Introduction

The analysis of biomolecules using electrospray ionization-mass spectrometry (ESI-MS) is often hindered by matrix effects due to the presence of phosphate buffers, urea, or inorganic salts [1, 2]. ESI-MS measurements of proteins in the presence of sodium chloride (NaCl) reveal extensive sodium ion adduction even for a low millimolar concentration [3]. The presence of NaCl also results in a decrease in both the stability of the electrospray and the yield of protonated ions [4]. The adduction of sodium ions to protein molecules distributes the signal over multiple mass-to-charge (m/z) peaks, reducing the signal-to-noise ratio of each protein feature and broadening the mass spectral peaks, resulting in a decrease in accuracy for mass measurement.

Since salt is ubiquitous in many biological samples, several strategies have been developed for desalting prior to ESI-MS analysis including nanoparticle-based microextraction [5], microdialysis [6], liquid chromatography [7], and ion-exchange chromatography [8, 9]. However, removal of salts can affect the structure of protein complexes. For example, a sigma activator protein (NtrC4) from Aquifex aeolicus requires millimolar concentrations of Mg2+, BeF3−, and adenosine 5′-diphosphate (ADP) sodium salt to form an active hexamer [10, 11]. Desalting processes that require multiple buffer exchange steps will denature the hexamer and may also produce a spurious signal in the ESI analysis due to inefficient removal of detergents [12].

Decoupling the sampling and electrospray ionization processes has enabled analysis of analytes from samples containing high salt concentrations. Fused-droplet electrospray ionization-mass spectrometry (FD-ESI-MS) involves ultrasonic nebulization of the sample solution to produce a fine aerosol which is then combined with electrospray-generated charged droplets [3]. Although this method showed a significantly higher salt tolerance in comparison with conventional electrospray (1.70 versus 0.17 M NaCl), interaction of protein molecules with electrosprayed methanol droplets containing 1% acetic acid denatures protein and shifts the charge state distribution (CSD) to higher charge in comparison with native state measurements. Probe electrospray ionization (PESI) uses a wire as both the sampling probe and electrospray emitter and has been used to detect biomolecules from solutions with high salt concentration [1]. The analysis of myoglobin using PESI displayed a salt tolerance, or ability to detect protein without ion suppression, of up to 250 mM in comparison with nano-electrospray ionization (nano-ESI) which has a salt tolerance of 50 mM. The high salt tolerance for PESI and nano-ESI is likely due to the formation of higher charge states given the fact that sodium ion adduction occurs mostly for low charge states [4, 13, 14]. Selective sampling of analytes rather than salt from a solution has been proposed to enhance detection in the PESI experiment in comparison with nano-ESI where the entire solution within the capillary is subjected to the nano-spray process resulting in analyte signal suppression given the higher salt concentration [1]. The investigation of drug mixtures (cocaine and diacetylmorphine) using desorption electrospray ionization (DESI) showed higher salt tolerance in comparison with ESI [15]. The salt tolerance in DESI was investigated by changing the sample substrate such as paper, polytetrafluoroethylene (PTFE), and a variety of coated glasses. The salt tolerance was dependent upon the surface being used, and PTFE was the optimal substrate for reducing ion suppression effects at higher salt concentration. A raw urine sample spiked with atrazine and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX) was successfully examined using extractive electrospray ionization (EESI), eliminating clean-up steps used to remove the matrix effects [16]. The stable signal intensity in EESI during salt-rich biological sample analysis was attributed to the difference in surface partitioning of polar and non-polar analytes. The non-polar analytes tend to compete for the surface while the polar analytes and salts are stabilized by increased solvation in the droplet interior.

Two-step ESI techniques decouple the vaporization and ionization process into separate processes to allow measurements that cannot be performed using either ESI or matrix-assisted laser desorption ionization (MALDI) alone. These methods include electrospray laser desorption ionization (ELDI) [17], matrix-assisted laser desorption electrospray ionization (MALDESI) [18, 19], and laser ablation electrospray ionization (LAESI) [20]. ELDI has been previously used to detect proteins dissolved in various solutions containing buffers, organic and inorganic salts, and strong acids and bases without undergoing solvent extraction, filtration, dialysis, or chromatographic separation [17]. The higher salt and detergent tolerance of ELDI in comparison with ESI-MS enabled the direct analysis of proteins from solution containing sodium dodecyl, octyl phenol ethoxylate, tetrabutyl ammonium bromide, etc. [17, 21].

