Negative Ion MALDI Mass Spectrometry of Polyoxometalates (POMs): Mechanism of Singly Charged Anion Formation and Chemical Properties Evaluation
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- Boulicault, J.E., Alves, S. & Cole, R.B. J. Am. Soc. Mass Spectrom. (2016) 27: 1301. doi:10.1007/s13361-016-1400-6
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MALDI-MS has been developed for the negative ion mode analysis of polyoxometalates (POMs). Matrix optimization was performed using a variety of matrix compounds. A first group of matrixes offers MALDI mass spectra containing abundant intact singly charged anionic adduct ions, as well as abundant in-source fragmentations at elevated laser powers. A relative ranking of the ability to induce POM fragmentation is found to be: DAN > CHCA > CNA > DIT> HABA > DCTB > IAA. Matrixes of a second group provide poorer quality MALDI mass spectra without observable fragments. Sample preparation, including the testing of salt additives, was performed to optimize signals for a model POM, POMc12, the core structure of which bears four negative charges. The matrix 9-cyanoanthracene (CNA) provided the best signals corresponding to singly charged intact POMc12 anions. Decompositions of these intact anionic species were examined in detail, and it was concluded that hydrogen radical-induced mechanisms were not prevalent, but rather that the observed prompt fragments originate from transferred energy derived from initial electronic excitation of the CNA matrix. Moreover, in obtained MALDI mass spectra, clear evidence of electron transfer to analyte POM species was found: a manifestation of the POMs ability to readily capture electrons. The affinity of polyanionic POMc12 toward a variety of cations was evaluated and the following affinity ranking was established: Fe3+ > Al3+ > Li+ > Ga3+ > Co2+ > Cr3+ > Cu2+ > [Mn2+, Mg2+] > [Na+, K+]. Thus, from the available cationic species, specific adducts are preferentially formed, and evidence is given that these higher affinity POM complexes are formed in the gas phase during the early stages of plume expansion.
KeywordsNegative Ion MALDI Polyoxometalates (POMs) In-source decay MALDI mechanisms Cationic adduct Fragmentation
Since the late 1980s, matrix-assisted laser desorption ionization (MALDI) has become a major ionization technique for mass spectrometric studies, especially regarding large molecule analysis that one encounters in the proteomics and polymer fields. Upon closer examination, the use of MALDI can be found in a rather wide variety of applications, including glycans analysis  and, more generally, for investigations of all types of large biomolecules . The small sample quantity required to perform an analysis has earned the MALDI technique renown as an exceptionally sensitive tool that is more tolerant of the presence of salts than other mass spectrometric ionization techniques (e.g., electrospray ionization). In addition, the possibility for label-free detection of a wide array of molecules, and the application of focused lasers for desorption/ionization at rather high spatial resolution, have attracted increasing attention for imaging mass spectrometry analysis . However, the major bottleneck of MALDI applications is the sample preparation step that initially involves finding an efficient matrix, and subsequently involves optimization of target preparation. Sample preparation is still largely performed empirically, even if many recipes are known and have undergone amelioration over time .
Even though much effort has been invested in increasing knowledge concerning details of MALDI processes, the comprehension of the exact mechanisms of the involved desorption/ionization is still on-going. Some models portray the MALDI process as commencing with the formation of huge aggregates of matrix  where analytes can be either precharged (“Lucky Survivor” model) , or can interact with charged matrix molecules to finish as naked intact protonated (or deprotonated for negative mode) species . Mechanistic studies concerning the ionization process are typically undertaken in the positive ion mode, and they mainly consider proton and/or electron transfers as being responsible for analyte charging.
Despite the fact that MALDI is much more used in the positive ion mode, it also seems important to develop studies in the negative ion mode that may improve the fundamental understanding of the ion formation process. The negative ion mode has been less frequently employed, with the exception of its application toward specific compound classes, such as DNA  , sugars , and lipids .
To widen the array of applications using negative mode MALDI analysis, we present results pertaining to polyoxoanionic species, [i.e., polyoxometalates (POMs)]. These polyanionic species are early-transition metal-oxygen structures that can be functionalized by organic and/or inorganic groups, thereby allowing the “tuning” of POM properties such as redox behavior, magnetic and acid-base characteristics, or even biocompatibility . The large variability of POM structures has resulted in a wide range of different properties and a corresponding range of potential chemical and biological applications. For example, POMs have found industrial applications for organic compound oxidation or dehydration , and they are promising tools for photovoltaic applications .
