MALDI MS and ICP MS Detection of a Single CE Separation Record: A ...

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MALDI MS and ICP MS Detection of a Single CE Separation Record: A Tool for Metalloproteomics Iva Tomalová,†,‡ Pavla Foltynová,‡ Viktor Kanický,†,‡ and Jan Preisler*,†,‡ †

Central European Institute of Technology (CEITEC), Masaryk University, Brno, Czech Republic Department of Chemistry, Faculty of Science, Masaryk University, Brno, Czech Republic

‡

S Supporting Information *

ABSTRACT: In this work, a novel approach based on off-line coupling of a single run of capillary electrophoresis (CE) separation to both matrix-assisted laser desorption/ionization (MALDI) and substrate-assisted laser desorption inductively coupled plasma (SALD ICP) mass spectrometry (MS) is presented. Using a liquid junction and subatmospheric deposition chamber, CE fractions were extracted from a separation capillary and collected as 20-nL droplets on a custom-built polyethylene terephthalate glycol (PETG) target plate coated with a 10-nm gold layer which guaranteed compatibility with both MALDI and SALD ICP techniques. The MALDI matrix solution was then added to the produced spots. After it was dried, the separation record was consecutively analyzed in MALDI MS and ICP MS instruments. Thus, both proteomic and metallomic information was obtained off-line from a single CE run. The concept was demonstrated by the analysis of a mixture of rabbit liver metallothionein isoforms. In an additional study, the droplets representing the archived separation record were alternately mixed with two different MALDI matrices to obtain complementary information on both the apoproteins and their complexes with metals from a single separation run. The presented technique is a viable alternative to online coupling of column separation to electrospray MS and nebulizer ICP MS.

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under analysis is entirely lost rendering species identification impossible. The molecule-specific analysis such as electrospray mass spectrometry (ESI MS) is therefore routinely applied in order to identify the unknown protein species.10,11 Owing to the soft character of ionization, ESI MS12 allows for the detection of multiply charged molecular ions, including noncovalent complexes, without any fragmentation.13 Regrettably, the sensitivity of ESI MS is strongly affected by the use of volatile salt buffers and solubilizing detergents, which are necessary to preserve metal-protein integrity and stability. Overall, combining ICP MS and ESI MS is often considered to be a dream team in this field, providing complementary data to comprehensive proteomic analysis.14 However, the use of replicate separation steps for both ICP and ESI MS analyses increases sample consumption, analysis time, and introduces potential errors. Replicate separation runs may not be fully reproducible. This problem can be solved by splitting the flow of effluent between ESI MS and ICP MS instruments which, in this case, must be placed side by side.15,16 Rogers et al. designed and constructed a dual-source time-of-flight (TOF) MS prototype equipped with both ESI and ICP ion sources.17 Straightforward online coupling is usually preferred in the metalloproteomic field with the exception of planar separation

etals play a crucial role in physiology and pathology of biological systems. While only a fraction of the metals in biological fluids and organs of living systems exists in a form of free cations or minerals, most of the metals are associated with various proteins or other ligands. It has been estimated that the metalloproteins comprise about one-third of all proteins. These proteins participate in the regulation of protein expression, in metal transport and homeostasis or detoxification processes.1,2 Therefore, the studies focused on the comprehensive characterization of the metal−protein or metal−metabolite complexes are becoming increasingly important. In the past decade, the research field of metalloproteomics has undergone rapid development. Advanced analytical approaches have been applied to enable characterization and identification of the metal−protein or metal−bioligand complexes usually existing in complex biological systems at trace level concentrations.3 Hyphenated techniques that combine separation with both sensitive and specific detection have proven as the most relevant.4 The most frequently used methods typically rely on the online coupling of high-resolution separation such as liquid chromatography (LC) or capillary electrophoresis (CE) with an inductively coupled plasma mass spectrometry (ICP MS).5−8 ICP MS can provide robust, highly sensitive, quantitative, both elemental and isotopic information on metals and several nonmetals.9 However, during the atomization and ionization process, the information on amino acid composition of proteins © 2013 American Chemical Society

Received: September 15, 2013 Accepted: November 27, 2013 Published: November 27, 2013 647