The use of laser electrospray mass spectrometry (LEMS) has enabled the quantitative analysis of complex mixtures without sample pre-processing at atmospheric pressure [22, 23]. LEMS couples non-resonant femtosecond (fs) laser vaporization (1013 W/cm2) with an electrospray ionization source for postionization. The successful detection of 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) spiked into whole blood sample and the detection of lipids and proteins from reduced-fat milk sample demonstrate that LEMS is capable of detecting analytes from complex mixtures with no pre-processing [24]. In addition, LEMS measurements on small molecule mixtures [25] and multicomponent protein mixtures [26] have shown that LEMS enables quantitative measurements up to ~ 2.5 orders of magnitude and over 4 orders of magnitude higher in concentration, respectively, in comparison with conventional electrospray where quantitative measurement of mixtures is not possible. The non-equilibrium partitioning of analyte on the surface of the charged droplet is likely responsible for the quantitative capability [25, 26]. This suggests that LEMS may be utilized to significantly reduce ion suppression effects in comparison with conventional ESI analysis, in particular for biomolecules from solution containing high salt concentration because the salt will partition into the droplet interior while the protein remains on the surface where excess charge resides.

In this study, the mass spectral features of either a single protein (lysozyme) or a mixture of the proteins lysozyme, cytochrome c, and myoglobin as a function of salt concentration are compared using LEMS and ESI-MS. The amount of protonated protein is measured as a function of salt concentration for both LEMS and ESI, and the average number of sodium adducts (< n >) bound to the 7+ charge state of lysozyme is calculated. The ability to identify protein components in a mixture using LEMS and ESI is compared as a function of salt concentration. Finally, a mechanism is presented for reduced salt adduction in the LEMS measurement in comparison with ESI.

Materials and Methods

Sample Preparation

Lysozyme, cytochrome c, myoglobin, and ammonium acetate (Sigma Aldrich, St. Louis, MO) were prepared in HPLC-grade water (Fisher Scientific, Pittsburgh, PA) to yield the final concentration of 1.0 × 10−3 M. A 1.0 M stock solution of sodium chloride was prepared in HPLC-grade water (Fisher Scientific, Pittsburgh, PA). For ESI measurements, an aliquot of the stock solution (single protein or protein mixtures) was diluted into 10 mM aqueous ammonium acetate to yield a final protein concentration of 1.0 × 10−5 M with the salt concentration ranging from 0.1 to 5.0 mM. For LEMS measurements, an aliquot of the stock solution (either individual or the protein mixtures) was diluted into water to yield a final protein concentration of 2.0 × 10−4 M with the salt concentration ranging from 2.5 to 250 mM. A 10 μL aliquot of the diluted protein solution was spotted onto a stainless steel plate and then subjected to laser vaporization into the electrospray (ES)-charged droplet stream consisting of aqueous ammonium acetate.

Laser Vaporization

A Ti:sapphire laser oscillator (KM Laboratories, Inc., Boulder, CO) seeded a regenerative amplifier (Coherent, Inc., Santa Clara, CA) that delivered 75 fs, 0.6 mJ laser pulses centered at 800 nm. The laser, operated at 10 Hz to couple with the ES ion source, was focused to a spot size of ~ 250 μm in diameter with an incident angle of 45° with respect to the sample using a 16.9-cm focal length lens, with an approximate intensity of 1 × 1013 W/cm2. The steel sample plate was biased to − 2.0 kV to compensate for the distortion of electric field between the capillary inlet and the needle caused by the sample stage. Aqueous protein sample (10 μL) deposited onto a steel substrate was vaporized by the laser pulse allowing for capture and ionization by an ES plume traveling perpendicular to the vaporized material.