Functionalized polyoxometalate anions commonly have a charge state varying between 2 and 12  and the tendency has been to perform mass spectrometric analyses using electrospray ionization (ESI). The obtained mass spectra typically display several charge states corresponding to various adducted forms of intact molecular ions. Analysis of POMs by MALDI is a challenge because of the numerous charges that the POMs possess, but singly charged ions have been observed for small molecules . MALDI and LDI analyses were reported for characterization of unfunctionalized POM cores in negative ion mode [13, 14], and polymers containing POM structures were even characterized in positive ion mode [15, 16]. However, to our knowledge, no fragile polyoxoanionic structures containing functionalization have been reported as intact molecular species using MALDI or LDI in the negative ion mode. A study concerning polyanionic dendrimer species has been published but despite the presence of multiple negative anionic substituents the molecules remain in salt form with cations associated with each negative moiety, plus one additional cation to render the dendrimers detectable as singly charged intact cations .
This article reports on MALDI investigations of functionalized POMs, with special focus placed upon a model POM, denoted POMc12, the ionization behavior of which was characterized in the presence of various matrixes and using different means of target preparation (e.g., salt addition). The effect of matrix choice and sample preparation on mass spectrum quality has undergone a detailed evaluation. Among the tested matrixes, 9-cyanoanthracene, which is a rather unusual matrix, even if its use was reported once by Limbach and Robins as a suitable matrix for crude oil analysis , proved its capability to readily desorb and ionize the model POM. The energetic behavior of this matrix, through the ability to both allow molecular ion detection and induce specific in-source fragmentations, was compared with other matrixes. These comparisons led to a general matrix classification according to the ability to induce in-source dissociations. The competition between cations to either produce an intact adduct ion or to produce cation-containing fragments was studied to establish a ranking of cationic attachment preferences.
The standard matrixes, α-cyano-4-hydroxycinnamic acid (CHCA), sinapic acid (SA), 3-hydroxypicolinic acid (HPA), 2′,4′,6′-trihydroxyacetophenone (THAP), trans-3-indoleacrylic acid (IAA), 2-(4-hydroxyphenylazo)benzoic acid (HABA), dithranol, 1,5-diaminonaphtalene (DAN), 2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-enylidene]malononitrile (DCTB) were purchased from Sigma-Aldrich (St. Quentin Fallavier, France) as high-purity MALDI matrix grade and were used without further purification. Aqueous solutions of salt additives (LiCl, NaCl, KCl, MgCl2(6H2O), CuSO4(5H2O), CoCl2(6H2O), AlCl3, CrCl3, FeCl3(6H2O), Fe(NO3)3(9H2O), GaCl3, and Fe(NO3)3 were prepared with a cation concentration of 0.5 M in deionized MilliQ water (Millipore, Darmstadt, Germany). The 9-anthracenecarbonitrile (CNA) 97% (CAS 1210-12-4) matrix was purchased from Sigma-Aldrich and purified by solubilization and precipitation with mixtures of deionized water and HPLC grade tetrahydrofuran (THF) or acetonitrile (CH3CN) in different proportions. The precipitate obtained underwent a final wash using HPLC grade ethanol and was left to dry at room temperature. Upon recrystallization, the powder, initially colored yellow-khaki, became yellow.
The polyoxometalates used throughout this study were synthesized at the IPCM  and were graciously donated for this study. POMc12 is built with 10 tungsten-oxides distributed around a central silicate nucleus, grafted with two linear alkylphosphonium chains. This quadruply negatively charged compound with original formula TMA3K[SiW10O36(POC12H25)2] was weighed in its salt form and was directly solubilized in acetonitrile at 1 g/L.
Sample Preparation for MALDI Analyses
Two kinds of MALDI plates were used in our analyses. The first is a stainless steel plate; the second is a polymer plate, pre-spotted with CHCA spots jointly developed by Bruker and Eppendorf (Bruker Daltonics, Inc., Bremen, Germany). The polymer plate of 1.25 mm thickness was cleaned with a similar procedure as was applied to the stainless steel plates. Washes were performed with a mix of deionized water and acetonitrile (~400 mL) followed successively by washes with acetic acid and then sodium hydroxide (1 M solutions). Afterwards, a wash with cyclohexane followed by ethanol was performed to remove hydrophobic compounds. A final CH3CN wash left the plate clean and dry.