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techniques such as nondenaturing gel electrophoresis18,19 or thin-layer chromatography20,21 that are naturally coupled offline to laser ablation (LA) ICP MS. Ballihaut et al. demonstrated the potential of the off-line coupling for multiple detection techniques.22 After locating the selenium-containing proteins in gel by LA ICP MS, selected proteins were extracted from the gel and identified by matrix-assisted laser-desorption/ ionization (MALDI) MS23 or nanoLC-ESI tandem mass spectrometry. The off-line coupling of column separation to LA ICP MS for metallomics is rarely performed.24 The benefit of the off-line interfacing strategy is the decoupling of the separation process from the detection one in both time and space.25,26 In combination with microcolumn separations, the effluent is deposited in the form of droplets or continuous streaks on a suitable target. Such a separation record is predestined for the most common off-line ionization techniques: MALDI or LA ICP. The decoupling of separation and ionization has the advantage of ruggedness from a single separation run and archiving that are routinely demonstrated in gel-based separations. It also overcomes many shortcomings of the online setup which originate from the constraints upon the required time duration of MS and MS/MS data acquisitions. Off-line storage of column effluent allows sample deposition to occur at the optimal speed matching that of high-resolution LC separation, while the mass spectrometer can operate with faster or slower data acquisition rates as needed to record the relevant MS and MS/MS information.25 In many cases, the separation record is not fully consumed in the initial MS analysis, so the fractions can be reanalyzed or used for additional analytical studies. In the last two decades, MALDI MS has been successfully coupled to both CE and LC separation using various deposition techniques such as contact deposition,27−30 piezospotting31 or electrospraying.32 MALDI TOF MS can analyze large numbers of deposited LC fractions in a short time interval33 and, compared to ESI MS, it is much less affected by the presence of acids (e.g., TFA), salts, and detergents in the effluents from separation columns. Because single-charged ions are generally formed in MALDI processes, MALDI gives simpler mass spectra. On the other hand, mass analysis of noncovalent interaction is known to be far more difficult using MALDI compared to ESI: in MALDI, the weak interaction has to survive both crystallization and the laser desorption/ionization event.34 Although MALDI has often been described as incapable of bringing any information about protein−metal complexes,3,35 several works reporting the opposite conclusion have been published.36−38 The success of detection of noncovalent complexes depends on pH, the type of applied MALDI matrix, or the way the spectra are accumulated.39,40 Recently, we have introduced an off-line coupling of CE to substrate-assisted laser desorption (SALD) ICP MS.41 The fractions of the CE effluent were stored on a polyethylene terephthalate glycol (PETG) sample plate in the arrangement that was earlier used for CE-MALDI MS.30 At relatively low laser power density (∼70 MW·cm−2), plate material acts as a strongly absorbing substrate and contributes to complete sample ablation/desorption necessary for quantitative elemental analysis. The potential of this method was demonstrated on a rapid, high-resolution separation of CrIII/CrVI species. In this work, we present a new approach for metalloproteomic studies based on an off-line coupling of microcolumn separation with multiple detection methods. A mixture

of rabbit liver metallothionein (MT) isoforms was chosen as a model system. Since these noncovalent metal-protein complexes are known to dissociate in acidic conditions,42 a pHneutral separation system was used to separate MTs by CE. The CE-MS off-line coupling interface utilized a liquid junction and subatmospheric deposition chamber. The fractions of CE effluent were deposited on disposable PETG targets plated with a thin gold film, which made them usable for both MALDI and SALD ICP MS analyses, and covered with a solution of MALDI matrix. MALDI MS consumed only a small amount of material from the individual dried droplets and the separation record was consequently subjected to SALD ICP MS thus providing quantitative elemental analysis. A design of the sample target and experimental conditions are discussed in detail.

