Ionization Orthogonal-Time

Article pubs.acs.org/ac

Infrared Matrix-Assisted Laser Desorption/Ionization OrthogonalTime-of-Flight Mass Spectrometry Employing a Cooling Stage and Water Ice As a Matrix Alexander Pirkl, Jens Soltwisch, Felix Draude,† and Klaus Dreisewerd* Institute of Medical Physics and Biophysics, University of Münster, Robert-Koch-Strasse 31, 48149 Münster, Germany S Supporting Information *

ABSTRACT: Although water ice has been utilized in the past as a matrix for infrared matrix-assisted laser desorption/ionization mass spectrometry (IR-MALDI-MS), it has not found a wider use due to limitations in the analytical performance and technical demands on the employment of the necessary cooling stage. Here, we developed a temperaturecontrolled sample stage for use with an orthogonal time-of-flight mass spectrometer (MALDI-o-TOF-MS). The stage utilizes a combination of liquid nitrogen cooling and counterheating with a Peltier element. It allows adjustment of the sample temperature between ∼−120 °C and room temperature. To identify optimal irradiation conditions for IR-MALDI with the water ice matrix, we first investigated the influence of excitation wavelength, varied between 2.7 and 3.1 μm, and laser fluence on the signal intensities of molecular substance P ions. These data suggest the involvement of transient melting of the ice during the laser pulse and primary energy deposition into liquid water. As a consequence, the best analytical performance is obtained at a wavelength corresponding to the absorption maximum of liquid water of about 2.94 μm. The current data significantly surpass the previously reported analytical features. The particular softness of the method is, for example, exemplified by the analysis of noncovalently bound holomyoglobin and of ribonuclease B. This is also the first report demonstrating the analysis of an IgG monoclonal antibody (MW ∼ 150 kDa) from a water ice matrix. Untypical for MALDI-MS, high charge states of multiply protonated species were moreover observed for some of the investigated peptides and even for lacto-N-fucopentaose II oligosaccharides. Using water ice as matrix is of particular interest for MALDI MS profiling and imaging applications since matrix-free spectra are produced. The MS and tandem MS analysis of metabolites directly from frozen food samples is demonstrated with the example of a strawberry fruit.

U

Although some reports have demonstrated that biomolecular ions (e.g., of peptides and small proteins1,11) can be generated by IR-MALDI-MS from frozen aqueous samples, the technique is not commonly used. One reason is that both the mass range and the so far achieved analytical sensitivity are well below that of standard UV-MALDI and/or electrospray ionization (ESI) mass spectrometry. The largest proteins that were successfully analyzed from water ice had molecular masses in the low 10 kDa range.1 Although Berkenkamp et al. showed that a mass range up to approximately 100 kDa was accessible by using semilyophilized proteins that were covered with a thin layer of water or a hydration shell, this sample preparation protocol is neither practical nor particularly sensitive. Considerably higher sensitivities down to the attomole range for the analysis of peptides have been reported for the closely related technique of laser-induced liquid bead/beam ion desorption mass spectrometry (LILBID-MS13,14). In this technique, small analytecontaining droplets are injected into the vacuum of the mass spectrometer and successively vaporized by a pulsed IR-laser beam. As with IR-MALDI, wavelengths of ∼3 μm are used that match the O−H stretch vibration band of water. Several reports have shown that LILBID provides soft desorption/ionization

sing water as a matrix for infrared matrix-assisted laser desorption ionization mass spectrometry (IR-MALDIMS) enables the analysis of biomolecules directly from aqueous solution1−3 and/or water-rich biological tissue.4−7 However, because of its high vapor pressure, the employment of a cooling stage is required if ions are generated at high vacuum conditions,8−10 e.g., in ion sources of “axial” time-of-flight (TOF) mass spectrometers (p < 10−6 mbar). If a fine vacuum ion source (p ∼ 1 mbar) is employed, evaporation of water from tissue may be slow enough to allow performing the MS analysis at room temperature. For example, IR-MALDI-MS in combination with a hybrid orthogonal extracting o-TOF instrument was used to analyze metabolites and lipids directly from plant leaves.7 However, freezing of samples has several advantages: (1) avoidance of excessive dehydration, which potentially deteriorates the analytical sensitivity of the analysis, (2) prevention of the evaporation of more volatile metabolites, (3) enabling the direct analysis of frozen liquid samples, and (4) structural conservation of water-rich (tissue) samples. As has been shown, the use of a cooling stage with atmospheric pressure (AP) MALDI mass spectrometry11 can be advantageous by preventing heating of samples by the frequently applied “hot counter gas flow”, which might cause rapid dehydration/evaporation of water or thermal degradation of biomolecules.12 © 2012 American Chemical Society

