Compelling Evidence for Lucky Survivor and Gas Phase Protonation: The Unified MALDI Analyte Protonation Mechanism
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- Jaskolla, T.W. & Karas, M. J. Am. Soc. Mass Spectrom. (2011) 22: 976. doi:10.1007/s13361-011-0093-0
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This work experimentally verifies and proves the two long since postulated matrix-assisted laser desorption/ionization (MALDI) analyte protonation pathways known as the Lucky Survivor and the gas phase protonation model. Experimental differentiation between the predicted mechanisms becomes possible by the use of deuterated matrix esters as MALDI matrices, which are stable under typical sample preparation conditions and generate deuteronated reagent ions, including the deuterated and deuteronated free matrix acid, only upon laser irradiation in the MALDI process. While the generation of deuteronated analyte ions proves the gas phase protonation model, the detection of protonated analytes by application of deuterated matrix compounds without acidic hydrogens proves the survival of analytes precharged from solution in accordance with the predictions from the Lucky Survivor model. The observed ratio of the two analyte ionization processes depends on the applied experimental parameters as well as the nature of analyte and matrix. Increasing laser fluences and lower matrix proton affinities favor gas phase protonation, whereas more quantitative analyte protonation in solution and intramolecular ion stabilization leads to more Lucky Survivors. The presented results allow for a deeper understanding of the fundamental processes causing analyte ionization in MALDI and may alleviate future efforts for increasing the analyte ion yield.
Key wordsCHCAClCCADeuterationDeuteronationEsterIonizationMatrixPlume reactionsProton transfer
Although matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) [1–4] has found widespread use in the analysis of biological and biochemical samples, the essential analyte ionization step is not yet fully understood. Variations of experimentally accessible parameters such as, e.g., laser fluence and irradiation wavelength as well as matrix and analyte properties led to a large variety of proposals and partial aspects regarding the underlying processes [2, 5–28]. For closer insights, the physicochemical reactions taking place were elucidated by determination of the gas phase basicities as well as proton and electron affinities of the involved matrix [29–36] and analyte [37–43] neutral and ion species and were complemented by theoretical considerations and computational chemistry [34, 44–49]. From these investigations, two conflicting theories predominantly discussed nowadays emerged: The Lucky Survivor theory [17, 50] and the gas phase protonation model [5, 16, 24, 28, 51]. Both will be briefly discussed in the following.
1.1 The Lucky Survivor Model
The Lucky Survivor model postulates that analytes are incorporated in the matrix crystals with their respective charge states preserved from solution. At typical preparation conditions, peptides and proteins, the preferentially investigated analyte classes, are protonated and would therefore be incorporated as positively precharged analytes together with their counterions. Incorporation of analytes in their respective solution charge state could be confirmed by means of pH indicator probes  and adduct detection of precharged analytes with anions of extremely strong acids originating from solution [53, 54]. The original model differentiates between two possibilities: In the first case, ablated clusters generated upon laser irradiation contain precharged analytes with their corresponding amount of counterions and are therefore without net charge. Cluster dissociation induces entire charge neutralization or generation of net neutral adducts in the case of stable anions which both cannot be detected. In the second case, clusters carry a net charge initiated by charge separation upon disruption of the matrix-analyte solid. A lack of counterions inhibits quantitative charge neutralization and leads to protonated analytes. Provided that these ions are not neutralized by absorption of photoelectrons or electrons from the metallic target [18, 55, 56], they can be detected as so-called Lucky Survivors.
However, the original Lucky Survivor model  had limitations in the description of analyte anion generation. Clusters with excess negative charges can quantitatively neutralize positively precharged analytes, but subsequent analyte deprotonation by counterion neutralization will be endergonic because the carboxylic acid groups of peptides and proteins are less acidic than common counterions such as trifluoroacetate or chloride. To account for this a refined Lucky Survivor model was developed , which postulates that matrix excess charges [ma + H]+ or [ma – H]– included in the clusters lead to generation of protonated analytes by counterion neutralization or to analyte deprotonation, respectively. Matrix anions generally consist of carboxylates or delocalized phenolates whose neutralized counterparts exhibit about the same acidity as the analyte carboxylic acid groups. Consequently, analyte deprotonation should not be very efficient, which is in agreement with the typically low intense anionic analyte signals achievable using acidic matrices [26, 49].
