-MS. The non-equilibrium partitioning of proteins on the surface of the charged droplets is proposed as the mechanism for the high salt tolerance phenomena observed in the LEMS measurements. Graphical Abstract ᅟ.

Assessment of Reproducibility of Laser Electrospray Mass Spectrometry using Electrospray Deposition of Analyte.

A nonresonant, femtosecond (fs) laser is employed to desorb samples of Victoria blue deposited on stainless steel or indium tin oxide (ITO) slides using either electrospray deposition (ESD) or dried droplet deposition. The use of ESD resulted in uniform films of Victoria blue whereas the dried droplet method resulted in the formation of a ring pattern of the dye. Laser electrospray mass spectrometry (LEMS) measurements of the ESD-prepared films on either substrate were similar and revealed lower average relative standard deviations for measurements within-film (20.9%) and between-films (8.7%) in comparison to dried droplet (75.5% and 40.2%, respectively). The mass spectral response for ESD samples on both substrates was linear (R2 > 0.99), enabling quantitative measurements over the selected range of 7.0 × 10-11 to 2.8 × 10-9 mol, as opposed to the dried droplet samples where quantitation was not possible (R2 = 0.56). The limit of detection was measured to be 210 fmol. Graphical Abstract ᅟ.

Isolating Protein Charge State Reduction in Electrospray Droplets Using Femtosecond Laser Vaporization.

Charge state distributions are measured using mass spectrometry for both native and denatured cytochrome c and myoglobin after laser vaporization from the solution state into an electrospray (ES) plume consisting of a series of solution additives differing in gas-phase basicity. The charge distribution depends on both the pH of the protein solution prior to laser vaporization and the gas-phase basicity of the solution additive employed in the ES solvent. Cytochrome c (myoglobin) prepared in solutions with pH of 7.0, 2.6, and 2.3 resulted in the average charge state distribution (Zavg) of 7.0 ± 0.1 (8.2 ± 0.1), 9.7 ± 0.2 (14.5 ± 0.3), and 11.6 ± 0.3 (16.4 ± 0.1), respectively, in ammonium formate ES solvent. The charge distribution shifted from higher charge states to lower charge states when the ES solvent contained amines additives with higher gas-phase basicity. In the case of triethyl ammonium formate, Zavg of cytochrome c (myoglobin) prepared in solutions with pH of 7.0, 2.6, and 2.3 decreased to 4.9 (5.7), 7.4 ± 0.2 (9.6 ± 0.3), and 7.9 ± 0.3 (9.8 ± 0.2), respectively. The detection of a charge state distribution corresponding to folded protein after laser vaporized, acid-denatured protein interacts with the ES solvent containing ammonium formate, ammonium acetate, triethyl ammonium formate, and triethyl ammonium acetate suggests that at least a part of protein population folds within the electrospray droplet on a millisecond timescale. Graphical Abstract ᅟ.

Nonresonant, femtosecond laser vaporization and electrospray post-ionization mass spectrometry as a tool for biological tissue imaging.

An ambient mass spectrometry imaging (MSI) source is demonstrated with both high spatial and mass resolution that enables measurement of the compositional heterogeneity within a biological tissue sample. The source is based on nonresonant, femtosecond laser electrospray mass spectrometry (LEMS) coupled to a quadrupole time-of-flight (QTOF) mass analyzer. No matrix deposition and minimal sample preparation is necessary for the source. The laser, translation stage, and mass spectrometer are synchronized and controlled using a customized user interface. Single or multiple laser shots may be applied to each pixel. A scanning rate of 2.0s per pixel is achieved. Measurement of a patterned ink film indicates the potential of LEMS for ambient imaging with a lateral resolution of ∼60µm. Metabolites including sugar, anthocyanins and other small metabolites were successfully mapped from plant samples without oversampling using a spot size of 60×70µm(2). Molecular identification of the detected analytes from the tissue was enabled by accurate mass measurement in conjunction with tandem mass spectrometry. Statistical analysis, non-negative matrix factorization and principle component analysis, were applied to the imaging data to extract regions with distinct and/or correlated spectral profiles.

Ambient Molecular Analysis of Biological Tissue Using Low-Energy, Femtosecond Laser Vaporization and Nanospray Postionization Mass Spectrometry.