Mass Spectrometry and Data Analysis

The non-resonant femtosecond laser pulse transfers the analyte into the gas phase for capture and ionization in an ES plume at atmospheric pressure. The flow rate for ES solvent was set at 2 μL/min by a syringe pump (Harvard Apparatus, Holliston, MA). The ESI needle was 6.4 mm above and parallel to the sample stage and was approximately 6.4 mm in front of the capillary entrance. The ES needle was maintained at ground while the inlet capillary was biased to − 4.5 kV to operate in positive ion mode. The postionized analytes were dried before entering the inlet capillary by countercurrent nitrogen gas at 200 °C flowing at 4 L/min. The charged sample was mass analyzed using a microTOF-Q II mass spectrometer (Bruker Daltonics, Billerica, MA).

Results and Discussion

Detection of Protein/Protein Mixtures from Solutions with Varying Salt Concentrations

The LEMS and ESI measurements for lysozyme are shown in Figure 1 for salt concentrations ranging from 0 to 100 mM and 0 to 5 mM, respectively. Higher lysozyme concentration was chosen for LEMS (200 μM) in comparison with ESI (10 μM) analysis as only about 3.6% of the vaporized analytes are captured in the electrospray plume, resulting in lower concentration of the protein being analyzed. The LEMS measurement for lysozyme vaporized without NaCl, shown in Figure 1a, reveals a narrow range of low charge states ranging from 6+ to 9+. The predominance of the 8+ charge state in Figure 1b–e indicates that the native state of lysozyme was preserved when vaporized from a solution with salt concentration up to 100 mM. Salt adduction to the protein ions for LEMS increases with decreasing charge. The 9+ feature reveals no adduction in any of the spectra, minimal adduction in 8+, and significant adduction in the 7+ through 4+ species (see Figure 1b–e; also shown in Supplementary Figure S1c–e, measurements taken at collision potential of 70 eV). Previous ELDI measurement of cytochrome c from solution containing high salt concentration using a denaturing electrospray solvent (acidic methanol solution) resulted in protein CSD peaked at 15+ with minimal salt adduction [21]. The increased salt tolerance for ELDI measurement is likely due to the formation of higher charge states where salt adduction to the protein molecule ion is minimal. Salt adduction to lower charge (folded) states is likely due to the increased salt concentration within the ES droplet during desolvation process in the charge residue mechanism [4]. The increasing salt concentration facilitates non-specific pairing of Na+ and Cl− with the protein ions. Sodium adducts are evident at higher collision potential (~ 70 V) while a lower potential of ~ 5 V results in a series of n(Na − H) and m(Cl + H) peaks in the mass spectrum. In the case of higher charge states, Konermann has proposed that the unfolded protein resides at the surface of the droplet, enabling charge ejection during the desolvation process when the salt concentration is lower [4]. The increase in local kinetic energy for the 8+ charge state in comparison with the 7+ and 6+ charge states (for a given collision potential) may also remove salt from the protein molecule through collisional processes resulting in the lower Na+ adduction for the 8+ charge state of lysozyme in comparison with the 7+ and 6+ charge states.

Figure 1
figure 1

Mass spectra representing laser-induced vaporization of 200 μM lysozyme with (a), no salt, (b) 2.5 mM NaCl, (c) 25 mM NaCl, (d) 50 mM NaCl, and (e) 100 mM NaCl into the ES of 10 mM aqueous ammonium acetate. Electrospray mass spectra of 10 μM lysozyme prepared in 10 mM aqueous ammonium acetate with (f) no salt, (g) 0.1 mM NaCl, (h) 1 mM NaCl, (i) 2 mM NaCl, and (j) 5 mM NaCl. The measurements were performed at collision potential of 15 eV. A feature with an asterisk (*) represents solvent