1.2 The Gas Phase Protonation Model
This model predicts neutral analytes in the gas phase originating either from incorporation in the matrix crystals as uncharged species or from quantitative charge recombination with the respective counterions in the case of precharged analytes. Gas phase collisions of neutral analytes with protonated [ma + H]+ or deprotonated [ma – H]– matrix ions lead to proton transfer reactions and to protonated or deprotonated analytes, respectively [16, 24, 51].
In addition, quenching of matrix ions at the expense of analyte protonation (also called the “matrix suppression effect” [15, 57]) could be detected by means of angular and time resolved intensity distributions of matrix and analyte ions [6, 7]. However, despite numerous investigations and indications regarding the analyte protonation, the final elucidation of the actual mechanism is still missing.
2.1 Materials and Reagents
α-Cyano-4-hydroxycinnamic acid (CHCA ) was purchased from Bruker Daltonics (Bremen, Germany), acetonitrile (ACN, Rotisolv HPLC gradient grade) was from Roth (Karlsruhe, Germany), and p-anisaldehyde (99+%) was obtained from Acros (Nidderau, Germany). MilliQ water was prepared by a Millipore (Schwalbach, Germany) water purification system. The peptides ITITPNK and ITITPNV were obtained from the Central Peptide Synthesis Unit of the German Cancer Research Center (DKFZ). All other chemicals were purchased from Sigma-Aldrich (Taufkirchen, Germany) and were of the highest grade available.
2.2 Synthesis of Undeuterated and Deuterated Matrix Esters
In a first step, cyanoacetic acid was esterified to cyanoacetic acid-tert-butylester-d9 according to the synthesis route given by Neises and Steglich . Briefly, 1 eq cyanoacetic acid was dissolved in sufficient amounts of dry CH2Cl2/ACN (1:3, vol/vol). Upon addition of 0.07 eq 4-dimethylaminopyridine and 1.25 eq tert-butanol-d10, the reaction mixture was cooled to 0 °C and 1.1 eq dicyclohexylcarbodiimid were added at small portions. After stirring for 5 min, the ice was removed and the reaction was stirred further 20 min at room temperature. After removing solid byproducts by filtration, the solvent was evaporated and the crude tert-butylcyanoacetate-d9 was purified by short-path distillation at 12 mbar and 84 °C. Yield: 68% of the theoretical value as clear oil. For subsequent syntheses of the α-cyanocinnamic acid ester derivatives, Knoevenagel condensation reactions using the deuterated or undeuterated cyanoacetic acid esters and the respective benzaldehyde (4-hydroxybenzaldehyde or 4-chlorobenzaldehyde) were performed according to the instructions given in the literature . Yields for the cyanocinnamic acid ester syntheses were between 82%–95% as white (4-chloro-α-cyanocinnamic acid (ClCCA )-ester) to yellow (CHCA-esters) crystals.
2.3 Sample Preparation and Measurement
Polished stainless steel substrates and standard dried droplet preparations were used for all experiments. For the MS and MS/MS matrix spectra, 0.5 μL of matrix solution (20 mM, 70% ACN) were spotted onto a target and air dried. For experiments applying analytes, mixtures of 0.5 μL of the matrix solution (20 mM, 70% ACN) and 1 μL of the analyte solution (10 μM, 30% ACN/0.1% TFA in the cases of the peptides ITITPNK and ITITPNV, 100 μM in all other cases) were spotted without premixing onto a target and air dried. Possible additions of acid or base are indicated at the appropriate places in the text. The reason for the comparably high analyte amounts required for the neutral matrix esters is assumed to be the lacking ion-pair formation possibility between deprotonated matrix and protonated analyte ions, which is hypothesized to be crucial for effective analyte incorporation .