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EXPERIMENTAL SECTION Chemicals. Solvents, acetonitrile and methanol, were purchased from Fluka (Seelze, Germany). A solution of NaOH (Merci, Brno, Czech Republic) was used for capillary treatment. Ammonia solution, ammonium bicarbonate, trifluoroacetic acid (TFA), and formic acid were purchased from Sigma (St. Louis, MO, USA). Acidic MALDI matrices α-cyano4-hydroxycinnamic acid (CHCA); 2,5-dihydroxobenzoic acid (DHB); and peptide calibration standard mixture (#206195) were purchased from Bruker Daltonics (Bremen, Germany). Nonacidic MALDI matrices 6-aza-2-thiothymine and p-nitroaniline (Lachema, Brno, Czech Republic) were purified using recrystallization before the MALDI experiments. Rabbit liver MT standards (MT-1 and MT-2) were obtained in acetylated forms from Enzo Life Sciences (Lausen, Switzerland). Certified reference element standard (100 mg·L−1) in 2% nitric acid was purchased from Analytika (Prague, Czech Republic). Polytetrafluoroethylene (DuPont Teflon PTFE DISP 30, Wilmington, DE, U.S.A.) was used for capillary tip coating. All reagents were of the highest available purity. Doubly distilled deionized water was used throughout all the experiments. Sample Plates. The PETG sheet (0.5 mm thick, Bayer sheet, Darmstadt, Germany) was cut into 50 × 25 mm rectangles and coated with layer of gold with a thickness of 1− 50 nm using vacuum sputter coater (SCD 500, Bal-TEC AG, Lichtenstein). This plate was mounted using a conductive copper tape onto an adapted massive MTP 384 target (Bruker Daltonics) and used for further experiments. CE with UV Detection. CE experiments were carried out using a laboratory-built CE system consisting of high voltage (0−30 kV) power supply (model CZE1000R, Spellman, Hauppauge, NY, USA); platinum wires served as the electrodes. CE separation was conducted in uncoated fused silica capillaries (Polymicro Technologies, Phoenix, AZ, U.S.A.) of 70 cm/63 cm in total/effective length and 75 μm in inner diameter. The capillaries were preconditioned with 1-M NaOH for 10 min and flushed with water for 30 min. After each sample was run, the capillary was regenerated using a 0.1-M NaOH flush for 10 min, followed by 3 min of flushing with the background electrolyte (20 mM NH4HCO3/HCOOH; pH 6.5−8.2). The samples were injected hydrodynamically from an unbuffered solution by pressure 2 kPa for 6 s. Electrophoresis was driven at −285 V·cm−1 giving current of 30 μA. Absorbance was measured at 205 nm using a variable wavelength detector Spectra 100 (Spectra Physics, Ontario, Canada). CE Fraction Collection for MALDI MS/SALD ICP MS. The fraction collection for the following MALDI MS/SALD ICP MS analyses was carried out in the manner reported 648

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Figure 1. Off-line coupling of CE(UV)-MALDI MS/SALD ICP MS. (a) Experiment workflow. (b) Effluent droplet deposition on an Au-PETG sample target with a PTFE-coated, tapered capillary tip. (c) CE fractions with MALDI matrix deposited on sample plate. (d) Attachment of the AuPETG plate to a standard MALDI target. (e) Sample plate after SALD ICP MS measurement.

detector by a pair of 1 m long optical fibers. A wavelength of 214 nm was used for absorbance measurements. Peptide standard mixture and MT-2 (0.1 mg·mL−1 in 20-mM NH4HCO3/HCOOH, pH 7.4) were used to optimize MALDI MS measurement parameters, MALDI matrix deposition process, and performance of the Au-PETG target. Their ∼20 or ∼25 nL aliquots were deposited onto stainless steel or AuPETG targets as separate droplets spaced by 1 mm for 20 nL droplets or by 2 mm for 25 nL droplets. The liquid flow was provided by infusion of the liquid into the subatmospheric deposition chamber. Aliquots of the same volume of the standard element solutions containing 1 mg·L−1 of Cd were deposited on the uncoated PETG and Au-PETG plates for SALD ICP MS performance comparison of the plates. A study of the effect of the MALDI MS measurements on the subsequent SALD ICP MS measurements was performed with 20-nL aliquots of MT-2 (0.1 mg·mL−1 in 20 mM NH4HCO3/HCOOH, pH 7.4). MALDI Matrix Deposition. After deposition of the CE fractions or direct deposition of the standard solution of peptides, MT-2 or Cd, each spot was overlaid with 26.5 mM CHCA solution (in 50% (v/v) ACN and 1% (v/v) TFA) in the subatmospheric deposition chamber. DHB solution (65 mM; in 50% (v/v) ACN and 0.05% (m/v) NH3 solution) was added to each aliquot for detection of metal−protein complexes. The volume of added matrix solution was equal to the volume of the deposited CE fraction/standard droplets, that is, 20 or 25 nL. MALDI TOF MS. All mass spectra were acquired using MALDI TOF/TOF mass spectrometer (Autoflex Speed, Bruker Daltonics) equipped with a 1-kHz, 355-nm ”smartbeam” Nd:YAG laser. The Au-PETG sample plate was mounted on an adapted massive MTP 384 sample target. The mass spectrometer was operated in the reflector positive ion mode with the accelerating, first plate, and reflector voltages at 19.00, 16.75, and 21.00 kV, respectively. The delayed extraction time was set to 200 or 250 ns for Au-PETG or stainless steel sample targets. Each mass spectrum was generated by averaging 100 laser shots from 10 different

