Enhanced MALDI MS Sensitivity by Weak Base Additives and Glycerol

Dec 10, 2013 - •S Supporting Information ... standards in MALDI MS, and new insights into the ... matrix compound and increased MALDI MS analytical...
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Enhanced MALDI MS Sensitivity by Weak Base Additives and Glycerol Sample Coating Rainer Cramer,*,† Michael Karas,‡ and Thorsten W. Jaskolla§ †

Department of Chemistry, University of Reading, Whiteknights, Reading RG6 6AD, United Kingdom Institute of Pharmaceutical Chemistry, Cluster of Excellence “Macromolecular Complexes”, Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438 Frankfurt, Germany § Institute of Hygiene, University of Münster, Robert-Koch-Str. 41, 48149 Münster, Germany ‡

S Supporting Information *

ABSTRACT: The concept of rationally designing MALDI matrices has been extended to the next “whole sample” level. These studies have revealed some unexpected and exploitable insights in improving MALDI sensitivity. It is shown that (i) additives which only provide additional laser energy absorption are best to be avoided; (ii) the addition of proton donors in the form of protonated weak bases can be highly beneficial; (iii) the addition of glycerol for coating crystalline samples is highly recommended. Overall, analytical sensitivity has been significantly increased compared to the current “gold” standards in MALDI MS, and new insights into the mechanisms and processes of MALDI have been gained.

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atrix-assisted laser desorption/ionization (MALDI)1 is well-known for being one of the two key ionization techniques in modern biological mass spectrometry (MS). It has recently attracted increased attention, mainly due to its imaging capabilities, its potential in biotyping, and the fundamental advances in ion production with respect to both ion quality and quantity, which includes the formation of stable and prolonged yields of multiply charged ions at high sensitivity2 and the systematic derivatization of well-proven classical matrix compounds for increased ion yields.3 The latter study demonstrated that targeted derivatization of α-cyanocinnamic acids, mainly by adding electron-withdrawing groups to the para position of the phenyl moiety, can lead to decreased proton affinity (PA) of the matrix compound and increased MALDI MS analytical sensitivity. Herein, we report an extension of this idea (of using rationally designed MALDI matrix compounds) to the entire MALDI sample by including matrix additives and (nonvolatile) solvents, considering the overall desorption and ionization process in MALDI MS and where changes in the MALDI sample composition deliver a competitive advantage in analytical performance. Our studies have led to several new approaches in improving MALDI ion yields by exploiting the concept of liquid MALDI samples and their advantages in investigating the MALDI process parameters. Liquid MALDI MS has recently experienced a renaissance as several groups have found sample systems that facilitate highly sensitive analysis of a variety of analytes and allow for enhanced ion formation and more reliable analyte quantification through prolonged and stable ion yields by using ionic liquid matrices (ILMs)4 and liquid support matrices (LSMs).5 In particular, © 2013 American Chemical Society

glycerol has been at the forefront of some remarkable new approaches in liquid MALDI MS, leading to vacuum-stable liquid samples and the formation of predominantly multiply charged ions.2 Interestingly, glycerol has been associated with MS ionization techniques for many years, going as far back as the origins of fast atom bombardment using neutral particle beams6 and liquid secondary ion mass spectrometry using ions for bombarding the liquid sample.7 Similarly, glycerol has been part of soft laser desorption for more than 25 years8 and a well-known matrix compound in IR-MALDI.9 It has also been investigated as a nonabsorbing and nonvolatile matrix component of UVMALDI samples. Some of the early studies used small graphite particles (2−150 μm) as UV absorbers and confirmed the beneficial nature of glycerol in UV-MALDI MS, initially without substantiated suggestions regarding its underlying mode of action10 but later with the assumption that it acts as a protonating agent.11 However, the latter is arguably in contradiction to one of the earlier results by Sunner et al.,10 where it was pointed out that a clear correlation between spectra with intense peptide and protein ions and low-intensity glycerol peaks exists, indicating that analyte protonation directly from protonated glycerol is unlikely. On the other hand, in a different study the absorbing (dye) compound in a binary absorber/glycerol matrix solution was also dismissed for direct analyte ion formation12which should also be true for metal particles as the absorber. However, Received: October 7, 2013 Accepted: December 10, 2013 Published: December 10, 2013 744