2.4 Mass Spectrometry
All MALDI MS measurements were carried out using a standard Voyager DE-STR TOF mass spectrometer (Applied Biosystems, Darmstadt, Germany) with a VSL-33737ND 337 nm nitrogen laser (LSI Laser Science, Newton, MA, USA) producing 3 ns laser pulses with a pulse repetition rate of 20 Hz and an approximately circular spot size of 50–100 μm in diameter. For pure CHCA as well as undeuterated and deuterated CHCA- and ClCCA-ester spectra, 10 single mass spectra with 500 laser shots each were accumulated. Also, for the experiments using the glycine monomer, dimer, and hexamer as well as the peptides ITITPNK and ITITPNV as analytes, all measurements were repeated 10-fold with 500 laser shots each. For all other mass spectra, four mass spectra with 500 laser shots each were accumulated. Laser fluences optimized for highest analyte S/N ratios were used for all approaches comprising analytes in which the absolute energy is not indicated. The optimum pulse energies were determined to about 1.5 μJ/pulse for CHCA, undeuterated and deuterated CHCA-tert-butylester and to about 1.9 μJ/pulse for ClCCA-tert-butylester-d9 using a calibrated energy probe (Nova laser power/energy meter; Ophir Optronics, Grosshansdorf, Germany) with a low-energy pyroelectric photodiode PE10 inserted in the laser beam path before entrance into the vacuum of the mass spectrometer. For analyzing pure matrices, the laser fluences were lowered by about 0.4 μJ for all used compounds, which were still about 10% above the respective threshold. Typical MALDI-TOF parameter settings for standard MS measurements were chosen and were polarity, positive; operation mode, reflector; accelerating voltage, 20 kV; grid voltage, 70%; low mass gate, off; delay time, 150 ns; shots per spectrum, 500; time resolution, 0.5 ns. For the peptides ITITPNK and ITITPNV, the grid voltage was set to 68.5% and the low mass gate to 550 Da. Data Explorer 4.5 (Applied Biosystems, Darmstadt, Germany) was used for analysis of the spectra. Peak detection was performed by using a S/N threshold of 10 after external calibration and advanced baseline correction with preset parameters. The chosen parameters for the MS/MS fragmentation of the CHCA-tert-butylester were as follows: laser fluence, 1.2 μJ/pulse; polarity, positive; operation mode, reflector; accelerating voltage, 20 kV; grid voltage, 68.5%; delay time, 150 ns; low mass gate, off; bin size, 0.5 ns; shots per segment, 500; precursor mass, 246 Da; post-source decay (PSD) mirror ratios, 1, 0.8, 0.75, and 0.5; guide wire for all segments, 0.05%.
The CHCA-tert-butylester-d9 degree of deuteration was determined using a modified ESI orthogonal time-of-flight mass analyzer (Mariner; Applied Biosystems, Framingham, MA, USA) equipped with a nanoelectrospray source and a heated transfer capillary. The mass spectra of the deuterated ester (2 mM in 80% ACN/0.1% TFA) were acquired in positive ion polarity for one minute at a spray potential of 800 V and a nozzle potential of 50 V. The temperature of the transfer capillary was set to 180 °C.
3 Results and Discussion
3.1 Discrimination Between Lucky Survivor and Gas Phase Protonation
For this purpose, a deuterated matrix compound, which enables exclusive generation of [ma + D]+ is necessary. According to Equation 1, generation of [ma + D]+ requires transfer of a D+ from a deuterated matrix radical cation ma•+ to a neutral matrix molecule. Polarized bonds as present in, e.g., deuterated hydroxyl- (–OD) or carboxylic acid (–COOD) groups will strongly alleviate bond breaking with transfer of D+ compared with nonpolar C–D bonds. Therefore, pronounced [ma + D]+ generation necessitates matrix compounds with, e.g., deuterated hydroxyl- and/or carboxylic acid groups. However, such deuterations are instable during conventional dried droplet (DD) sample preparation due to D/H-exchange reactions in protic solvents, with analyte functionalities or also the humidity of the air. The use of deuterated solvents for avoiding loss of matrix deuteration will cause at least partial H/D exchanges of the analyte which has to be prevented since in this case the origin of analyte deuteration cannot be unequivocally determined. Consequently, the strategy was to synthesize deuterated matrix compounds without solution-phase D/H-exchange by using stable C–D bonds. Upon excitation by laser irradiation, the matrix to synthesize has to undergo a rearrangement leading to the generation of polarized –OD or –COOD groups for subsequent generation of [ma + D]+.