earlier.41 Briefly, the separation capillary was connected to a deposition capillary via a grounded liquid junction43 fabricated from polycarbonate filled with the background electrolyte. PEEK liners (Valco Instruments Co., Houston, TX) were inserted into holes drilled in the polycarbonate block to precisely align the separation and deposition capillaries, leaving a gap of approximately 50 μm between the capillaries. The fused silica deposition capillary (Polymicro Technologies; 18 cm length, 26, 30, and 50 μm i.d., all 360 μm o.d.) inserted in a laboratory-built subatmospheric deposition chamber (kept at 20 kPa pressure) served for collection of effluent fractions onto a sample target fastened to an XY-stage. Flat cut or tapered fused silica capillaries were tested for deposition. The tapered capillaries were fabricated by mechanical grinding using a laboratory-built grinding machine. The deposition ends of all capillaries were coated with PTFE in accordance with recommendations from the manufacturer to prevent tip wetting and undesirable carry-over effects. The optimum operation conditions (capillary i.d., buffer, separation voltages, and injection mode) were taken from the CE-UV study described above. CE effluent was spotted on the sample plate as discrete 2-s fractions along a serpentine trail with 1-mm spacing in both directions. A fiber optic UV detector mounted 7 cm upstream of the outlet end of the separation capillary was used for the immediate monitoring of the separation process. The detector was constructed similarly to that described previously.44,45 Briefly, the above-mentioned Spectra 100 detector was equipped with optical fiber holders in place of the original flow cell and the amplification of the transimpedance amplifier of the detector was increased by a factor of 20 to compensate for the light intensity loss in the optical fibers. The fiber optic detection cell was constructed out of two aluminum disks (diameter 40 mm; thickness 6 mm) and the SMA adapter (905/906; Amphenol, Danbury, CT, U.S.A.). The capillary passed through the center of the SMA adapter via 0.4-mm holes drilled in the walls; two stainless steel tubes were used as the guides for the capillary. The cell was connected with the 649

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undesirable memory effects and allows long-term archiving of the CE (or LC) separation records. Target Performance Evaluation. The MALDI MS performance of the Au-PETG target was evaluated using 20nL aliquots of standard solution containing peptide calibration standard and MT-2 spotted and covered with small aliquots of solvents containing MALDI matrix. In terms of sensitivity, the Au-PETG target showed comparable results to the stainless steel one: for a mixed solution of MT-2 isoforms, the detection limits were 2 and 1 μg·mL−1 for the Au-PETG and stainless steel targets, respectively, which correspond to the low femtomole range for individual isoforms. The delayed extraction time had to be adjusted from 250 to 200 ns for the Au-PETG target, nevertheless, 80−95% of mass resolution values attained using stainless steel targets were repeatedly achieved. As expected, certain amount of flexibility in the PETG target and a less than perfect mounting affected the mass accuracy in the described arrangement. Mass error did not exceed 200 ppm when traveling over a 3-cm distance along the surface of the Au-PETG target using the external calibration. The MTP slide adapter II (Bruker Daltonics), originally designed for MS imaging, was also tested as a possible target frame. The missing bottom support, however, resulted in inefficient Au-PETG plate alignment causing a mass error up to 750 ppm over 3 cm and loss of peak resolution across the sample target. In SALD ICP MS measurements of 12 spots with deposited standard Cd solution (20 nL, 1 mg·L−1), a slight increase in the integrated 111Cd ion signal from (110 ± 8) ×103 to (130 ± 8) × 103 arbitrary units (a.u.) was observed when the Au-PETG targets were used instead of bare PETG plates. A possible reason for this increase is the lower amount of ablated organic material and thus lower ionization suppression. The addition of CHCA (26.5 mM) as a MALDI matrix caused an insignificant change in the integrated 111Cd ion signal to (124 ± 8) × 103 a.u., most likely due to the same suppression phenomenon. One the basis of long-term observations, matrix addition resulted in a higher relative standard variation of the integrated 111 Cd ion signal acquired from spots deposited on Au-PETG; the relative standard variation increased typically from 6% to 10%. Model System Selection. Rabbit liver metallothioneins (MTs) were selected as a model system for investigation of the overall performance of off-line coupling of CE with MALDI MS/SALD ICP MS analyses. MTs form a class of small, evolutionary highly conserved metalloproteins characterized by a high cysteine content (up to 30% residues) that allows noncovalent binding of a wide range of transition and heavy metal ions.46 MT isoforms differ in only a few amino acids and thus high performance separation methods, such as CE, have often been used for the MT separation.47,48 The nanoliter scale injection volume corresponding to picomolar quantity together with molecular weight of 6−7 kDa poses a challenge for MALDI TOF MS in terms of both sensitivity and resolving power. Choice of Separation Conditions. MT speciation by CE has been addressed in literature many times. However, most works relied on simple UV detection or online coupling with ESI MS or ICP MS detection.48 In our case, the use of the three coupled systems, CE, MALDI MS, and SALD ICP MS, imposed certain restrictions regarding the choice of the electrophoretic buffer: the prerequisite for the MS detection of protein-metal complexes is preserving the MT native