Full size image

The ESI-MS measurements for lysozyme in aqueous ammonium acetate solution with NaCl concentrations ranging from 0 to 5 mM are shown in Figure 1f–j. The ESI mass spectrum of lysozyme in ammonium acetate, without NaCl, reveals a narrow range of low charge states ranging from 6+ to 9+ in Figure 1f. The spectra corresponding to ESI measurements in the presence of salt (Figure 1g–j) again shows that increasing salt adduction occurs for the 8+, 7+, and 6+ charge states with decreasing charge. In the ESI measurement, the adduction is significantly enhanced in comparison with LEMS for similar concentrations of salt (e.g., Figure 1b, i). Peak broadening due to sodium ion adduction to the 6+ and 7+ charge states for salt concentration of ≥ 1 mM makes the detection of protonated lysozyme ([L + 7H]7+ and [L + 6H]6+) challenging. In particular, peak broadening limited the detection of 6+ charge state of lysozyme for salt concentration of ≥ 1 mM. This is in agreement with the previous nano-spray measurement of ubiquitin where peak broadening of [U + 6H]6+ and [U + 5H]5+ charge states hindered the detection of protonated protein ions when analyzed from solution containing 1.0 mM of NaCl without the addition of solution additives that facilitate desalting of proteins [2].

The LEMS and ESI measurements for an equimolar mixture of lysozyme, cytochrome c, and myoglobin in salt concentration ranging from 0 to 250 mM are shown in Figure 2. The LEMS measurement of protein mixtures without NaCl vaporized into an ES solvent consisting of aqueous ammonium acetate, shown in Figure 2a, reveals a narrow range of low charge states ranging from 7+ to 10+ for lysozyme, 6+ and 8+ for cytochrome c, and 7+ to 9+ for myoglobin, respectively. The CSDs of lysozyme, cytochrome c, and myoglobin vaporized from concentrations of NaCl ranging from 75 to 250 mM (Figure 2b–d) are similar to the 0 mM salt solution and correspond to the folded protein charge state distribution. This suggests that the native states of the proteins were preserved when vaporized from solutions with high salt concentration. Previous LEMS measurement of aqueous myoglobin into the electrospray solvent containing aqueous ammonium acetate resulted in intact holo-myoglobin features suggesting that the non-covalently bounded protein complexes can be transferred intact into the electrospray plume [27]. This is presumably due to shorter time scale at which the energy is deposited into the system in comparison with the time required for protein unfolding. The majority of salt adduction to the protein mixture occurred for the 6+, 7+ and 8+ charge states of lysozyme, for the 6+, 7+ and 8+ charge states of cytochrome c, and for the 7+, 8+, and 9+ charge states of myoglobin (see Figure 2b–d; also shown in Supplementary Figure S2b–d). The majority of salt adduction occurred at the 6+ and 7+ charge states in the case of lysozyme during single protein analysis (see Figure 1b–e; also shown in Supplementary Figure S1b–e).

Figure 2
figure 2

Mass spectra representing laser-induced vaporization of 200 μM protein mixtures (lysozyme (L), cytochrome c (C), and myoglobin (M)) with (a) no salt, (b) 75 mM NaCl, (c) 125 mM NaCl, and (d) 250 mM NaCl into the ES of 10 mM aqueous ammonium acetate. Electrospray mass spectra of 10 μM protein mixtures prepared in 10 mM aqueous ammonium acetate with (e) no salt, (f) 0.5 mM NaCl, (g) 2 mM NaCl, and (h) 5 mM NaCl. The measurements were performed at collision potential of 15 eV