3.2 A Matrix for Exclusive Generation of the Deuteronated Matrix Species [ma + D]+
From experiments carried out earlier in the systematic investigation of the role of functional groups on matrix properties of α-cyanocinnamic acid derivatives , we recollected that matrix esters typically yield two prominent ion signals, which are the protonated intact molecule and the protonated free acid as a fragment ion. Fragmentation of the ester to the acid can be proven by PSD analysis of the protonated ester, which yields nearly exclusive generation of the protonated free CHCA acid (compare Supplemental Information (SI) Figure 1 for the PSD spectrum of protonated CHCA-tert-butylester). It was tested whether a matrix ester, which is per-deuterated at the ester group, behaves in the expected way, thus forming the discriminating test reagent matrix needed. A possible contamination by the free matrix acid as a synthesis byproduct can be easily detected or excluded by the inspection of the ester mass spectra since present free acids will produce radical molecular ions, see SI Figure 2. The low-resolved signal shifted to a somewhat higher m/z-range in SI Figure 2b cannot be detected in the upper spectrum of the free acid and is most probably due to PSD generated matrix acid from the matrix ester after ion acceleration. Similar low-resolved signals can also be detected for all other investigated esters. This suggests that the ester fragmentation is not finished until acceleration voltage is applied but already begins in the very early phase of the expanding plume. For more detailed discussion concerning the matrix ester fragmentation see SI Data 1.
The only source of deuterons for generation of the deuteronated tert-butylester-d9 and subsequently for the deuteronated acid [CHCA-COOD + D]+ is the deuterated ester group. For energetic reasons, it could be expected that the CHCA-tert-butylester-d9 radical cation transfers a proton from the 4-hydroxyl group instead of a deuteron from a more stable C–D bond of the ester group. Nevertheless, deuteron abstraction could cause a subsequent loss of isobutene-d8 and generation of [CHCA – H]• according to Equation 1. Although the exact reasons are not known in detail, it can be assumed that abstraction of a deuteron is energetically favored due to entropy and/or that the larger surface of the tert-butyl group compared with the hydroxyl group increases the probability for deuteron abstraction instead of that of a proton. Consequently, the missing detectability of the ester radical cations in the mass spectra of Figure 1 might be explained by a high efficiency of this reaction. In order to completely exclude the involvement of the hydroxyl-hydrogen and to verify the role of matrix proton affinity , ClCCA-tert-butylester-d9 was synthesized as a second deuterated test matrix. It was not determined whether gas phase protonations and deuteronations exhibit identical reaction rates.
3.3 Neutral Analytes A
3.4 Medium Basic Analytes A/[A + H]+
The reason for using the ClCCA-tert-butylester-d9 instead of the CHCA-analog was to avoid the possibility of D/H-exchange reactions between deuteronated nicotinamide [A + D]+ and the 4-hydroxyl group of the CHCA matrix as alternative explanation for generation of the protonated nicotinamide. Although such D/H-exchanges could not be detected for the previously discussed deuteronated analytes p-anisaldehyde or 4′-hydroxyacetophenone using CHCA-tert-butylester-d9, application of ClCCA-tert-butylester-d9 clearly proves the existence of the Lucky Survivor process since no acidic hydrogens are available in ClCCA-tert-butylester-d9 or [ClCCA-COOD + D]+ capable of explaining an alternative generation of [A + H]+.
Activation of the gas phase protonation process requires higher amounts of the protonating matrix species [ma + H]+ or [ma + D]+ in the case of analyte deuteronation, respectively. According to Equation 1, the yield of these matrix ions depends on the amount of matrix radical cations, which in turn depends on the applied laser fluence. Increase of the laser fluence, consequently, activates gas phase deuteronation as is verified by the mass spectra of Figure 3b, which are all recorded at acidic conditions. With increased laser fluence going along with stronger formation of [ma + D]+ ions, an increase in deuteronated nicotinamide [A + D]+ is observed. The absolute intensity of [A + H]+ also increases with rising laser fluence, which can be traced back to a stronger ablation of matrix material with embedded precharged analytes [A + H]+ leading to a more intense counterion separation and/or to a more efficient counterion neutralization of [A + H]+ by [ma + D]+. Thus, also the contribution of the Lucky Survivor mechanism increases upon higher laser fluences, although the gain is less pronounced than in the case of gas phase protonation or deuteronation.