positions across the spot, resulting in a total 1000 laser shots. The CE separation records were acquired fully automatically using WARP-LC 1.2 software (Bruker Daltonics) using the custom geometry with 1-mm spacing. The mass spectra were minimally smoothed using a Savitzky−Golay filter (1 cycle, smoothing window width of m/z 0.2); baseline was subtracted. SurveyViewer software (Bruker Daltonics) was used for visualization of CE separation and data analysis. SALD ICP MS. After MALDI MS analysis, the Au-PETG plate with sample spots was inserted into an ablation system (model UP 213, New Wave Research, Inc., Fremont, CA, U.S.A.) consisting of Q-switched Nd:YAG laser operating at 213 nm, movable ablation cell (model SuperCell) and built-in microscope/CCD camera system for visual control of samples during desorption. Sample spots were scanned by the laser beam with the beam waist adjusted to ∼250 μm. Sample spots were desorbed at 0.7 J·cm−2 laser fluence, at 10-Hz frequency, and 200 μm·s−1 scan rate. The regime for the serpentine scanning raster with 200-μm spacing was selected in accordance to the spot diameter to cover the entire sample area. The ablation cell was flushed with a carrier gas (helium, 1.0 L min−1 flow rate), which transported the aerosol to an ICP MS (model 7500 CE, Agilent Technologies, Inc., Santa Clara, CA, U.S.A.). A sample gas flow of argon was admixed to the helium carrier gas subsequent to the LA cell (0.6 L·min−1). Both the flush and laser warm-up times were set to 4 s. The ions (13C and 111Cd) were measured with the integration time 0.1 s per isotope at a frequency 3.3 Hz. The 13C ion signal was monitored to indicate desorption and to set the interval for integration of the 111Cd ion signal accordingly.

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RESULTS AND DISCUSSION We propose a novel approach for complementary molecular and element-specific detection that combines two methods developed previously, CE-MALDI MS30 and CE-SALD ICP MS.41 Compared to the original CE-SALD ICP MS arrangement, in which the deposition of the separated fractions was followed directly by the SALD ICP MS detection step, MALDI MS detection requires an additional application of the MALDI matrix, modifications to the sample plate and an adjustment of the liquid junction interface to gain maximum sensitivity. The experiment workflow is shown in Figure 1. Sample Target Design. The previous approach employed the deposition of droplets of the CE effluent onto a suitable substrate (PETG) essential for efficient sample desorption that was followed by ICP MS analysis. To ensure electrical conductivity of the PETG plate necessary for MALDI TOF MS, the plates were sputtered with a thin conductive metal layer. Gold was chosen as the most suitable metal for its availability in appropriate purity, high chemical inertness and good electrical conductivity. Being neither a biogenic nor toxic element, it is rarely monitored in biological systems. As one of the higher atomic number elements, which occur naturally with a single isotope, it does not cause serious spectral interferences in ICP MS. A gold layer thickness of ∼10 nm was found to be optimal to retain the target transparency for visual sample inspection. Also, it did not prevent ablation of substrate material (PETG) after laser irradiation; no signal reduction was observed by comparison of the integrated signal of Cd standard solution on the uncoated PETG plate and Au-PETG plates. Furthermore, it allowed MALDI MS measurements at high laser repetition rates without any observable surface charging effects. Disposability of the plates eliminates the risks of 650