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it cannot be excluded that CxHy+ clusters generated in graphite− liquid matrix mixtures are involved in the protonation process.10 Studies like the ones above clearly demonstrate that only little is known about the contribution of all sample components to analyte ionization as well as their importance in absorbing the laser energy and making this energy available for both the desorption and ionization process. We have focused on the opportunities that the concept of liquid MALDI samples can generally offer, in particular with respect to the flexibility in combining/mixing various matrix components, by investigating the beneficial nature of glycerol and the need in MALDI of providing sufficiently high laser energy absorption, analyte dispersion, and importantly, proton transfer efficiency. We have looked at some newly synthesized MALDI matrix compounds and their application and enhancement toward an overall rationally designed MALDI sample with the goal to obtain some significant additional improvement of the MALDI desorption and ionization process by taking the concept of a rationally designed matrix to the next (whole sample) level. Although our approach is far from fully treating the MALDI sample holistically, it has already revealed some unexpected and exploitable insights. In short, one of the main guiding principles of our present study was to achieve an absolute improvement of MALDI sensitivity by focusing on the best-known solid UV-MALDI matrices for providing highest sensitivity for peptide analysis and how their employment can be further enhanced by a more suitable overall sample environment through optimal sample morphology, analyte incorporation, energy absorption and desorption characteristics, and proton availability for analyte protonation.

recorded in positive ion mode if not otherwise noted. MALDITOF parameters were the following: reflector as operation mode; accelerating voltage of 20 kV; grid voltage of 64.8%; extraction delay time of 150 ns; no low mass gate for m/z 10− 500 spectra, low mass gate at m/z 650 for m/z = 800−3000 spectra and low mass gate at m/z 750 for m/z = 900−3000 spectra; bin size of 0.5 ns; minimum S/N of 10; monoisotopic peaks for mass peak filter; no baseline correction; 500 shots per spectrum. Laser fluences were optimized for highest analyte S/N in all cases and were about 2.5 μJ/pulse for α-cyano-4hydroxycinnamic acid (CHCA), 2.8 μJ/pulse for 4-chloro-αcyanocinnamic acid (ClCCA) preparations using positive-ion mode and 3.5 μJ/pulse for negative-ion mode analyses, 3.5 μJ/ pulse for 4-chloro-α-cyanocinnamic acid amide (ClCCAamide), 3.6 μJ/pulse for α-cyano-2,4-difluorocinnamic acid (DiFCCA), 4.0 μJ/pulse for α-cyano-2,4-difluorocinnamic acid amide (DiFCCAamide), 7−10 μJ/pulse for F3CCCA without base, and about 8−14 μJ/pulse for α-cyano-4-trifluoromethylcinnamic acid (F3CCCA) with pyridine as base. The low mass range spectra m/z = 10−500 of Figure 1 were internally mass calibrated by means of the monoisotopic masses of [N(Me)3 + H]+ (60.08078 Da), [35ClCCA + H − H2O]+ (190.00542 Da), [35ClCCA + H]+ (208.01598 Da), and [35ClCCA + K]+ (245.97187 Da), if detectable. For the higher mass range spectra m/z = 800/900−3000 of the BSA digest experiments, the monoisotopic masses of the protonated tryptic BSA digest peptides YLYEIAR (927.4934 Da), LVNELTEFAK (1163.6307 Da), HLVDEPQNLIK (1305.7161 Da), TVMENFVAFVDK (1399.6926 Da), RHPEYAVSVLLR (1439.8118 Da), LGEYGFQNALIVR (1479.7954 Da), DAFLGSFLYEYSR (1567.7427 Da), KVPQVSTPTLVEVSR (1639.9378 Da), MPCTEDYLSLILNR (1724.8346 Da), RPCFSALTPDETYVPK (1880.9211 Da), LKPDPNTLCDEFKADEK (2019.9692 Da), RHPYFYAPELLYYANK (2045.0279 Da), and SHCIAEVEKDAIPENLPPLTADFAEDKDVCK (3511.6720 Da) were used for internal calibration, if detectable. The absorption measurements, MP2 calculations, Mascot database searches, and a list of materials and reagents are described in the Supporting Information. Health and Safety Considerations. Concentrated hydrochloric acid is highly corrosive and should be handled accordingly and neutralized with aqueous sodium bicarbonate solution. Diphenylamine is poisonous and very toxic to aquatic organisms with long-lasting effects. It has to be handled with care and its disposal typically requires licensed/approved companies.