Received: March 27, 2012 Accepted: June 1, 2012 Published: June 1, 2012 5669

dx.doi.org/10.1021/ac300840b | Anal. Chem. 2012, 84, 5669−5676

Analytical Chemistry

Article

the OPO laser system has a relatively large divergence of >1 mrad and therefore a reduced focusability, a telescope (expansion ratio, 3:1) was used to expand the beam for reduction of focal spot sizes. Care was taken to adjust the position of the telescope for compensation of the wavelength dispersion. The laser beam was focused onto the sample at an angle of 30° using an Infrasil lens with a nominal focal length of 200 mm. The focal beam cross section (laser spot diameter) was determined using a knife-edge method to ∼235 × 470 μm2 (Figure S-1 in the Supporting Information22). Coarse beam attenuation was achieved using a set of Schott glass substrates while counter-rotating ZnS plates served for fine-control of the laser pulse energy. Laser pulse energies were monitored online using a custom-made pyroelectric detector onto which a fraction of the light energy was directed using a beam splitter. This detector was calibrated against the laser pulse energy delivered to the sample by placing a commercial energy meter at the sample position. The second orthogonal extracting mass spectrometer (similar in design to the prototype o-TOF-MS) was a QSTAR pulsar i (AB SCIEX). In this case, the beam of an Er:YAG laser (Speser, Spektrum Laser, Berlin, Germany), providing an emission wavelength of 2.94 μm, a pulse duration of ∼150 ns, and a pulse repetition rate of 2 Hz, was coupled into the oMALDI 2 ion source by replacing its standard UV optics and vacuum window with CaF2 substrates. The focal laser spot size of the Er:YAG laser was about 110 × 220 μm2 (1/e2-definition). In both instruments, ions were generated in a N2 buffer gas environment of ∼1.5 mbar and extracted by an electrical field of 30−45 V/mm applied between sample plate holder and ion extraction cone. The lower m/z cutoff of the ion transfer quadrupoles was set to a minimal value of ∼130 except for the analysis of proteins where it was increased to ∼3000 to enhance the ion transmission. After passing the transfer quadrupole(s) of the hybrid instruments, ions were accelerated by electrical potentials of 10 kV (o-TOF) or 4 kV (QSTAR) into the time-of-flight parts of the mass spectrometers. Lowenergy collision-induced dissociation (CID) of selected precursor ions for tandem mass spectrometry was performed with the QSTAR using Ar as the collision gas at a pressure of about 5.3 × 10−5 mbar. All presented mass spectra were recorded in the positive ion mode. Cooling Stage. A photograph of the sample stage mounted to the oMALDI 2 source is shown in Figure 1a and its layout

conditions, a feature that is beneficial for the analysis of noncovalently bound complexes.13−15 An advantage of the IRlaser based methods compared to ESI-MS is that the ion generation process is generally less affected by the presence of buffers. Biomolecules can more easily be investigated under near-physiological pH solution conditions or even by using strong concentrations of detergents like urea.16 A disadvantage of the LILBID method is that it is, at present, not compatible with state-of-the-art high-resolving mass spectrometer platforms. In ambient “IR-laser mass spectrometry” a sizable increase in sensitivity has recently been achieved by intersecting the IR-laser generated material plume by an ESI beam for laser ablation electrospray ionization (LAESI17,18) as well as by atmospheric pressure chemical ionization (APCI19). The achieved gain, however, is likely offset by a reduced ion transmission in AP instruments. Finding an MS method that would combine the advantages of the different methodologies (IR-MALDI, LILBID, and ESI) and which would allow analyzing aqueous samples under near-physiological conditions could provide a milestone for biomolecular mass spectrometry. In this report we first describe experiments aimed at identifying optimal sample preparation and IR-laser irradiation conditions. Second, we present novel applications of IRMALDI-MS employing a water ice matrix. The analyses became possible by utilizing a temperature-controlled sample stage which we developed for the oMALDI 2 ion source (AB SCIEX, Concord, CA). At optimal irradiation conditions (λ = 2.94 μm), we investigated the IR-MALDI-o-TOF-MS analysis of oligosaccharides, peptides, and proteins with molecular weights as large as 150 kDa (monoclonal antibody). The “softness” of the method was probed by the analysis of glycosylated/ phosphorylated ribonuclease B (RNase B) and of holomyoglobin. A piece of strawberry fruit flesh was studied as an example for the analysis of plant tissue.

■

EXPERIMENTAL SECTION Materials. Substance P was from Bachem (Bubendorf, Switzerland), lacto-N-fucopentaose II (LNFP II) was from Dextra Laboratories (Reading, U.K.). All other chemicals were from Sigma-Aldrich (Schnelldorf, Germany). Most samples were prepared without further treatment; a subset of samples (as identified in the figure captions) was desalted prior to the analysis using Biospin chromatography columns (Bio Rad, München, Germany). Compounds were dissolved in distilled water (Milli-Q, Merck Millipore, Billerica, MA) water to the desired concentrations. Strawberry samples (Fragaria ‘Elsanta’) were purchased from a local market. Mass Spectrometers and Lasers. Two o-TOF-type mass spectrometers were employed, each equipped with an oMALDI 2 ion source. The prototype o-TOF mass spectrometer used in most of the experiments has been described in detail previously.20,21 The oMALDI 2 source of this instrument contains a custom-made port to adopt the IR laser beam. In the present study, a wavelength-tunable optical parametric oscillator laser system (model KNbO3 OPO-C1064, GWU Lasertechnik, Erftstadt, Germany) served for generation of gaseous bimolecular ions. The tuning range of this system is λ = 2.71−3.14 μm. Over the course of the experiments, two similar Nd:YAG-lasers were used as pump sources for the OPO system. The first was as a SpitLight laser (InnoLas, Krailing, Germany), the second a Surelite II (Continuum, Darmstadt, Germany). Both lasers generated IR-OPO-pulses of ∼6 ns width at a pulse repetition rate of 10 Hz. Because the beam of

Figure 1. Custom-made MALDI cooling sample stage: (a) photograph showing the stage mounted to the oMALDI 2 ion source and (b) schematic cross section (electrical contacts are not shown).