We therefore conclude that the nature of the analyte (basicity) as well as the preparation and measurement conditions (pH and laser fluence) determines the dominant analyte protonation pathway which, however, is not discernible in normal MALDI mass spectrometric experiments.
3.5 Basic (Positively Precharged) Analytes [A + H]+
The overwhelming majority of analytes investigated with MALDI MS are peptides and proteins. It was therefore of interest whether the Lucky Survivor or the gas phase protonation process is the dominant ionization pathway for these analyte classes.
One can argue that according to line 3 of Scheme 1, a more reactive matrix with a higher proton and deuteron transfer efficiency should also favor counterion neutralization of the precharged analyte [A + H]+ leading not only to an increase in gas phase protonation but also of the Lucky Survivor process. Although this might be true for anions of very strong acids, neutralization of normally involved anions such as, e.g., chloride, trifluoroacetate, or matrix anions is strongly exoergonic, also taking into account an energy barrier to overcome due to coulomb forces between the charges . Consequently, more reactive matrices should not distinctively enhance such processes. The detectability of deuteronated analytes [A + D]+ raises the question of the origin of the neutral analytes required for this purpose. At acidic pH values, the basic peptide ITITPNK with a pI of 10.1 exists nearly quantitatively as protonated species in solution. Therefore, the detected [A + D]+ intensity cannot be due to deuteronation of the remaining neutral analytes but can only be explained by deuteronation of the pool of neutral analytes generated upon neutralization of precharged [A + H]+. This possibility will be elucidated in the following.
3.6 Neutralization of Precharged Analytes
Therefore, the only reasonable explanation for the decreasing [A + D]+ amounts in the cases of the glycine dimer and hexamer are likewise decreasing amounts of the corresponding deuteronable neutral analytes. As was discussed for the gas phase, intramolecular peptide and protein folding upon analyte protonation during incorporation in the matrix crystal or already in solution can stabilize the protonated ions. Consequently, more stable [A + H]+ ions such as the protonated glycine hexamer will have a higher probability to survive, which means they should undergo a more efficient Lucky Survivor process compared with less stable [glycine + H]+. Therefore, the neutralized analyte amounts of the glycine dimer and especially hexamer will be lower, which results in only low gas phase deuteronation intensities. Vice versa, less stable protonated glycine undergoes a more efficient neutralization leading to a higher probability for subsequent gas phase deuteronation.
In summary and in addition to the effects discussed in the chapter “Medium Basic Analytes A/[A + H]+”, the efficiency of the Lucky Survivor further depends on the ability of the precharged analytes to stabilize their charge and therefore on their respective size and composition. This reflects the common experience that larger  and more basic analytes [74–76] exhibit higher ion yields.