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EXPERIMENTAL SECTION Synthesis of Halogenated Matrices. The halogenated CCA-derivatives were synthesized according to the instructions given in Jaskolla et al.13 For synthesis of the CCA-derivatives, cyanoacetic acid amide was used for condensation instead of cyanoacetic acid. MALDI Sample Preparation. MALDI samples were prepared by mixing 0.5 μL of matrix solution with 0.5 μL of analyte solution on target and subsequent drying under ambient conditions. In general, DD matrix solutions were prepared by dissolving the matrix compounds to 20 mM in 1:1 (v/v) ACN:20 mM NH4H2PO4 or 70% ACN while LSM solutions were prepared as published earlier.5,14 Further details can be found in the Supporting Information. MALDI MS Data Acquisition. Mass spectra were acquired on a Voyager DE-STR TOF mass spectrometer (Applied Biosystems, Darmstadt, Germany) with a VSL-337ND nitrogen laser (LSI Laser Science, Newton, MA) emitting a Gaussian beam profile with a pulse length of 3 ns at 337 nm. The pulse repetition rate was 20 Hz, and the laser spot size was approximately circular with a diameter of 50−100 μm. Each mass spectrum resulted from the accumulation of data acquisitions from 500 single laser shots. The laser fluences were optimized for each matrix for highest analyte signal-to-noise ratios (S/N). Absolute pulse energies were determined by measuring the pulse energy before the laser beam entered the vacuum of the mass spectrometer using a calibrated energy probe (Nova laser power/energy meter, Ophir Optronics, Grosshansdorf, Germany) with a low-energy pyroelectric photodiode PE10. Data Explorer 4.5C (Applied Biosystems, Darmstadt, Germany) was used for spectra analysis. All mass spectra were

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RESULTS AND DISCUSSION To start with, we collected some fundamental data about the physicochemical properties of various candidate components for an optimal, potentially liquid, UV-MALDI sample. Table S-1 summarizes this data and includes the important parameters, including proton affinity, pKA, and molar absorption coefficient. Absorption of the basic and acidic components was measured in both their neutral and (de)protonated form by adding a strong nonabsorbing acid or base, respectively. As most of the previously rationally designed halogenated UV matrix compounds are derivatives of the highly effective CHCA matrix compound, which show lower absorption in their neutral form at the typical MALDI wavelengths of 337 and 355 nm due to hypsochromic absorption band shifts, it was investigated whether their use in a liquid MALDI sample by the addition of a suitable base would be beneficial through (i) bathochromic shifts due to matrix deprotonation and/or (ii) additional and thus 745

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Table 1. Extinction Coefficients for Individual Matrix Compounds and Their Values in LSMs Using 3-AQ as Base compound

MWavg of neutral [g/mol]