depicted in Figure 1b. The stage is mounted via two hinges and can be exchanged with the regular sample stage holder of the oMALDI 2 ion source within 2 min. Components added to the custom-made cooling stage comprise a liquid nitrogen tank, a hollow brass cylinder that acts as a coldfinger, a Peltier element (model MS2,192,14,20,15,25,11, Telemeter Electronic, Donau5670



Article

Figure 2. (a) Heat maps displaying the signal intensities of substance P ions generated from a water ice matrix as a function of OPO laser wavelength and laser fluence. The combined signal intensities of singly, doubly, and triply protonated molecular ions were used to derive the contour plot. Substance P was prepared in a concentration of 10−4 mol/L. Data were normalized such that each data point represents the same number of laser pulses; typically, 200−500 laser pulses were applied per data point, depending on laser fluence (material consumption). Because of the increased (bulk) amounts of material that are ablated at fluences significantly above the ion detection threshold these data points were not recorded, resulting in white areas in the heat maps. The reciprocal absorption profiles (α−1; corresponding to the laser penetration depth) of liquid water at 295 K and water ice at 100 K (Reprinted from refs 25 and 26. Copyright 1989 (ref 25) and 1969 (ref 26) American Chemical Society) are also shown. (b) Signal intensity of singly protonated substance P ions [M + H]+ as a function of laser fluence at a wavelength of 2.94 μm. The solid line represents a power law fit of the form I ∼ Fm to the experimental data with m = 4.9 (I, signal intensity; F, fluence, m, best-fit parameter).

wörth, Germany), a platinum resistance thermometer (Pt100, RS Components, Mörfelden-Walldorf, Germany), a multiple electrical feed-through (for supply of sample plate voltage, voltages for Peltier element, and readout for the Pt100 thermosensor), as well as a rubber vacuum sealing. The stage uses a combination of liquid nitrogen cooling and counterheating by means of the Peltier element. The sample temperature T0 can be adjusted between ∼−120 °C and room temperature. The lower limit is reached by liquid N2 cooling with the Peltier element switched off. Counter heating using the Peltier element allows adjustment of T0 within seconds and a precision of better than 1 °C, an important feature for a convenient sample transfer (see below). Using the Peltier element in cooling mode led only to a minor temporary further reduction of the sample temperature before the heat produced at the “warm” side of the Peltier element balanced this cooling gain. Therefore, the Peltier element was only used in mild heating mode during the MS measurements to produce a constant sample temperature T0 of −86 °C. At a back pressure of ∼1 mbar of N2, temperatures below approximately −60 °C are sufficient to prevent notable sublimation of water. Using the Peltier element without liquid N2 cooling produced only a minimum sample temperature T0 of >−30 °C. However, over several months of operating the stage, three Peltier elements failed, which we attribute to freezing of water in the area of its semiconductor junctions. To avoid such problems in the future, we will test resistance heating as an alternative, more robust technical means. A custom-made stainless steel sample plate could be mounted directly on the Peltier element using strong permanent magnets (neodymium iron boron, Webcraft, Gottmandingen, Germany). For improved thermal contact, heat sink paste (WLP 035, Bürklin, Oberhaching, Germany) was applied between the sample plate and the Peltier element. An electrode for electrical contact and a Pt100 sensor are pressed against the bottom of the sample plate using springs. For preparation of liquid samples, the plate contained a set of half-spherical wells of ∼0.5 mm radius. The stainless steel sample plate was utilized in the fundamental studies investigating the wavelength dependence of the IR-MALDI

process. In all other measurements, a thin copper plate of 0.2 mm thickness was mounted onto the Peltier element using thermally conductive glue. The 1 mm-thick infrasil sample substrates were mounted on top of this plate using heat sink paste. Because infrasil is transparent at the laser wavelengths studied here, a significant suppression of background was observed with this sample target. The ion extraction potential was supplied to the copper plate and its temperature measured by a Pt100 element, affixed with thermally conductive glue. Using the latter geometry, a minimum sample temperature of ∼−100 °C was achieved, which is 20 °C above that achievable for the above stainless steel setup; the difference is likely due to the limited thermal conductivity of the glass substrate and the additional heat transfer between the Peltier element and copper plate. Sample Preparation and Transfer Protocol. Aliquots of ∼0.3 μL of liquid samples were transferred to a stainless steel sample plate (only used in the wavelength study with substance P) or an Infrasil glass substrate (used in all other studies with liquid samples). Infrasil is transparent between 2.7 and 3.1 μm and thus produces no background ions if irradiated by the beam of the utilized optical parametric oscillator (OPO) laser system. Whole strawberry fruits were shock-frozen by dipping them into liquid nitrogen. Thin slices were cut from the deep-frozen fruits using a razor blade and were thaw-mounted on glass slides. Glass slides (or stainless steel plates) were dipped into liquid nitrogen for >30 s and were then rapidly mounted onto the copper plate (or directly onto the Peltier element). At this stage, the top side of the Peltier element was kept at a temperature of approximately 0 °C such that the heat sink paste was well spreadable (the minimum temperature allowing spreading of the paste is about −5 °C). Directly after closing the door of the sample introduction system and initiating the pump-down sequence of the oMALDI 2 control software (oMALDI server; AB SCIEX), the heating of the Peltier element was turned off and the sample plate temperature dropped to below −60 °C within seconds. This protocol ensured that the ice did not melt and prevented excessive condensation from forming. During the MS experiments, the 5671



Article

hinge cover was briefly warmed using a blow-drier, in order to prevent that the enclosed rubber sealing became excessively cold and lost its sealing properties. A reduced tightness of the sealing became eventually notable by a gradual increase of the source pressure. Safety Considerations. Contact of skin with liquid nitrogen can cause severe frostbite. Protective clothing including safety glasses need to be worn. Evaporation of pure nitrogen can displace oxygen in laboratory air and cause an asphyxiation hazard. Sufficient air circulation is necessary. Exposure of eyes/skin to laser radiation poses a severe health hazard. Safety goggles and protective clothing need to be worn while working with free laser beams.