In order to minimize any differences in behavior due to solubility differences for the various matrixes, a special ternary solvent capable of solubilizing all the used matrices at a concentration of 10 g/L was employed. This solvent is composed of CH3CN-THF-water in a 5/4/1 (v/v/v) volumic proportion. This solvent mixture ensured that all tested matrixes were soluble and that introduced salt remained soluble. All the matrixes were dissolved and vortexed at 10 g/L without any acidification. Aqueous salt solutions were introduced in matrix solution in order to obtain matrixes with 5 mM cation concentrations using the following salts: LiCl, NaCl, KCl, MgCl2(6H2O), CuSO4(5H2O), CoCl2(6H2O), AlCl3, CrCl3, FeCl3(6H2O), Fe(NO3)3(9H2O), and GaCl3. Mixtures prepared initially with FeCl3 showed unexpected adduct species that presumably contained chloride, so the nitrate equivalent was used instead, and no further counter-ions were observed or studied, even though counter-ions have previously shown some influence on MALDI results . Moreover, salt precipitation was observed with iron(III) nitrate when matrix solution was prepared in advance, so matrix solutions were prepared just prior to analyses and were deposited on the MALDI plate, along with analytes, without delay. For the cation competition study, solutions containing two salts were obtained by mixing two previously prepared matrix solutions, ending in CNA matrix solutions containing 2.5 mM concentrations of each salt.
Analytes and matrixes were mixed and spotted using the dried droplet method . A 1 μL drop of matrix was spotted on the plate; each deposition was repeated two times for qualitative studies, or three times when peak intensities had to be considered for fragmentation yield calculations.
MALDI-TOF MS experiments were performed on an Autoflex III instrument (Bruker Daltonics, Inc., Bremen, Germany), equipped with a SmartBeam Nd:YAG laser emitting at 355 nm. Typically, 200 laser shots were accumulated to obtain a mass spectrum; each shot had a duration of 3 ns and a spot size diameter of approximately 50 μm. The repetition rate was 20 Hz. An extraction delay of 80 ns was used when employing detection in the reflectron mode, and ions under m/z 850 were not recorded in either ion mode, so as to preserve detectors. For high mass calibration (i.e., m/z 900 to m/z 3500), a calibration mix comprised of six peptides in a CHCA matrix was employed. The mass error for an obtained calibration curve was less than 10 ppm.
The employed laser power value can be attenuated by use of an adjustable diaphragm, and it can be varied downward from a maximum value of 100 μJ. In order to compare the fragmentation yields obtained for various spot preparation methods, the laser power is expressed in comparison with the specific matrix’s threshold laser power. Typically, a laser power of approximately 1.5 times the laser threshold value was used for each matrix to obtain in-source decay (ISD). No other parameters were modified because the optimum delay time observed for ISD fragments seems to be in agreement with standard MALDI-TOF/MS experiments .
MS/MS experiments using LIFT mode (Bruker Daltonics) were performed using Argon as collision gas (pressure about 10–6 mBar). Alternatively, MALDI-TOF/TOF tandem mass spectra were acquired using the Applied Biosystems 4700 Proteomics Analyzer (ABSciex, Concord, Ontario, CA). MALDI-TOF mass spectra of the samples were acquired in reflector mode using delayed extraction and a focus mass set at 2100 u, with a laser power just above the threshold for desorption/ionization and the low mass cut-off set at m/z 1000. MALDI-TOF/TOF fragmentation experiments were carried out either without addition of collision gas (residual pressure 2 × 10−8 Torr, favoring metastable decompositions), or under CID conditions (N2 at 2 × 10-7 Torr, with a collision energy of 1 keV).