ε337 nm ε355 nm [L mol−1 cm−1]

molar ratio of matrix anion: [3-AQ + H]+/ 3-AQ

ε = ∑εici/∑ci of LSM with 3-AQa ε337 nm ε355 nm

3-AQ [3-AQ + H]+ [CHCA−H]− [ClCCA−H]− [DiFCCA−H]− [TriFCCA−H]− [F3CCCA−H]−

144.17 144.17 189.17 207.61 209.15 227.14 241.17

3770, 2370 2270, 3800 17 100, 5500 580, ∼0 280, ∼0 ∼0, ∼0 25, ∼0

N/A N/A 1:1:2.94 1:1:3.32 1:1:3.36 1:1:3.73 1:1:4.02

N/A N/A 6500, 3680 3250, 2800 2950, 2570 3000, 2670 3090, 2760

a Although the combined concentration-dependent absorption of all absorbers Σεici was measured, ε = ∑εici/∑ci has been used as pseudovalue for the average extinction coefficient of the LSM constituents, and to facilitate the direct comparison with the pure matrix values in column 3.

Table 2. MALDI MS Analysis of a 100 fmol BSA Digest Using LSMs and Various Concentrations of the UV Absorber DCM LSM with ClCCA and pyridine Mascot search results [ClCCA−H]

‑

4.6 nmol

DCM

molar ratio of matrix anion-to-DCM

sequence coverage

matched peptides

score

1 nmol 100 pmol 10 pmol

4.6:1 46:1 460:1

35% 53% 70% 76%

20 36 60 61

36 32 44 72

LSM with TriFCCA and pyridine Mascot search results [TriFCCA−H] 4.2 nmol

‑

DCM

molar ratio of matrix anion-to-DCM

1 nmol 100 pmol 10 pmol

4.2:1 42:1 420:1

sequence coverage

matched peptides

score

60%

no hit no hit no hit 43

66

All substance amounts refer to 1 μL of MALDI sample, which was prepared by mixing 0.5 μL of analyte solution with 0.5 μL of diluted LSM. See Supporting Information for LSM preparation and Mascot search details.

overall absorption reflects very well the dominant absorption of 3-AQ as the matrix carboxylates contribute only marginally or not at all (see Figure S-1). In these cases, the absorption maximum is unanimously around 345 nm, which can be explained from an overlap of the absorption maxima of neutral and protonated 3-AQ at ∼337 and ∼370 nm, respectively, taking into account that the molar base/matrix ratio is around 4:1 to 5:1, resulting in only ∼20−25% protonated 3-AQ. Further, using pyridine as base with no absorption at the relevant wavelengths, the LSM absorption profiles very closely reflect the absorption of the carboxylate anions (i.e., a hypsochromic shift of ≈15−25 nm), indicating that also CHCA exists as carboxylate in pyridinebased LSM samples. From the above results, it can be concluded that liquid MALDI sample absorption can be set to values of interest based on theoretical considerations using the components’ individual absorption data as long as the correct ionic form is considered. However, the question remains whether an increase in overall absorption is beneficial for the MALDI process such as lowering the laser energy/fluence threshold for ionization (defined as the minimum deposited laser energy, at which the ion intensities of interest were distinguishable from the surrounding noise level (S/N ≥ 5)) or activation of otherwise nonabsorbing matrices. To answer this question, we investigated TriFCCA, which in any form has an extremely low solution absorption at 337 or 355 nm. Here, it has to be considered that the CCA absorption profiles are significantly broadened in the solid state due to intermolecular forces (see Figure S-2).15 As a result, ablation and ionization