Molecular peptide ion signals increase exponentially with fluence F of the OPO laser beam (Figure 2b). Empirically, this increase can be fit to a function of the form I ∼ Fm, where I is the ion signal intensity and m is a best-fit parameter. At 2.94 μm, a best fit is obtained for m = 4.9 (solid line in Figure 2b). Similar strong ion signal-fluence dependences were determined before in numerous IR-MALDI28,29 and UV-MALDI studies.30−32 These studies and other work demonstrate that complex desorption/ionization mechanisms underlie pulsed IRlaser induced material ablation and the ionization processes.28−37 Therefore, the above simple empirical equation and the derived exponent m are at best a practical measure for a characterization of the ion signal intensity-fluence relationship. In contrast to previous studies, in particular those in which a shallow water layer (water of hydration) was used as a matrix and in which large signal scattering was observed,1 a significant improvement in the reproducibility is obtained, even if the data do not fully reproduce the quality reported for liquid UVMALDI matrixes.38 The standard deviation in the signal intensities produced for a fixed fluence from different, identically prepared sample spots is about 40%. This is, for example, demonstrated by the high-fluence data points of Figure 2b. This variation in the ion yields may be attributed small-scale differences in the crystal structures and exact irradiation conditions effecting the microscopic ablation process. Qualitatively, the mass spectra were not affected if the initial sample temperature T0 was varied in the range of ∼−120 °C to −60 °C by using the Peltier element in the heating mode. However, ion counts increase slightly with T0 for a given fluence F (Figure S-2 in the Supporting Information). Presumably, this finding can be attributed to the additional amount of light energy that is necessary at a lowered T0 for heating of the sample to the phase transition temperature(s). Similar observations were made previously in a UV-MALDIMS study utilizing a cooling stage39 and were also predicted theoretically.40 Analytical Features. Analysis of Peptides. An IR-MALDI mass spectrum of a peptide mix generated from water ice at the optimal wavelength of 2.94 μm is displayed in Figure 3a. Peptides are predominantly detected as protonated species and in lower abundances as sodium/potassium adducts. Next to the singly charged molecular ions, high abundances of doubly protonated molecules are detected for two out of the five test compounds, namely, [Sar1,Ala8]-angiotensin II and substance P. Remarkably, the highest signal for substance P molecular ions is found for the doubly protonated form. Triply charged substance P species are also observed though at lower abundance. In contrast, sizable abundances of multiply charged molecules are not obtained for the other three test peptides. This difference in behavior could reflect different physicochemical properties of the compounds. Future studies will address this issue in detail. The IR-MALDI-o-TOF mass spectra obtained from the water ice matrix are distinguished by an exceedingly low chemical background as is demonstrated by the inset to Figure 3a. This indicates that analyte fragmentation is almost absent. In contrast, a certain degree of “post-source” decay or “in-source” decay-type of fragmentation typically accompanies the UV-MALDI-MS analysis of peptides.21 Moreover, in the recorded m/z range above ∼130, the spectra are devoid of water cluster-derived ion signals. Both factors facilitate the analysis of complex sample mixtures.

■

RESULTS AND DISCUSSION Signal Intensities As a Function of Laser Wavelength and Pulse Energy. Numerous studies have shown that ion yields in IR-MALDI mass spectrometry exhibit a strong dependence on the excitation wavelength and the optical absorption of the matrix.23,24 Generally, the best MS performance in terms of a maximal ion yield and, concomitantly, lowest ion detection threshold fluences, is obtained at wavelengths at which the matrix exhibits a high optical absorption.23 To investigate the desorption/ionization mechanisms underlying IR-MALDI-o-TOF mass spectrometry using ice as a matrix, we recorded the summed signal intensities of singly, doubly, and triply charged molecular substance P ions as a function of laser wavelength and laser fluence. A heat map representing this data cube is displayed in Figure 2a; essentially the same heat maps were obtained if only the individual differently charged substance P ion species were evaluated (not shown). Between 2.71 and ∼3.0 μm, the wavelength-dependent “threshold fluences” (representing, e.g., the ion detection threshold or fluences generating a certain ion signal intensity/ion count slightly above) essentially follow the reciprocal of the optical absorption characteristics of liquid water (gray solid line in Figure 2a). For example, the minimum in the threshold fluence found in the range of ∼2.90−2.95 μm corresponds well to the maximum in optical absorption α (or minimum in laser penetration depth d = α‑1) of liquid water at ∼2.93 μm (at 295 K). At the short wavelength side of the absorption band, a slight “blue-shift” of the contour lines relative to the low-intensity IRabsorption characteristics can be discerned. This observation may be attributed to transient heating of the water during the laser pulse.23,24 Similar shifts between low-intensity IRabsorption curves of the components and the wavelengthdependent MS-characteristics have been observed before for a range of IR-MALDI matrices (see, e.g., ref 23). A less pronounced second local threshold fluence minimum is observed at ∼3.1 μm, but only for low laser fluences/ion counts. Presumably, this can be attributed to the maximum in optical absorption of water ice (second solid line in Figure 2a). This assumption is corroborated by observations made by Focsa et al.27 In this work a marked match between the absorption characteristics of ice and OPO-laser irradiationinduced ablation yield of water clusters was found. In contrast to the present study, the OPO laser fluence was kept below that for direct generation of ions while the water clusters were probed by photoionization. Together, the data suggest the occurrence of transient melting of the ice during the laser pulse and predominant absorption of the light energy into liquid water at the higher IR-MALDI-laser fluences. 5672