Direct analysis of plant and animal tissue samples by laser electrospray mass spectrometry (LEMS) was investigated using low-energy, femtosecond duration laser vaporization at wavelengths of 800 and 1042 nm followed by nanospray postionization. Low-energy (<50 µJ), fiber-based 1042 nm LEMS (F-LEMS) allowed interrogation of the molecular species in fresh flower petal and leaf samples using 435 fs, 10 Hz bursts of 20 pulses from a Ytterbium-doped fiber laser and revealed comparable results to high energy (75-1120 µJ), 45 fs, 800 nm Ti:Sapphire-based LEMS (Ti:Sapphire-LEMS) measurements. Anthocyanins, sugars, and other metabolites were successfully detected and revealed the anticipated metabolite profile for the petal and leaf samples. Phospholipids, especially phosphatidylcholine, were identified from a fresh mouse brain section sample using Ti:Sapphire-LEMS without the application of matrix. These lipid features were suppressed in both the fiber-based and Ti:Sapphire-based LEMS measurements when the brain sample was prepared using the optimal cutting temperature compounds that are commonly used in animal tissue cryosections.

Internal energy deposition for low energy, femtosecond laser vaporization and nanospray post-ionization mass spectrometry using thermometer ions.

The internal energy of p-substituted benzylpyridinium ions after laser vaporization using low energy, femtosecond duration laser pulses of wavelengths 800 and 1042 nm was determined using the survival yield method. Laser vaporization of dried benzylpyridinium ions from metal slides into a buffered nanospray with 75 µJ, 800 nm laser pulses resulted in a higher extent of fragmentation than conventional nanospray due to the presence of a two-photon resonance fragmentation pathway. Using higher energy 800 nm laser pulses (280 and 505 µJ) led to decreased survival yields for the four different dried benzylpyridinium ions. Analyzing dried thermometer ions with 46.5 µJ, 1042 nm pulse-bursts resulted in little fragmentation and mean internal energy distributions equivalent to nanospray, which is attributable to the absence of a two-photon resonance that occurs with higher energy, 800 nm laser pulses. Vaporization of thermometer ions from solution with either 800 nm or 1042 nm laser pulses resulted in comparable internal energy distributions to nanospray ionization.

Increasing protein charge state when using laser electrospray mass spectrometry.

Femtosecond (fs) laser vaporization is used to transfer cytochrome c, myoglobin, lysozyme, and ubiquitin from the condensed phase into an electrospray (ES) plume consisting of a mixture of a supercharging reagent, m-nitrobenzyl alcohol (m-NBA), and trifluoroacetic acid (TFA), acetic acid (AA), or formic acid (FA). Interaction of acid-sensitive proteins like cytochrome c and myoglobin with the highly charged ES droplets resulted in a shift to higher charge states in comparison with acid-stable proteins like lysozyme and ubiquitin. Laser electrospray mass spectrometry (LEMS) measurements showed an increase in both the average charge states (Zavg) and the charge state with maximum intensity (Zmode) for acid-sensitive proteins compared with conventional electrospray ionization mass spectrometry (ESI-MS) under equivalent solvent conditions. A marked increase in ion abundance of higher charge states was observed for LEMS in comparison with conventional electrospray for cytochrome c (ranging from 19+ to 21+ versus 13+ to 16+) and myoglobin (ranging from 19+ to 26+ versus 18+ to 21+) using an ES solution containing m-NBA and TFA. LEMS measurements as a function of electrospray flow rate yielded increasing charge states with decreasing flow rates for cytochrome c and myoglobin.

Direct analysis of intact biological macromolecules by low-energy, fiber-based femtosecond laser vaporization at 1042 nm wavelength with nanospray postionization mass spectrometry.

A fiber-based laser with a pulse duration of 435 fs and a wavelength of 1042 nm was used to vaporize biological macromolecules intact from the condensed phase into the gas phase for nanospray postionization and mass analysis. Laser vaporization of dried standard protein samples from a glass substrate by 10 Hz bursts of 20 pulses having 10 µs pulse separation and <50 µJ pulse energy resulted in signal comparable to a metal substrate. The protein signal observed from an aqueous droplet on a glass substrate was negligible compared to either a droplet on metal or a thin film on glass. The mass spectra generated from dried and aqueous protein samples by the low-energy, fiber laser were similar to the results from high-energy (500 µJ), 45-fs, 800-nm Ti:sapphire-based femtosecond laser electrospray mass spectrometry (LEMS) experiments, suggesting that the fiber-based femtosecond laser desorption mechanism involves a nonresonant, multiphoton process, rather than thermal- or photoacoustic-induced desorption. Direct analysis of whole blood performed without any pretreatment resulted in features corresponding to hemoglobin subunit-heme complex ions. The observation of intact molecular ions with low charge states from protein, and the tentatively assigned hemoglobin α subunit-heme complex from blood suggests that fiber-based femtosecond laser vaporization is a "soft" desorption source at a laser intensity of 2.39 × 10(12) W/cm(2). The low-energy, turnkey fiber laser demonstrates the potential of a more robust and affordable laser for femtosecond laser vaporization to deliver biological macromolecules into the gas phase for mass analysis.