overall higher absorption provided by the base. From absorption measurements (see Table S-1 and Figure S-1) under deprotonating conditions using pyridine as nonabsorbing base at 337 and 355 nm, it is evident that the absorption profiles of CHCA and its derivatives ClCCA, DiFCCA, α-cyano-2,4,6trifluorocinnamic acid (TriFCCA), and F3CCCA (for structures see Table S-2) show a hypsochromic shift of ≈15−25 nm due to the carboxylate formation. However, under aqueous conditions, these halogenated derivatives can only be deprotonated to their carboxylate form, while CHCA itself can be further deprotonated to the dianionic carboxylate-phenolate form by stronger bases, which induces a bathochromic shift of ≈60 nm (see Table S-1 and Figure S-1). Neither of these shifts is desirable as the absorption maxima for all anionic matrices shift away from the aforementioned MALDI wavelengths, leading in all cases except for CHCA at 355 nm to no improvement in absorption. Similarly, the investigated bases pyridine, 3-aminoquinoline (3AQ), 6-aminoquinoline (6-AQ), and 6-amino-2-methylquinoline (AMQ) show lower absorption at these wavelengths (except for 3-AQ at 355 nm) when protonated using trifluoroacetic acid (TFA), due to a bathochromic shift of their absorption profile. For LSM samples consisting of deprotonated acidic matrix and a (3-fold) mass excess of partially protonated base as well as glycerol, the additional absorption of the investigated protonated (and neutral) bases (except nonabsorbing pyridine) leads to an increase in the overall absorption of the MALDI sample for all investigated matrices but CHCA (see Table 1). Absorption measurements of acetonitrile-diluted LSMs with 3-AQ as base show that for ClCCA, DiFCCA, TriFCCA, and F3CCCA the 746

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Figure 1. MALDI mass spectra from liquid and crystalline sample areas of an LSM preparation with a 10 fmol tryptic BSA digest as analyte and ClCCA as matrix acid and trimethylamine as matrix base.

concentration and sample volume unchanged. Table 2 shows the results for the analysis of a tryptic digest of 100 fmol bovine serum albumin (BSA), revealing a clear analytical deterioration with increasing DCM concentration. The above results show that laser energy absorption in MALDI samples, particularly in LSMs, can be effectively increased through the addition of strong absorbers but also that these do not necessarily lead to lower laser energy thresholds for MALDI ionization. More importantly, the additional absorbers and their laser energy absorption lead to lower matrix and analyte ionization. Although there is the possibility of a change to lower protonation efficacy due to the introduction of the additional absorber, our experiments for Table 2 have minimized this possibility through the use of homogeneous LSMs and the swap of similarly strong bases (pKa of 3-AQ and pyridine is 4.86 and 5.14, respectively)16 as well as using a dye at concentrations well below the ones for the base pyridine. Thus, it appears that

become also possible for TriFCCA and F3CCCA at 337 nm and at increased laser irradiation. Using pyridine as base, the LSM based on TriFCCA required laser threshold energies of 2.5 ± 0.2 μJ/pulse for matrix and 7.5 ± 0.3 μJ/pulse for analyte ionization, while for 3-AQ as base and competing absorber (using the same molar ratio to the matrix anions), the thresholds were 3.5 ± 0.1 and 12.0 ± 0.6 μJ/pulse, respectively. This result clearly indicates that higher overall absorption of the MALDI sample is not necessarily beneficial for the desorption/ionization process. Similar results were obtained with the other acids (see Table S-3), although for these, sample morphologies between the pyridine-based LSMs and the 3-AQbased LSMs were slightly different. Further, pyridine-based LSMs were prepared with different additions of the strong UV absorber 4-(dicyanomethylene)-2methyl-6-(4-dimethylaminostyryl)-4H-pyran (DCM; ε337 nm = 20 000; ε355 nm = 18 600), keeping as before the matrix anion 747