Article

Figure 3. IR-MALDI-o-TOF mass spectra of a peptide mix acquired from (a) a water ice and (b) from a glycerol matrix at T0 = −86 °C. The peptides bradykinin fragment 1-7 (P1), [Sar1,Ala8]-angiotensin II (P2), angiotensin II (P3), substance P (P4), and neurotensin (P5) were prepared at a concentration of 8 pmol/μL, each. Approximately 1 pmol of the peptide mix was consumed for generation of each mass spectrum. The spectra were recorded with the OPO laser system tuned to 2.94 μm and by applying 600 laser pulses at a fluence of ∼4500 J/m−2 (water) and ∼8500 J/m2 (glycerol), respectively; the factor of 1.9 in the fluences accounts for the different absorption coefficients of water (1.3 × 104 cm−1 25) and glycerol (6.5 × 103 cm−1 41) at 2.94 μm at room temperature.

Glycerol is probably the most commonly used IR-MALDI matrix. Because of its chemical structure, it exhibits a similar polarity as water and a high optical absorption around 3 μm. Compared to H2O, glycerol exhibits a much lower vapor pressure and can, therefore, be used under high-vacuum conditions without cooling. In order to compare spectra acquired from the water ice matrix with mass spectra generated with the standard matrix glycerol, we prepared the same peptide mix at −86 °C, where glycerol exhibits a glassy state, and adjusted the laser pulse energy such that comparable ion signal strengths were obtained. Analysis of the peptide mix using glycerol provided similar overall peptide ion abundance compared to desorption/ ionization from the water ice (Figure 3b). However, notably higher abundances of multiply charged molecules are obtained from the ice matrix (Figure 3). Using glycerol as a matrix, only the doubly protonated substance P species is recorded in sizable abundance. Significantly, a more pronounced chemical background is produced with the glycerol matrix (see low mass range and inset in Figure 3b). Analysis of Proteins. To determine whether the method is also suitable for the analysis of larger biomolecules, we investigated a set of proteins ranging in molecular weight from ∼5 to 150 kDa. Mass spectra of ribonuclease B (RNase B, MW ∼ 15 kDa), holo-myoglobin (MW ∼ 18 kDa), and a

Figure 4. IR-MALDI-o-TOF mass spectra of three proteins acquired from a water ice matrix: (a) RNase B (MW ∼ 15 kDa; ∼18 pmol were consumed to record the mass spectrum, the analyte solution was desalted prior to the analysis); (b) holo-myoglobin (MW ∼ 17.5 kDa; ∼ 3 pmol consumed, no desalting of sample); (c) monoclonal antibody (IgG; MW ∼ 150 kDa; ∼ 6 pmol consumed, analyte solution was desalted prior to analysis). Denoted sample amounts represent those prepared for one sample spot (these were typically fully consumed to produce the mass spectra). The spectra were recorded with the OPO laser system tuned to 2.94 μm; laser fluences were adjusted for optimal spectral quality. Man, mannose; P, H3PO4.

monoclonal antibody (IgG; MW ∼ 150 kDa), all acquired from pure water ice matrix, are displayed in Figure 4. A mass spectrum of bovine serum albumin (BSA; MW ∼ 66 kDa) is shown in Figure S-3 in the Supporting Information. The spectra reveal several noteworthy features. First, this is the first report to our knowledge showing that proteins exhibiting molecular weights in greater than 100 kDa can be desorbed/ ionized from frozen water preparations. A relatively low acceleration voltage of 10 kV is applied for the TOF analysis 5673