Full size image

ESI measurements were performed for equimolar mixtures of lysozyme, cytochrome c, and myoglobin (10 μM of each) from aqueous ammonium acetate solution containing NaCl concentrations ranging from 0 to 5 mM, as shown in Figure 2e–h. The ESI mass spectra of protein mixtures with no added NaCl revealed charge states ranging from 7+ to 10+ for lysozyme, 7+ and 8+ for cytochrome c, and 8+ and 9+ for myoglobin, as shown in Figure 2e. The ESI measurements for salt concentrations of 0.5 and 2 mM NaCl (Figure 2f, g) show significant salt adduction for the 7+ and 8+ charge states of lysozyme, the 7+ and 8+ charge states of cytochrome c, and the 8+ and 9+ charge states for myoglobin. The ESI measurement of protein mixtures at salt concentration of 5 mM resulted in charge states ranging from 7+ to 14+ for cytochrome c and suppressed the detection of the lower charge states of myoglobin (8+ and 9+ charge states) and lysozyme (7+ and 8+ charge states) by significant salt adduction, as shown in Figure 2h. The observation of unfolded charge state distribution for cytochrome c is likely due to the increase in electrospray droplet temperature at higher salt concentration due to the non-volatile nature of the salt. The formation of higher charge states were also observed in a previous experiment when the electrospray solvent contained higher salt concentration [1]. In that study, the addition of 50 mM NaCl into the 10 μM myoglobin solution resulted in a CSD ranging from 10+ to 22+ (peaked at 12+ and 20+), while the lower salt concentration (5 mM NaCl) resulted in a CSD ranging from 7+ to 18+ (peaked at 11+). Identification of individual proteins from multicomponent mixtures is difficult using ESI when salt is present in the sample solution at concentration > 5 mM due to ion suppression effects [28] and spectral congestion [29] arising from overlapping features. Direct analysis of protein mixtures using ESI in the presence of inorganic salts is complicated because of peak broadening due to adduction. This is not the case for LEMS where salt concentrations up to 250 mM do not result in protein structural change or significant adduction, and thus, the identification of individual protein from the multicomponent mixture is possible.

Salt Adduction to Proteins: LEMS Versus ESI-MS

The adduction of sodium ion to protein was compared for LEMS and ESI-MS measurements to provide insight into the ionization mechanisms of each experiment. Sodium adduction to the protein molecule can be expressed using Equation 1 [4],

$$ {\left[\mathrm{M}+{\mathrm{zH}}^{+}+n\left({\mathrm{Na}}^{+}\hbox{--} {\mathrm{H}}^{+}\right)+m\left({\mathrm{Cl}}^{-}+{\mathrm{H}}^{+}\right)\right]}^{z+}+70\ \mathrm{eV}\ \mathrm{CID}\to {\left[\mathrm{M}+\left(z\hbox{--} n\right){\mathrm{H}}^{+}+{n\mathrm{Na}}^{+}\right]}^{z+}+m\mathrm{HCI} $$
(1)

where M is the protein molecule, z is the number of protons (H+), n and m are the number of Na+ and Cl− adducts, respectively, z is the charge state of the protein, and z+ is the overall charge on the protein molecule. The lower collision potential of 15 eV used in this measurement resulted in a series of n(Na+ − H+) and m(Cl− + H+) features that complicate the mass spectra [4]. Increasing the collision potential to 70 eV resulted in the complete loss of HCl, and Na-adducted protein ions are predominately formed as shown in Figures 3 and 4. The removal of HCl is mainly attributed to an in-source reaction [30, 31] where protein ions are subjected to collision-induced dissociation (CID). The increase in collision potential also resulted in the loss of H2O from lysozyme as shown in Supplementary Figure S3.

Figure 3
figure 3

High-resolution LEMS mass spectra of 200 μM lysozyme [L + 7H]7+ with (a) no salt, (b) 2.5 mM NaCl, (c) 25 mM NaCl, (d) 50 mM NaCl, and (e) 100 mM NaCl into the ES of 10 mM aqueous ammonium acetate. High-resolution electrospray mass spectra of 10 μM lysozyme [L + 7H+]7+ prepared in 10 mM aqueous ammonium acetate with (f) no salt, (g) 0.1 mM NaCl, (h) 1 mM NaCl, (i) 2 mM NaCl, and (j) 5 mM NaCl. The measurements were performed at collision potential of 70 eV. Sodium adducts are indicated as n = 0, 1, 2, etc