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vs DD, 50; protein sequence coverage: LSM + NH3, 83% vs DD, 70%; Mascot score: LSM + NH3, 225 vs DD, 128), which has been previously shown to be superior to the commonly used CHCA matrix. However, no or highly inferior spectra were obtained from liquid sample areas. Here, it is important to note that protonated ammonia is stabilized in solution by multiple hydrogen bondings, which strongly lower its reactivity. This stabilization also explains why ammonia has a relatively low pKB value of 4.79. As MALDI protonation of peptides seems to occur mostly in charged clusters,20 the lower reactivity of solvated ammonia should be particularly well-observable with MALDI samples, from which many (larger) clusters or even droplets are obtained, for example, liquid samples (cf. ref 2), and thus explains the above-noted differences in analyte ionization. Indeed, glycerol/ammonium adduct ions were strongly detectable from the liquid and less or none from the crystalline sample areas using LSMs prepared with ammonia (see Figure S-3). Consequently, we thought to improve the overall efficacy of the analyte ionization process by using bases of extremely low basicity, which are less stabilized by hydrogen bonding. Although the reactivity of extremely weak bases is most likely still lower than that of [matrix acid + H]+, the idea was that this disadvantage is more than outweighed by providing the base as quantitatively protonated species (although most of it might neutralize upon ablation), whereas laser irradiation of neutral matrices leads to overall matrix ion yields of only 10−6 to 10−4.21 Thus, we investigated diphenylamine, which has a low basicity in both solution and the gas phase, that is, a high pKB (13.2) and low PA (∼900 kJ/mol), and is still protonatable in aqueous solvents. Due to a lack of absorption of diphenylamine at 337 and 355 nm, we added ClCCA as absorber. As the formulation of ClCCA/ diphenylamine-based LSMs resulted in samples of very badly smeared-out films, we eliminated the glycerol from the samples, which led to fully crystalline MALDI samples. Please note that concentrated HCl solution was needed to protonate/dissolve diphenylamine for these matrix formulations, resulting in neutral ClCCA and diphenylammonium chloride. Figure 2 displays the negative and positive ion mode comparison of the mass spectra of a tryptic BSA digest obtained with a standard ClCCA DD preparation and with the ClCCA/diphenylamine preparation. In both ion modes, the spectra of this newly designed matrix system are superior to those obtained from pure ClCCA DD preparations, which is also reflected in the peptide mass fingerprinting results in Table S-4. Optimization of this new binary matrix system led to an optimal ClCCA-to-diphenylamine molar ratio of 1.6, outperforming the conventional ClCCA DD preparation with 70% ACN as solvent as well as the control ClCCA DD preparation using the same solvent system that was necessary for the binary matrix system preparation, that is, conc. HCl/ACN (1:3; v/v) (see Figure S-4). In line with the above results, suggesting that a liquid MALDI sample with a base prohibits the production of primary matrix acid ions and thus the classic foundation for efficient analyte ionization, MALDI sample preparations such as the DD preparation resulting in solid samples are still preferred in the community. This is most likely due to the achievable higher absolute analyte signal intensity in general. However, LSMs have been reported to show superior analytical sensitivity for specific types of analytes such as glycans and glycopeptides22 and in cases where the occurrence of unspecific (cluster and matrix) background ions has a detrimental effect for the analysis (e.g., in protein digest analyses using peptide mass fingerprinting).14,23