Article

compared to other instruments. As a result, the o-TOF instrument is less sensitive for the detection of high mass ions. Enhanced protein signal intensities might be possible if a higher ion extraction potential could be applied or a high mass detector employed.42,43 The RNase spectrum displayed in Figure 4a reflects the particular softness of the method and the distribution of differently glycosylated/phosphorylated isoforms of the highly mannosylated enzyme. Similar RNase B ion patterns were previously determined in ESI-MS experiments,44 suggesting that under the IR-MALDI conditions the loss of posttranslational modifications is not forming a major pathway. The mass spectrum of holo-myoglobin (Figure 4c) reveals that even intact noncovalently bound protein complexes can be detected. Previously, only a few examples showed this possibility for IR-MALDI-MS using a glycerol matrix.45−47 The abundance ratio between the holo and apo ion species is approximately 2:3 for the singly charged molecule. In line with previous ESI-MS results, a change in the charge state distribution toward lower charge states is observed for the holo form.48,49 This observation provides evidence that the ion species do not represent nonspecific gas phase complexes. An ESI mass spectrum of the sample generated from water containing 10% MeOH showed higher abundances (90−95%) of intact holo-myoglobin ions (data not shown). It is currently not clear whether the increased loss of the heme group under the IR-MALDI conditions is primarily due to partial cleavage of the noncovalent complex in the IR-MALDI process or whether it occurs as a consequence of the freezing of the sample. Despite the partial loss of the prosthetic group, this initial result indicates a potential use of the method for screening of receptor−ligand interactions. One drawback of the method is the presence of a sizable adduct formation between larger proteins and presumably alkali salts, resulting in a tailing of the protein ion signals toward higher m/z values. A sizable tailing persisted after desalting of the analyte solutions using spin columns (see, e.g., the mass spectra of IgG and BSA plotted in Figure 4c and Figure S-3 in the Supporting Information). Improved desalting strategies would be helpful. Adduct formation with water molecules/ clusters, on the other hand, seems to play at best a minor role, in contrast to a more sizable adduct formation between analyte and matrix that is often observed if a glycerol is used as a matrix for IR-MALDI-o-TOF mass spectrometry.45,20 Analysis of Oligosaccharides. Previous studies have shown that IR-MALDI mass spectrometry in combination with a glycerol or water matrix is well-suited for the analysis of oligosaccharides.50−52 An IR-MALDI-o-TOF mass spectrum that was obtained from 40 pmol of lacto-N-fucopentaose II (LNFP II) is displayed in Figure 5. In contrast to standard UVand IR-MALDI-MS studies using the glycerol matrix, the oligosaccharides desorbed/ionized from water ice are predominantly detected as [M + H]+ and [M + NH4]+ species and only in minor abundance as [M + Na]+ and [M + K]+ adducts. Furthermore, doubly charged molecular ions of the neutral milk oligosaccharides are detected in sizable abundance. Additionally, only a minor loss of the labile bound fucose group is observed in the IR-MALDI-o-TOF spectra from water ice. Limits of Detection and Comparison to Related Desorption/Ionization Techniques. Essentially the same limits of detection (LOD) of a few hundred femtomoles are found for the IR-MALDI-MS analysis of peptides and oligosaccharides (data not shown). In contrast, in standard

Figure 5. IR-MALDI-o-TOF mass spectrum of lacto-N-fucopentaose II (LNFP II) acquired from a water ice matrix. A total of 40 pmol of oligosaccharides was consumed for generation of the mass spectrum. The spectra were recorded with the OPO laser system tuned to 2.94 μm; laser fluences were adjusted for optimal spectral quality.

UV-MALDI mass spectrometry, peptides are typically detected with a sensitivity that is several orders of magnitude higher. For peptides and denatured proteins, the current LOD of a few hundred femtomoles cannot currently compete with that achievable by UV-MALDI mass spectrometry; however, LODs are more compatible for the oligosaccharides. In light of the low detection efficiency of the o-TOF mass spectrometer for higher mass ions, we did not attempt to identify a more precise LOD for the protein samples. Under the given experimental conditions, the LOD of large proteins roughly falls into the 1 pmol range. Analysis of Food Samples. The employment of the cooling stage facilitates the IR-MALDI-MS analysis of waterrich biological tissue and might thus in the future be useful for MS imaging of “native” tissue slices containing endogenous tissue water. To test this possibility, we acquired a mass spectrum from a ∼1 mm thick frozen slice cut from a strawberry fruit (Figure 6a). Some ion signals are tentatively assigned with their possible identity, based on exact mass measurement and comparison with the METLIN databank (http://metlin.scripps.edu/); experimental and theoretical m/z values of the compounds are provided in Table S-1 in the Supporting Information. A low-energy collision-induced (CID) tandem mass spectrum of the ion at m/z 271, representing the plant pigment pelargonidin, an anthocyanidin, is shown in Figure 6b. Labeled ion signals represent fragment ion species that were previously identified in an ESI tandem MS study.53 The data demonstrate that structural characterization by lowenergy CID-MS of compounds desorbed/ionized by direct IRMALDI from tissue is possible.

■

CONCLUSIONS Employing a cooling stage in combination with o-TOF mass spectrometry provides some noteworthy analytical features, several of which are novel and/or surpassing those reported previously. The IR-MALDI-MS analysis of biomolecules using a standard commercial QSTAR instrument as mass analyzer becomes possible directly from frozen aqueous solutions and structurally conserved water-rich biological tissue. The analysis is distinguished by a particular softness, which seems to be approaching that achieved by ESI and LILBID mass spectrometry and, e.g., allowed to detect noncovalently bound holo-myoglobin. It remains to be tested whether even larger noncovalently complexes could also be amenable to the 5674



Article

unusual features better as well as to further improve the analytical potential of the technology.

■

ASSOCIATED CONTENT

S Supporting Information *

Additional material as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +49-2518356726. Fax: +49-251-8355121. Present Address †

Physikalisches Institut, University of Münster, WilhelmKlemm-Str. 10, 48149 Münster.

Author Contributions

The first two authors contributed equally to this work. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS We wish to thank GWU Lasertechnik, InnoLas Laser, Alexandre Loboda and AB SCIEX, Stefan Berkenkamp and Sequenom, as well as Franz Hillenkamp for support of the study. We are grateful to Werner Ens and Vic Spicer of the University of Manitoba for providing tofmulti software and Thorsten Deilmann for custom-made changes to the program. We thank Gottfried Pohlentz for the ESI-MS analysis of holomyoglobin and numerous helpful discussions, as well as Kristina Neue, Michael Mormann, Alfredo J. Ibáñez-Gabilondo, Joanne Y. Yew, and Thorsten W. Jaskolla for providing samples and/or for helpful discussions. This work was supported by the funds “Innovative Medical Research” of the University of Münster Medical School (Grants DR520805 and DR511003) and by the German Science Foundation (Grant DR416/8-1).