Full size image
Figure 4
figure 4

High-resolution LEMS mass spectra of 200 μM protein mixtures (cytochrome c [C + 7H]7+ and lysozyme [L + 8H]8+) with (a) no salt, (b) 75 mM NaCl, (c) 125 mM NaCl, and (d) 250 mM NaCl into the ES of 10 mM aqueous ammonium acetate. High-resolution electrospray mass spectra of 10 μM protein mixtures prepared in 10 mM aqueous ammonium acetate with (e) no salt, (f) 0.5 mM NaCl, (g) 2 mM NaCl, and (h) 5 mM NaCl. The measurements were performed at collision potential of 70 eV. Sodium adducts are indicated as n = 0, 1, 2, etc

Full size image

To compare the Na+ ion adduction as a function of salt concentration for both LEMS and ESI-MS measurements, we focus on the 7+ charge state of lysozyme as shown in Figure 3. LEMS analysis of lysozyme with salt concentration of 25 mM NaCl revealed salt adducts ranging from 1 to 13 (Figure 3c). The dominant feature in the mass spectra collected with a CID potential of 70 eV is the protonated peak of lysozyme [L + 7H]7+. Conversely, ESI analysis of lysozyme with 1 mM NaCl resulted in the Na = 3 adduct dominating with sodium-adducted protein peaks ranging from 1 to 16 (Figure 3h). The sodium adducts resulted in peak broadening for ESI-MS measurements (Figure 1h). The average number of sodium ions adducts < n > can be calculated [4] as follows:

$$ <n>=\frac{\sum_{n=0}^N\ \left(n\ast {I}_n\right)}{\sum_{n=0}^N{I}_n} $$

where I n is the peak intensity of the Na+ adducts and N is the maximum number of Na+ observed for each charge state. The average number of sodium ions bound to the 7+ charge state of lysozyme (<n > ) for the LEMS measurement are 1.71, 5.23, 5.26, and 5.11 for the salt concentration of 2.5, 25, 50, and 100 mM NaCl, respectively. The Na+ adduction did not increase between 25 and 100 mM NaCl concentrations in the LEMS analysis. The average number of sodium ions bound to the 7+ charge state of lysozyme for the ESI measurement for salt concentrations of 0.1, 1, 2, and 5 mM NaCl are 2.65, 6.44, 7.57, and 8.48, respectively. Conventional electrospray measurements showed increasing salt adduction with increasing salt concentration with a corresponding decrease in intensity for the protonated peak of the protein. The measurements revealed significantly reduced sodium adduction for LEMS measurements in comparison with ESI-MS for all charge states.

For the mass spectral analysis of multicomponent protein mixtures, the collision potential was increased to 70 eV to eliminate HCl to form predominately Na-adducted protein ions [4]. To compare the sodium adduction to the protein molecule for LEMS and ESI-MS measurements, the 7+ charge state of cytochrome c (m/z 1766.5), 8+ charge state of lysozyme (m/z 1789.1), and 8+ charge state of myoglobin (m/z 2122.4) were chosen. Salt adduction to the 7+ charge state of cytochrome c overlaps with the protonated ([L + 8H]8+) lysozyme peak. LEMS measurements revealed well-resolved, protonated features for cytochrome c ([C + 7H]7+) and lysozyme ([L + 8H]8+) for salt concentration of 250 mM NaCl, as shown in Figure 4d. This suggests minimal adduction/overlap and limited ion suppression of the protonated protein ions even in the presence of high salt concentration using laser vaporization to transfer sample into the ES droplets. Conversely, in ESI-MS measurements, the mass spectra revealed congested features and suppression of protonated ions ((C + 7H)7+ and (L + 8H)8+) for NaCl concentrations ranging from 0.5 to 5 mM, as shown in Figure 4f–h. The ESI measurements for cytochrome c and lysozyme with 0.5 mM NaCl revealed that the Na = 2 and Na = 1 adducts were the dominant features with sodium adducts ranging from 1 to 6 and 1 to 11, respectively (Figure 4f). At a salt concentration of 5 mM, unresolvable spectral features for both cytochrome c and lysozyme are observed due to excessive salt adduction. Adducted protein features were observed for 0 mM NaCl in both LEMS and ESI measurements of cytochrome c and lysozyme, presumably due to impurities in the solvents (Figure 4a, e).