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Effect of MALDI MS Measurements on Consequent SALD ICP MS Ones. The sample consumption during MALDI MS detection was examined using 20-nL aliquots of the MT-2 (0.1 mg·mL−1 in 20 mM NH4HCO3/HCOOH, pH 7.4) overlaid with CHCA solution. Five sets, each consisting of 10 spots, were exposed to 0, 500, 1000, 2500, and 5000 laser shots in steps of 100 shots across the whole sample spots in the MALDI TOF mass spectrometer and then analyzed by SALD ICP MS. The laser power was set to the same value as used in other MALDI MS measurements. The values of the integrated 111 Cd ion signal gradually decreased from (545 ± 56) × 103 to (474 ± 39) × 103 a.u. from 0 to 5000 laser shots, but they did not differ substantially. A total number of 1000 laser shots was therefore selected as a compromise between sufficient MALDI mass spectra quality in automated runs and minimal sample consumption before subsequent SALD ICP MS measurement. CE(UV)-MALDI MS/SALD ICP MS. A 14 ng MT mixture corresponding to 2.2 pmol in total was injected and separated under the optimal conditions. The separation process was monitored online by a fiber optic UV detector and fractions at migration time tmig = 7−12 min were collected in 2-s intervals on the Au-PETG target, and covered with MALDI matrix solution. The total number of the collected fractions was 150. The separation record was then subjected to consecutive MALDI MS and SALD ICP MS analyses. The UV trace, MALDI MS and ICP MS electropherograms recorded from a single separation are shown in Figure 2. It should be noted that

structure. Since metals are known to dissociate in acidic conditions, physiological pH range (6−8) was investigated. With regard to the maximum sensitivity, MALDI MS requires the use of volatile buffers. Another limitation is the electric current for CE separation; it is well-known that Joule heating of the capillary negatively affects the separation efficiency. To the best of our knowledge, no CE-MALDI MS of MTs has been reported earlier and thus, the separation system was designed accounting for these requirements as well as earlier published works on CE-ICP MS and CE-ESI MS of MTs.10,49,50 NH4HCO3/HCOOH buffer (20 mM) was preferred to other buffer solutions as it provides buffering capacity in the required pH range and sufficient volatility. Using CE-UV, the highest separation efficiency was obtained at pH 7.4 (data not shown). CE-MS Interface. The off-line hyphenation of CE to mass spectrometry analysis was performed by using a liquid junction and subatmospheric deposition chamber. The optimization of the interface focused on preserving the high separation efficiency of CE and minimizing the postcolumn dilution. Capillaries with inner diameters 26, 30, and 50 μm were tested for effluent deposition. The use of 50 μm (i.d.) capillaries caused excessive dilution and thus negatively affected the method sensitivity that has proven to be the challenging factor primarily for MALDI MS; 26-μm (i.d.) capillaries were troublesome because of their relatively frequent clogging. The 30-μm (i.d.) capillaries with the length of 18 cm were found to be the optimal choice for effluent deposition. For 2-s fractions, the approximate aqueous droplet volume 20 nL was calculated using Poiseuille’s equation for given capillary i.d., length and pressure difference between the atmosphere and the subatmospheric chamber. The ∼50-μm spacing between the separation and deposition capillary in the liquid junction was found to be optimal as it guaranteed efficient analyte transfer and, at the same time, eliminated induction of the laminar flow thus preserving the high sensitivity and separation efficiency of CE. MALDI Matrix Deposition. It is well-known that the selection of matrix and sample preparation techniques are two imperatives for successful MALDI MS experiments. CHCA was chosen as the most appropriate matrix in terms of sensitivity and reproducibility; at given conditions (26.5 mM CHCA in 50% ACN and 1% TFA; drying at 20 kPa, room temperature), homogeneous spots applicable for automated MALDI MS were formed. Most reported CE-MALDI MS experiments rely on matrix prespotted or precoated targets,27,51,52 on the matrix employed in the CE buffer53 or in the sheath liquid for droplet formation.54 In our implementation, prespotting resulted in poor mass spectra; we put this outcome down to the limited solubility of CHCA in the water-based environment of the CE effluent. Therefore, the reverse order of the matrix-sample deposition was applied, which also enabled additional acidification by TFA to increase MALDI MS sensitivity. This order, however, resulted in non-negligible analyte carry-over up to ∼15% that was observed in the MALDI mass spectra when the 25-nL aliquots of standard MT-2 solution deposited with 2-mm spacing were covered by a solution containing CHCA with 1mm spacing using the flat cut capillaries. Therefore, tapered tips coated with PTFE, which prevented the tip wetting, were tested. With these tips, no analyte carry-over was noticed in the MALDI mass spectra and the SALD ICP MS integrated 111Cd ion signal revealed in average only 1.2% carry-over, never exceeding 3.8%. Moreover, the droplet formations on the narrowed capillary tip and deposition process were easier to control visually (Figure 1b).