Figure 6. (a) IR-MALDI-MS analysis of a piece of strawberry fruit flesh. Selected ion signals are assigned with their putative identity based on exact mass values; cf. Table S-3 in the Supporting Information for a list of experimental and theoretical m/z values and tentative assignment of compounds. Mass spectra were acquired using the QSTAR mass spectrometer and an Er:YAG laser. Approximately 360 laser shots were applied over an area of ∼1 mm2. (b) Low-energy CID tandem MS spectrum of plant pigment pelargonidin; labeled ion signals correspond to species that were previously identified in a ESI tandem MS study.53 To record the MS/MS spectrum, 2400 laser shots were applied.

■

REFERENCES

(1) Berkenkamp, S.; Karas, M.; Hillenkamp, F. Proc. Nat. Acad. Sci. U.S.A. 1996, 93, 7003−7007. (2) Laiko, V. V.; Taranenko, N. I.; Berkout, V. D.; Yakshin, M. A.; Prasad, C. R.; Lee, H. S.; Doroshenko, V. M. J. Am. Soc. Mass Spectrom. 2002, 13, 354−361. (3) Rapp, E.; Charvat, A.; Beinsen, A.; Plessmann, U.; Reichl, U.; Seidel-Morgenstern, A.; Urlaub, H.; Abel, B. Anal. Chem. 2009, 81, 443−452. (4) Dreisewerd, K.; Draude, F.; Kruppe, S.; Rohlfing, A.; Berkenkamp, S.; Pohlentz, G. Anal. Chem. 2007, 79, 4514−4520. (5) Li, Y.; Shrestha, B.; Vertes, A. Anal. Chem. 2007, 79, 523−532. (6) Shrestha, B.; Vertes, A. Anal. Chem. 2009, 81, 8265−8271. (7) Ibáñez, A .J.; Scharte, J.; Bones, P.; Pirkl, A.; Meldau, S.; Baldwin, I. T.; Hillenkamp, F.; Weis, E.; Dreisewerd, K. Plant Meth. 2010, 6, 14. (8) Hunter, J. M.; Lin, H.; Becker, C. H. Anal. Chem. 1997, 69, 3608−3612. (9) Baltz-Knorr, M. L.; Schriver, K. E.; Haglund, R. F. Appl. Surf. Sci. 2002, 197, 11−16. (10) Leisner, A.; Rohlfing, A.; Berkenkamp, S.; Hillenkamp, F.; Dreisewerd, K. J. Am. Soc. Mass Spectrom. 2004, 15, 934−941. (11) Von Seggern, C. E.; Gardner, B. D.; Cotter, R. J. Anal. Chem. 2004, 76, 5887−5893. (12) Schneider, B.; Lock, C.; Covey, T. J. Am. Soc. Mass Spectrom. 2005, 16, 176−182. (13) Wattenberg, A.; Sobott, F.; Barth, H. D.; Brutschy, B. Int. J. Mass Spectrom. 2000, 203, 49.

IR-MALDI MS analysis if an optimized instrumentation was employed, including, for example, a high mass detector. Compared to previous studies in which water was used as a matrix for IR-MALDI-MS at ambient pressure2 or under highvacuum conditions,1,10 the accessible mass range is extended by about an order of magnitude and the analytical sensitivity is improved. It is likely that ion extraction conditions in the oMALDI 2 ion source contribute to these improvements. For instance, a large number of low-energy collisions between analyte-containing water clusters and buffer gas molecules could lead to favorable ion formation. It is speculated that the employment of suitable postionization techniques could lead to further improvement in sensitivity by making use of the vast majority of neutral particles that are ablated in the process. In combination with a fine-vacuum ion source, the use of a second pulsed high-power light source, either emitting UV54 or IRphotons10 could provide a means to enhance the ion yields for both small photoionized molecules and larger compounds ionized in a secondary MALDI process.10 Another noteworthy feature is the detection of highly charged peptide and oligosaccharide ions, a feature that can facilitate structural analysis by tandem mass spectrometry. Clearly, additional future experiments are needed to better understand these 5675



Article

(48) Loo, J.; Giordani, A. B.; Muenster, H. Rapid Commun. Mass Spectrom. 1993, 7, 186−189. (49) Gross, D. S.; Zhao, Y. X.; Williams, E. R. J. Am. Soc. Mass Spectrom. 1997, 8, 519−524. (50) Von Seggern, C. E.; Moyer, S. C.; Cotter, R. J. Anal. Chem. 2003, 75, 3212−3218. (51) Von Seggern, C. E.; Moyer, S. C.; Cotter, R. J. J. Am. Soc. Mass Spectrom. 2003, 14, 1158−1165. (52) Dreisewerd, K.; Berkenkamp, S.; Kölbl, S.; Peter-Katalinić, J.; Pohlentz, G. J. Am. Soc. Mass Spectrom. 2006, 17, 139−150. (53) Kammerer, D.; Carle, R.; Schieber, A. Rapid Commun. Mass Spectrom. 2003, 17, 2407−2412. (54) Hanley, L.; Zimmermann, R. Anal. Chem. 2009, 81, 4174−4182.