The LEMS measurement for the 8+ charge state of myoglobin revealed salt adducts ranging from 1 to 14 for salt concentration of 75 mM NaCl (Supplementary Figure S4b). The dominant feature in the mass spectra is the protonated peak of apo-myoglobin [M + 8H]8+. The formation of apo-myoglobin is due to collision-induced unfolding of myoglobin upon increasing the collision energy to 70 eV. The ESI analysis of myoglobin with 0.5 mM NaCl resulted in the Na = 3 adduct being the largest feature with sodium adduct features ranging from 1 to 15 (Supplementary Figure S4f). Again, the LEMS experiment enabled measurements of protein molecules for samples with higher salt concentration because of reduced adduct formation.

To test whether the higher protein concentration (200 μM) used in the LEMS measurements resulted in increased salt tolerance and reduced adduct formation in comparison with ESI-MS (where a protein concentration of 10 μM was used), we performed an experiment where the concentrations of lysozyme in both LEMS and ESI analyses were adjusted to be 50 μM. The salt concentration was varied from 0 to 100 mM for the LEMS measurements and 0 to 5 mM for the ESI measurements. The mass spectra obtained are shown in Supplementary Figures S5 and S6. Similar to the analysis of 200 μM lysozyme using LEMS, a protonated protein feature (Supplementary Figure S6d) was obtained up to the salt concentration of 100 mM NaCl. Conversely, salt concentration greater than 0.1 mM NaCl resulted in salt-adducted features dominating the protonated feature of lysozyme (50 μM in concentration, Supplementary Figure S6f) in electrospray analysis. These results indicate that the adduct formation in LEMS and ESI measurements is independent of the protein concentration.

We also investigated the effect of an emitter size on salt tolerance using a nano-spray emitter with the inner diameter of 50 μm and a flow rate of 400 nL/min. The nano-spray mass spectra of lysozyme prepared in 2.5 mM NaCl concentration resulted in Na = 3 adduct being the dominant feature with sodium-adducted protein peaks ranging from 1 to 11 (inset of Supplementary Figure S7e). The nano-spray analysis of lysozyme with 50 mM NaCl resulted in an unresolved mass spectral feature at the CID potential of 70 eV indicating that the presence of high salt concentration results in ion suppression effects in nano-spray ionization under these experimental conditions. Conversely, the protonated lysozyme feature was obtained in the LEMS measurements up to the salt concentration of 100 mM. Our preliminary studies using nano-spray emitter with inner diameter of 50 μm did not show increased salt tolerance in comparison with electrospray emitter with 124 μm inner diameter. However, a recent study has shown that the use of a nano-spray emitter with tip size of less than ∼ 1.4 μm results in higher salt tolerance and reduced adduct formation [32]. Future studies will compare the performance of such emitters with LEMS measurements.

These measurements suggest that in the LEMS experiments, the Na+ ions are somehow excluded from interacting with the protein molecules. The reduced Na+ ion adduction in LEMS measurements in comparison with ESI may be due to the non-equilibrium nature of laser-vaporized proteins interacting with the ES droplets. We have previously proposed [25, 26, 33] that the partitioning of analytes into the ES droplets is limited by the time that laser-vaporized analytes spend on the droplet surface prior to entering into the MS inlet capillary. The time for interaction between the vaporized protein and the charged droplet is estimated to be < 100 ms [33], and this time will therefore dictate the partitioning of analytes based on their respective sizes and solution enthalpy. Considering a simple diffusion process, the mixing time, t, of two analytes depends upon their diffusion coefficient (D) and the distance separating the diffusing solutes (d), where t = d2/6D. Given the fact that the diffusion coefficient of analytes depend on their size and shape, we anticipate that Na+ and Cl− will diffuse into the interior of the droplet much more quickly than the protein in the LEMS process, thus limiting the opportunity for the chemistry described by reaction 1 if the droplet lifetime is short compared to the protein diffusion time. The diffusion constant for Na+ is 1.6 10−9 m2/s, and assuming d = 1 μm, the diffusion time is (1 × 10−6 m)2/6 × 1.6 10−9 m2/s = 104.2 μs. This time is short compared to the lifetime of the droplet ~ 10 ms, and thus, the salt ions can partition into the interior of the droplet. The diffusion constant for hemoglobin is 6.9 × 10−11 m2/s, suggesting a 23× longer diffusion time. The proteins are also much more hydrophobic than salt ions and thus will tend to remain on the surface of the charged droplet [34, 35]. This results in a lower salt concentration on the surface of the newly formed electrospray droplet from which a gaseous protein ion is released. This also results in a higher concentration of protein on the surface of the electrospray droplet where the charge resides. Thus, the non-equilibrium nature of the partitioning of salt and protein between droplet surface and interior will reduce adduction in the LEMS measurement.