additional strong absorbers are principally counterproductive for the MALDI process and should be avoided. Next, we studied the effect of base addition to acidic MALDI matrices, in particular to LSMs, with regard to its contribution to protonation. Several bases, of which some are typically used in the formulation of LSMs and ILMs, such as aniline, 3-AQ, and pyridine, were investigated. In most cases, LSM preparations consisted of liquid as well as crystalline sample areas. In general, strong [matrix base + H]+ signals were detected, while only very small signals (relative to solid dried droplet samples without base addition) of matrix acid•+ and [matrix acid + H]+ were obtained. In fact, these small matrix acid signals were only obtained from areas where the samples were crystalline, whereas virtually no positive ion signals from the matrix acids were obtained from liquid sample areas. The detection of matrix acid ion signals was concomitant with greater analyte ionization, indicating that these matrix ions (i.e., [matrix acid + H]+) are more reactive and important for the analyte protonation process than [matrix base + H]+ ions (see Figure 1). A comparison of various LSMs using the matrix acid ClCCA and the matrix bases trimethylamine, 6AQ, 3-AQ, and pyridine, respectively, showed that in all cases the analyte ion signal intensity was far greater at crystalline sample areas than at liquid sample areas. These results are in agreement with the far lower proton affinities of the used matrix acids (790− 830 kJ/mol) compared to the bases (930−970 kJ/mol). For “liquid” MALDI sample preparations such as ILMs or LSMs, it can be assumed that the matrix acids solely exist in the form of solvated matrix anions (liquid areas) or as part of ion crystals with [matrix base + H]+ ions (solid areas). Upon ablation, the solvated matrix ions either survive or neutralize, whereas the direct ion−ion interaction within the solid crystals allows for additional reactions to occur such as the detected generation of matrix acid•+ and [matrix acid + H]+. Acid−base neutralization followed by photoionization of neutralized matrix molecules seems to be unlikely due to the unfavorable irradiation parameters. Lippa et al. observed photoelectron detachment from a multitude of matrix anions with vertical detachment energies of about 1 eV, which can be easily provided at irradiation wavelengths of 337 and 355 nm with photon energies of 3.68 and 3.49 eV, respectively.17 The energy of electrons generated in MALDI was estimated to be ≤2 eV,18 which is well below the threshold of 15 eV up to which low-energy capture of neutral matrices leads to formation of matrix anions.19 Consequently, electron capture of positively precharged matrix bases in ion crystals of LSMs are most likely. Therefore, we propose for liquid MALDI sample preparations an alternative ionization pathway as detailed in Scheme 1. Interestingly, LSMs with ammonia (PA: 850 kJ/mol) showed far better performance in the analysis of a 10 fmol tryptic digest of BSA from the crystalline sample areas than a dried droplet (DD) preparation of ClCCA (no. of matched peptides: LSM + NH3, 61 Scheme 1. Proposed MALDI Ionization Pathway at the Solid Ion Crystals of “Liquid” Sample Preparations

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Figure 2. Comparison of ClCCA-based DD MALDI sample preparations using a tryptic BSA digest. When diphenylamine was used, the diphenylamineto-ClCCA molar ratio was 2.

experiments in which the amides of some of the above CHCA derivatives have been investigated (see Figure S-6). The addition of glycerol led in all cases to greater analyte S/N for the analysis of BSA digests and thus to greater protein sequence coverages and database search scores (see Table S-5) despite the fact that LSMs in general did not perform that well when data was acquired from the liquid sample areas. This S/N increase is arguably due to a reduction of unspecific matrix cluster ions as part of the chemical background noise. As shown in Figure 3, the addition of glycerol to the matrices CHCA, ClCCA, and DiFCCA, leading to glycerol-coated crystalline DD samples, substantially improved the analytical sensitivity in peptide mass fingerprinting analyses. Interestingly, in these measurements, no (quasi)molecular ion signals for glycerol were detected, thus suggesting that glycerol is not directly involved in the analyte ionization process, which is in agreement with Sunner et al.10

Thus, we specifically investigated the effect of the addition of glycerol as a formidable vacuum stable solvent that provides the liquid support for the LSMs. Earlier results already showed that some newly designed solid UV matrices that did not perform well were perfectly adequate as matrix compounds when used in ILMs or LSMs,24 displaying good analytical sensitivity for protein digest analysis. Here, we focused on preparations based on F3CCCA, a matrix acid that generates strong matrix clusters, using pyridine as base (in contrast to 3-AQ that was used in the earlier studies) and found that the addition of glycerol led to background (mainly matrix clusters) ion reduction and greater analyte ion signal-to-noise ratios (see Figure S-5), which is in agreement with earlier conclusions.14,25 Even for DD preparations was the addition of glycerol highly beneficial (see Figure S5), making it an interesting component for any future MALDI sample design. This can also be seen in another set of 749