Figure 2. (a) CE-UV (214 nm) electropherogram, (b) MALDI MS extracted ion electropherograms for selected m/z, (c) 2D MALDI MSelectropherogram, and (d) SALD ICP MS-electropherogram for m/z = 111 of a single CE run of MT isoform mixture. 651

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Figure 3. Typical MALDI mass spectrum of MT-1 acquired from the CE separation record (tmig = 8.35 min) on an Au-PETG sample target. Inset shows isotopic pattern of the individual MT2d subisoform as an example.

the fiber optic detector was positioned 7 cm upstream from the liquid junction whereas CE effluent for both the off-line MS detectors was taken at the end of the separation capillary. The time scale in UV electropherogram was therefore adjusted by multiplying the tmig by a factor of 70/63 for easy comparison of all electropherograms. In both the MS electropherograms, the time scales were adjusted by subtracting the time needed for analyte transfer through the deposition capillary, which was calculated from Poiseuille’s equation. The UV electropherograms (Figure 2a) revealed, similar to electropherograms reported elsewhere,10,55 two dominant peaks and a minor peak which can be assigned to MT-1 and MT-2 isoforms on the basis of the migration times of the individual species. Nevertheless, the positive identification of the peaks merely from the UV trace is impossible. Using the CHCA as a MALDI matrix under acidic condition, MALDI MS data (Figure 2b and 2c) revealed the information about MT apoforms. The peaks observed in the UV electropherograms were identified as MT-1 and MT-2 based on their molecular weight. The first two peaks (tmig = 8.4 and 8.7 min) were identified as MT-1 encompassing MT1a, MT2d and MT2e isoforms detected at m/z 6145.2, 6215.3, and 6241.3 (the most dominant peak in the isotopic patterns). The last peak (tmig = 9.2 min) was assigned as MT-2 containing isoforms MT2a, MT2b and MT2c which were detected at m/z 6125.2, 6146.2, and 6155.2. Although CE did not have the power to separate MT-1 or MT-2 individual subisoforms in uncoated capillaries, the importance of CE separation is evident; the MT1a and MT-2b with the most dominant peaks at m/z 6145.2 and 6146.2, respectively, would overlap with their isotopic patterns without CE separation. A typical MALDI mass spectrum of MTs acquired from separation is shown in Figure 3. The detected forms corresponded well to the MT specification from supplier (see Supporting Information for MT specification). This arrangement provides certain advantage over the online coupling for the detection of MT-apoforms. Mounicou et al., whose work dealt with MT analysis using the online hyphenation of CE at neutral pH to both ICP MS or ESI MS,10 described that the efforts of effluent postcapillary acidification with acidic makeup flow failed because of insufficient mixing and the perturbation of the separation process. Only the MT-metal complexes were detected in the online arrangement. With the MT capacity of binding from 0 to 7 various metals with close atomic masses, the acquired mass

spectra were difficult and in some cases even impossible to interpret without a correlation with molecular masses of the corresponding MT apoforms. SALD ICP MS data (Figure 2d) of the integrated 111Cd ion signal were found to be in agreement with the UV and MALDI MS detection. Since the 111Cd ion trace followed the UV signal record, it has been used for evaluation of effluent and matrix deposition. This indicated that the separation efficiency was virtually unaffected. No substantial peak tailing as a result of a memory effect of the deposition capillary that was reported elsewhere56 or analyte carry-over during the matrix deposition step were observed in the SALD ICP MS electropherogram. Cadmium content was quantified using the calibration curve method. The reference Cd standard solution was diluted in water to concentrations of 0.1, 1.0, and 10 mg·L−1; 20 nL fractions of each solution were deposited in 10 replicates for each concentration on the same Au-PETG target and covered with a CHCA solution under the optimized conditions described above. The total Cd amount determined from the resulting calibration curve (R = 0.9997) was 600 ± 90 pg that corresponded well to the expected value on the basis of manufacturer’s specification as well as with the MT capacity to bind 0−7 metal ions. Detection of Metal−Protein Complexes. To fully exploit the potential of the off-line coupling and gain even more information from a single separation record, nonacidic MALDI matrices 6-aza-2-thiothymine or p-nitroaniline and acidic matrices CHCA or DHB adjusted to neutral pH with ammonia solution were tested for detection of MT-metal complexes. DHB yielded the best results in terms of sensitivity and ability to detect noncovalent complexes. Aliquots (∼25 nL) of DHB adjusted with ammonia solution to neutral pH and CHCA acidified with 1% TFA were then applied on odd and even fractions of the single separation record, respectively. The same conditions as described above were used for MT separation and injection. The MALDI mass spectra of MT apoforms from CE fractions covered with CHCA matrix were acquired in an automated run in order to locate the zones with MTs. Afterward, because of the inhomogeneous DHB−analyte cocrystallization, the mass spectra of protein−metal complexes were acquired manually within the MT zones. The separation record was then subjected to SALD ICP MS measurement, which revealed the element specific information. The SALD ICP MS electropherogram and representative MALDI mass spectra from the two adjacent CE fractions are shown in Figure 652