A previous study investigating the ionization mechanism of proteins in ESI-MS revealed a completely different salt adduction pattern for folded and unfolded proteins [4]. The folded protein revealed higher salt adduction, which is in agreement with the charge residue model (CRM) where the non-specific interaction between salt and protein increases as the solvent evaporates. The unfolded protein however revealed minimal to no salt adduction which is attributed to the difference in ionization mechanism for folded and unfolded proteins in ESI. Unfolded proteins are likely expelled from the ES droplet via the chain ejection model (CEM) [36] prior to the increase in salt concentration within the ES droplets resulting in minimal to no salt adduction for higher charge states. The reduced salt adduction for proteins in LEMS measurement suggests that protein ionization in LEMS likely occurs in a manner similar to the chain ejection model. However, the salt adduction in LEMS is significantly lower than that for ESI, suggesting that the proteins do not equilibrate into the interior of the droplet. The polar analytes (e.g., salt) equilibrating into the droplet interior due to increased solvation will thus be isolated from proteins resulting in minimal salt adduction in LEMS measurements.

The preferential interaction of laser-vaporized protein with the highly charged electrospray droplets in LEMS measurements has been proposed to enhance the detection of protonated protein ions in comparison with conventional ESI measurements [37]. Protein residing in the droplet interior in the ESI-MS measurement has to partition onto the droplet exterior for ionization. This enhances interaction of protein with salt in the case of ESI resulting in an increased salt adduction.

Conclusions

The analysis of lysozyme, and a mixture of lysozyme, cytochrome c, and myoglobin, in solution with varying salt concentrations revealed a significantly higher salt tolerance for LEMS measurements in comparison with conventional electrospray with respect to suppression of the protonated ion signal and the ability to distinguish mixture components. The average sodium adducts 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, whereas conventional electrospray measurements for lysozyme from solutions 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. The reduced salt adduction in the LEMS experiment is likely due to the non-equilibrium nature of partitioning of laser-vaporized proteins between the surface and interior of the charged electrospray droplets. The reduced interaction time of the laser-vaporized analytes with the ES droplets presumably allows small ions like Na+ and Cl− to partition into the droplet interior on the time scale of transit to the capillary leaving the much larger protein on the droplet surface where excess charge resides. This results in a lower salt concentration on the surface of the ES droplets, which leads to the formation of a higher fraction of protonated protein ions in comparison with conventional ESI measurements. Conversely, proteins in electrospray measurements have to partition from the droplet interior to the droplet’s surface for ionization which results in an increased interaction of protein with salt in the ES droplet from which an ionized protein is released.

We note that the lower ion abundance of LEMS measurement in comparison with ESI-MS is due to the low capture efficiency (~ 3.6 ± 1.8%) of the laser-vaporized analyte by the electrospray plume. The use of methods similar to previous studies [38, 39] to increase the transfer efficiency of laser-vaporized analyte into the electrospray plume will enable analysis of lower concentrations of samples. For instance, it has been reported that the use of continuous flow solvent probe as the plume capture source in LAESI measurement resulted in the increase in transfer efficiency of the laser-vaporized material from 2 to 50% [38]. Similar designs will significantly improve the LOD of a two-step ionization technique like LEMS that couples laser ablation with electrospray postionization mass spectrometry.