3.7 Alternative Explanation Possibilities for Generation of Deuteronated Analytes [A + D]+
Deuteronation of the neutral analytes p-anisaldehyde and 4′-hydroxyacetophenone clearly proves the existence of gas phase deuteronation. However, H/D-exchange reactions of [A + H]+ with deuterated matrices might also account for generation of [A + D]+. Conversion of [A + H]+ to [A + D]+ upon collision with neutral deuterated matrix esters seems to be improbable because of their stable C–D bonds. Therefore, only collisions of [A + H]+ with neutral deuterated matrix acids generated upon ester cleavage seem to be able to generate [A + D]+. Gas phase backbone H/D exchanges can lead to multiply deuterated analytes, causing broadening or complete shifting of the isotope pattern [77–79]. Nevertheless, the vast majority of the deuteronated analytes exhibit attachment of only one deuteron, which does not reflect the possibility of multiple H/D exchanges but is in accordance with the result of gas phase deuteronations (compare Figures 3, 4 and 5). Additionally, no D/H exchanges have been observed in the case of the deuteronated analytes p-anisaldehyde and 4′-hydroxyacetophenone with the hydrogen of the CHCA-tert-butylester-d9 hydroxyl group which contradicts efficient H/D-plume exchanges, see Figure 2 and SI Figure 4. Application of ClCCA-tert-butylester-d9 and its deuteronated fragmentation product [ClCCA-COOD + D]+ increase the analyte deuteronation efficiency compared with the CHCA-counterpart, as shown in Figure 4 and SI Figure 6. This cannot be explained by more efficient H/D-exchanges of protonated analytes since ClCCA is more acidic than CHCA  and the exchange rates decrease with reagent acidity [77, 80]. It is, however, in accordance with a higher deuteron transfer capability due to the lower proton affinity of ClCCA [28, 60, 63]. Further, taking into account that glycine hexamers produce less [A + D]+ than the shorter glycine analytes, even though they exhibit a larger collision cross-section and, therefore, should undergo more efficient H/D-exchanges, it can be concluded that the amount of [A + D]+ generated upon H/D exchanges of protonated analytes is low.
3.8 The Unified MALDI Analyte Protonation Mechanism
As emphasized in the chapter “Neutralization of Precharged Analytes,” the proton affinities of the amino-terminus and of the amide backbone as least basic proton acceptors are still much higher than that of common matrices. Therefore, subsequent neutralizations of protonated analytes are possible only with deprotonated ions such as, e.g., [ma – H]–. Consequently, it appears unlikely that the generation of deprotonated analyte ions [A – H]– occurs according to the Lucky Survivor process of positively precharged analytes, which necessitates 2-fold analyte deprotonation with participation of [ma – H]– but rather to deprotonation of neutral analytes resulting from charge neutralization upon cluster decay, see Figure 6. The gas phase basicities of deprotonated α-amino acids  are clearly higher than that of the CHCA matrix anion , which leads to endergonic analyte deprotonation according to the equilibrium model for charge transfer between matrix and analyte ions [5, 44]. In addition to this, the assumed exclusion of the important Lucky Survivor pathway for [A – H]– generation further substantiates the positive/negative analyte ion yield ratio > 1 typically achieved with acidic matrices [26, 49].
The two long since postulated MALDI analyte protonation pathways known as the Lucky Survivor and the gas phase protonation model could both finally be experimentally proven. Several parameters favoring either the Lucky Survivor or the gas phase protonation were detected and investigated in detail. In addition to laser fluence, matrix and analyte proton affinity and the protonated-to-neutral analyte ratio embedded in the matrix crystal also the analyte composition and size affects the protonation pathway. With increasing size, the investigated peptides were preferably protonated according to the Lucky Survivor mechanism. We ascribe this to more pronounced intramolecular foldings of larger analytes during incorporation in the matrix crystal or already in solution going along with higher survival probabilities of the intramolecular solvated precharged species. It can be expected that for the same reason the survival probabilities of larger precharged peptides and proteins are even higher. In the case of the commonly used matrix CHCA or its deuterated counterpart, respectively, the investigated peptide analytes were ionized to about 90% according to the Lucky Survivor process. However, the efficiency of the gas phase protonation process strongly increases by using more reactive matrices with lower proton affinities such as ClCCA. Under these conditions, both protonation pathways exhibited nearly same probabilities. The detection of protonated analyte ions in cases of deuterated matrices without any exchangeable hydrogens confirmed the previously claimed incorporation of analytes in their respective solution charge states [52–54].
In a way, it could be expected that both postulated ionization pathways are true, taking into account the multitude of investigations regarding these models. Consequently, it stands to reason that both Lucky Survivor and gas phase protonation are parts of one greater overall mechanism, which we tried to account for with the presented Unified MALDI Analyte Protonation Mechanism. Therefore, this work finally ends a long-lasting discussion about how at least a partial aspect of one of the most important techniques in current molecular analysis works.
The use of halogenated MALDI matrices is subject of a German patent and international patent application.
The authors acknowledge financial support from the WI Bank for economic and public infrastructure development.