(14) Morgner, N.; Hoffmann, J.; Barth, H. D.; Meier, T.; Brutschy, B. Int. J. Mass Spectrom. 2008, 277, 309−313. (15) Morgner, N.; Zickermann, V.; Kerscher, S.; Wittig, I.; Abdrakhmanova, A.; Barth, H. D.; Brutschy, B.; Brandt, U. Biochim. Biophys. Acta Bioenerg. 2008, 1777, 1384−1391. (16) Steiner, K.; Pohlentz, G.; Dreisewerd, K.; Berkenkamp, S.; Messner, P.; Peter-Katalinić, J.; Schäffer, C. J. Bacteriol. 2006, 24, 7914−7921. (17) Nemes, P.; Vertes, A. Anal. Chem. 2007, 79, 8089−8106. (18) Shrestha, B.; Vertes, A. Anal. Chem. 2009, 81, 8265−8271. (19) Coon, J. J.; Harrison, W. W. Anal. Chem. 2002, 74, 5600−5605. (20) Dreisewerd, K.; Müthing, J.; Rohlfing, A.; Meisen, I.; Vukelić, Z.; Peter-Katalinić, J.; Hillenkamp, F.; Berkenkamp, S. Anal. Chem. 2005, 77, 4098−4107. (21) Soltwisch, J.; Souady, J.; Berkenkamp, S.; Dreisewerd, K. Anal. Chem. 2009, 81, 2921−2934. (22) Soltwisch, J.; Dreisewerd, K. Rapid Commun. Mass Spectrom. 2011, 25, 1266−1270. (23) Menzel, C.; Dreisewerd, K.; Berkenkamp, S.; Hillenkamp, F. Int. J. Mass Spectrom. 2001, 207, 73−96. (24) Dreisewerd, K.; Berkenkamp, S.; Leisner, A.; Rohlfing, A.; Menzel, C. Int. J. Mass Spectrom. 2003, 226, 189−209. (25) Bertie, J. E.; Ahmed, M. K.; Eysel, H. H. J. Phys. Chem. 1989, 93, 2210−2218. (26) Bertie, J. E.; Labbé, H. J.; Whalley, E. J. Phys. Chem. 1969, 50, 4501−4520. (27) Focsa, C.; Mihesan, C.; Ziskind, M.; Chazallon, B.; Therssen, E.; Desgroux, P.; Destombes, J. L. J. Phys.: Condens. Matter 2006, 18, S1357−S1387. (28) Feldhaus, D.; Menzel, C.; Berkenkamp, S.; Hillenkamp, F.; Dreisewerd, K. J. Mass Spectrom. 2000, 35, 1320−1328. (29) Rohlfing, A.; Menzel, C.; Kukreja, L. M.; Hillenkamp, F.; Dreisewerd, K. J. Phys. Chem. B 2003, 107, 12275−12286. (30) Rohlfing, A.; Leisner, A.; Hillenkamp, F.; Dreisewerd, K. J. Phys. Chem. C 2010, 114, 5367−5381. (31) Dreisewerd, K.; Schürenberg, M.; Karas, M.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1996, 154, 171−178. (32) Westmacott, G.; Ens, W.; Hillenkamp, F.; Dreisewerd, K.; Schürenberg, M. Int. J. Mass Spectrom. 2002, 221, 67−81. (33) Leisner, A.; Rohlfing, A.; Rö hling, U.; Dreisewerd, K.; Hillenkamp, F. J. Phys. Chem. B 2005, 109, 11661−11666. (34) Fan, X.; Murray, K. K. J. Phys. Chem. A 2010, 114, 1492−1497. (35) Apitz, I.; Vogel, A. Appl. Phys. A: Mater. Sci. Process. 2005, 81, 329−338. (36) Vogel, A.; Venugopalan, V. Chem. Rev. 2003, 103, 577−644. (37) Zhigilei, L. V.; Garrison, B. J. J. Appl. Phys. 2000, 88, 1281− 1298. (38) Cramer, R.; Corless, S. Proteomics 2005, 5, 360−370. (39) Schürenberg, M.; Dreisewerd, K.; Kamanabrou, S.; Hillenkamp, F. Int. J. Mass Spectrom. Ion Processes 1998, 172, 89−94. (40) Zhigilei, L. V.; Garrison, B. J. Appl. Phys. A: Mater. Sci. Process. 1999, 69, S75−S80. (41) Fan, X.; Little, M. W.; Murray, K. K. Appl. Surf. Sci. 2008, 255, 1699−1704. (42) Dreisewerd, K.; Schürenberg, M.; Röhling, U.; Hillenkamp, F. Proceedings of the 50th ASMS Conference on Mass Spectrometry and Allied Topics, Orlando, FL, June 2−6, 2002. (43) Seyfried, B. K.; Siekmann, J.; Belgacem, O.; Wenzel, R. J.; Turecek, P. L.; Allmaier, G..; Turecek, P. J. Mass Spectrom. 2010, 45, 612−617. (44) Winger, B. E.; Hofstadler, S. A.; Bruce, J. E.; Udseth, H. R.; Smith, R. D. J. Am. Soc. Mass Spectrom. 1993, 4, 566−577. (45) Dreisewerd, K.; Rohlfing, A.; Spottke, B.; Urbanke, C.; Henkel, W. Anal. Chem. 2004, 76, 3482−3491. (46) Von Seggern, C. E.; Cotter, R. J. J. Mass Spectrom. 2004, 39, 736−742. (47) Dybvik, A.; Norberg, A. L.; Schute, V.; Soltwisch, J.; PeterKatalinić, J.; Vaarum, K.; Eijsink, V.; Dreisewerd, K.; Mormann, M.; Sørlie, M. Anal. Chem. 2011, 83, 4030−4036. 5676


Ionization Orthogonal-Time

Recommend Documents