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Figure 3. Peptide mass fingerprinting results from Mascot searches of mass spectral data of BSA digests using CHCA, ClCCA, and DiFCCA with and without glycerol as matrix. Matrices were prepared as LSMs without the addition of base. When glycerol was also eliminated, the LSM preparations resembled DD preparations.

energy absorption are best to be avoided, (ii) the addition of proton donors in the form of protonated weak bases can be highly beneficial, and (iii) although liquid sample areas typically lead to lower analyte ion signals if bases are added (due to the loss of the most reactive proton donors), the addition of glycerol for coating LSM and standard DD preparations is certainly recommended. From our experience, glycerol addition can be easily made and also included in established protocols. So far, we have shown that a MALDI matrix composition consisting of ClCCA as matrix acid and diphenylamine as matrix base outperforms the recently rationally designed solid MALDI matrix ClCCA on its own (i.e., without further additives such as weak bases). Together with the improvements through the addition of glycerol, we have demonstrated the advantage of a wider holistic MALDI sample design, resulting in further sensitivity increases compared to when the focus is just on one matrix component alone.

In general, if a matrix is used for LSM preparations, acidic matrices are dissolved in glycerol up to the amount of available counter base and the solubility of the ion pair, with the remainder forming solid areas. If no base is added, neutral matrix crystals are obtained which are optically similar to DD preparations but covered with glycerol. Although further research is necessary to elucidate the underlying effects induced by glycerol coating, it can be assumed that the UV-transparent glycerol coat leads to no or only little changes in the absorption process in combination with ablation of a higher amount of material/clusters. With a potentially higher plume collision probability, more efficient matrix cluster reductions can be expected since the coablated glycerol exhibits presumably the same initial velocity as the matrix itself, thus avoiding collisional cooling.

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CONCLUSIONS In summary, for future MALDI sample designs, we have shown the following: (i) additives which only provide additional laser 750

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(20) Jaskolla, T. W.; Karas, M. J. Am. Soc. Mass Spectrom. 2011, 22, 976−988. (21) Mowry, C. D.; Johnston, M. V. Rapid Commun. Mass Spectrom. 1993, 7, 569−575. Quist, A. P.; Huthfehre, T.; Sundqvist, B. U. R. Rapid Commun. Mass Spectrom. 1994, 8, 149−154. (22) Fukuyama, Y.; Nakaya, S.; Yamazaki, Y.; Tanaka, K. Anal. Chem. 2008, 80, 2171−2179. Mank, M.; Stahl, B.; Boehm, G. Anal. Chem. 2004, 76, 2938−2950. (23) Zabet-Moghaddam, M.; Heinzle, E.; Lasaosa, M.; Tholey, A. Anal. Bioanal. Chem. 2006, 384, 215−224. (24) Towers, M. W.; Jaskolla, T. W.; Karas, M.; Cramer, R. In Proceedings of the 58th ASMS Conference on Mass Spectrometry, Salt Lake City, UT, May 23−27, 2010; American Society for Mass Spectrometry: Santa Fe, NM; p TP25 576. (25) Cramer, R.; Corless, S. Proteomics 2005, 5, 360−370.

ASSOCIATED CONTENT

S Supporting Information *

Supplementary experimental section, tables (Tables S-1 to S-5), and figures (Figures S-1 to S-6) as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax:+44 118 378 6331. Tel.: +44 118 378 4550. Author Contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes

The authors declare the following competing financial interest(s): T.W.J. and M.K. are inventors of German and international patents regarding the use of halogenated matrices. The use of halogenated MALDI matrices is the subject of patents WO 2009/026867 and WO 2011/107076.

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ACKNOWLEDGMENTS This research was supported by the Alexander-von-Humboldt foundation (RC) and the DFG (TWJ; Grant No. JA2127/1-1). We thank Stavroula Markoutsa for preparing the tryptic BSA digest.

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REFERENCES

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dx.doi.org/10.1021/ac403228d | Anal. Chem. 2014, 86, 744−751