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Figure 4. (a) SALD ICP MS electropherogram for m/z = 111 with corresponding MALDI mass spectra of (b) MT-1 apoforms including MT1a, MT2d, and MT2e subisoforms at m/z = 6145.2, 6215.3, and 6241.3, respectively, and (c) MT-1-Cd complexes acquired after application of CHCA/ TFA and DHB/NH3.

plate modifications are required to further improve mass accuracy of MALDI TOF MS measurements. To the best of our knowledge, this is the first report on CE-MALDI MS of MTs. We believe that the described off-line arrangement is a viable alternative to the more traditional online coupling using ESI and ICP techniques. The decoupling of the detection and separation steps using an archived separation record does not demand the operation of all instrumentation at a given place and time. It also offers greater analytical versatility through postcolumn sample treatment: for instance, it allows postcolumn acidification of effluent without concerns about its effect on CE separation. Importantly, we demonstrated that the alternate addition of different MALDI matrices to a sequence of sample aliquots containing a record of the CE separation provides unique complementary information.

4. While sensitive analysis of MT apoforms was achieved with CHCA (Figure 4b), the application of DHB under neutral conditions enabled the detection of metal-protein complexes. In addition to the peaks corresponding to MT apoforms, complexes of MTs with up to four Cd ions were detected, see Figure 4c. The shape of SALD ICP MS electropherogram (Figure 4a) is slightly deformed compared to the one in the above due to the use of two different MALDI matrices. Spot-tospot SALD ICP MS reproducibility was observed to be more affected when DHB was applied as a MALDI matrix; the relative standard deviation of the integrated 111Cd ion signal of ten 20-nL replicates of 1 mg·mL−1 Cd standard solution increased to 26%. Also, a higher number of laser shots and higher laser power had to be applied during the MALDI MS measurement when DHB was used instead of CHCA. Nevertheless, the characteristic pattern typical for an MT electropherogram is apparent.

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ASSOCIATED CONTENT

S Supporting Information *

CONCLUSION The present study demonstrates a novel analytical approach of employing three detection techniques: UV absorption, MALDI MS, and ICP MS, to characterize a single CE separation run. Off-line storage of CE separation was vital in obtaining complementary information using both MALDI MS and ICP MS techniques. Fractions of the effluent from the separation capillary were collected in a subatmospheric chamber on a PETG target coated with a 10-nm Au layer. A single Au-PETG target could accommodate up to 800 fractions, several tens of nanoliters each, from microcolumn separations. While the conductive Au layer provided compatibility with MALDI TOF MS, the PETG material acted as an efficient substrate in the subsequent SALD ICP MS analysis. The feasibility of the offline concept was demonstrated in a successful characterization of MT isoforms separated by CE and analyzed by MALDI MS and SALD ICP MS. The latter ionization technique delivered additional quantitative information about the metal distribution without performing repetitive separation runs or sample treatment. Although all the material in the separated fractions was consumed after the final SALD ICP MS step, the on-target digestion or laser-induced fluorescence as well as the combination of split-flow with other online detection techniques can be implemented to obtain further information from a single separation record. More elaborate ways of plate mounting inside a MALDI TOF mass spectrometer or target

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

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AUTHOR INFORMATION

Corresponding Author

*Address: Kamenice 5, 62500 Brno, Czech Republic. Tel.: +420-549-496-629. Fax: +420-549-492-494. E-mail: preisler@ chemi.muni.cz. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was conducted in CEITECCentral European Institute of Technology with research infrastructure supported by project CZ.1.05/1.1.00/02.0068 financed from The European Regional Development Fund. The authors gratefully acknowledge financial support from the Czech Science Foundation (GAP206/12/0538) and the Program of “Employment of Newly Graduated Doctors of Science for Scientific Excellence” (CZ.1.07/2.3.00/30.009) co-financed from European Social Fund and the state budget of the Czech Republic. The authors also thank Pavel Krásenský for his help with instrumentation development and service, as well as Bruker Daltonics software support team for providing customized 653

dx.doi.org/10.1021/ac402941e | Anal. Chem. 2014, 86, 647−654

Analytical Chemistry

Article

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