Results and Discussion
A variety of polyoxometalates (POMs), including POMc12, were subjected to MALDI-TOF experiments. MALDI mass spectra were recorded from m/z 900–3500, and each prepared sample was tested in both positive and negative ion modes. Above m/z 3500, no identifiable signals were observed, whereas below m/z 900, ion matrix interferences combined with abundant low mass fragments could saturate the detector in both +/– polarity modes. The molecular mass of POMc12, without counter-ions is 2875 Da (form with four negative charges) and the monoisotopic peak is centered in the middle of the wide isotopic pattern. This hybrid compound, combining organic and inorganic structures, acts as a catalytic reagent to oxidize alkenes in microemulsions. It is able to sequester electrons provided by hydrogen peroxide, and subsequently deliver them to epoxy compounds . With the exception of one instance where a mixture of POMc12 and Li+ produced an ion corresponding to [POMc12 + 5Li]+, no other combination of POM/salt allowed the detection of intact POM signals in the positive ion mode. The results that follow all pertain to the negative ion mode and, unexpectedly, all the signals observed revealed singly charged species, despite the inherent polyanionic nature of POMs. Moreover, the numerous isotopes of tungsten, combined with the fact that each analyte contains 10 tungsten atoms, invariably produced a wide isotopic pattern for each species present. The readily visible nature of this isotopic pattern, characteristic of each POM, presents an advantage for identifying POM species. However, it also introduces a drawback in the case of ions having close m/z values because of the overlap of the respective isotopic patterns. When extensive superposition of isotope patterns occurs, there is a difficulty to unequivocally attribute a unique formula to each peak.
Method Development for POMs Analysis in Negative Ion MALDI MS
Upon achieving success with singly charged cations, and bearing in mind that there are four negative charges held by the analyte, a wide range of cations with different charge states were tested. The separate use of many different cations permits the observation of molecular singly charged adduct species, (e.g., [POMc12 + 3Li]–, [POMc12 + Ga]–, or [POMc12 + Al]–, see Figure 1). However, as often occurs in MALDI analysis, no multiply charged species were detected , regardless of the employed instrumental parameters and target preparation used (including the presence of additives).
Signals obtained using the CNA matrix without salt addition led to the production of a weak signal at m/z 2860, which is assigned as a singly charged fragment ion [POMc12-O + H]-, and numerous other overlapping signals (as evidenced by the extended isotope patterns observed) at higher m/z values (data not shown). These unresolved signals that are clearly constituted of tungsten cores could be considered to represent adducts of POMc12 that incorporate various cations that are present in the bulk. Note that POMc12 is initially synthesized with potassium and trimethylammonium (TMA) counter-ions. Routine ESI-MS analysis performed in our laboratory  revealed the presence of these cations in adduct species of intact POMs. Surprisingly, MALDI analysis in the negative ion mode never showed molecular adduct ions containing TMA as counter-ions. This result contrasts with what was observed for a different POM structure in positive ion mode by Mayer et al. . Using the CNA matrix doped with aluminum chloride, the MALDI mass spectrum of POMc12 (Figure 1) displays an abundant and unique molecular ion species [POMc12 + Al]–; this cation was thus chosen to optimize sample preparation. Molar ratios of Al3+:POMc12 between 10 and 1000 provided similar results (data not shown). A ratio below 10 did not induce sufficient adduct ion formation, whereas ratios higher than 1000 led to salt precipitation in the apolar solvents. The final three-component ratio of (matrix):(added cations):(analyte) was approximately of 1600:160:1 for mixes containing one salt, or 1600:80:1 for mixes containing two added salts (each salt present in a proportion of 80). An excess of cations served to ensure the production of the desired abundant intact POM adduct ion. Notably, two different MALDI plates were used during our analyses: one made of stainless steel and the second an isolating polymer (see Experimental section). The results were quite comparable for the two plates. Fragmentation yield and, more generally, signal intensities, were higher on the polymer plate, but no clear intact species desorption trends or in-source fragmentation trends could be correlated to the plate composition. Herein, all the presented results were obtained using a stainless steel MALDI plate.
Two Categories of Matrixes: Group 1 and Group 2
MALDI In-Source Decay (ISD) of POMc12, Mechanism of Decomposition
Fragment ions observed by decompositions of POMc12 are divided into two parts. The first part concerns high mass fragments (m/z >2500) which still contain a portion of the organic functionalization. Among these decomposition products, the fragment ion centered at m/z 2627 [HSiW10O34POC12H25]– corresponds to the loss of one alkylphosphonium group ([O3PC12H25]2–). This fragment ion is observed for all targets (Figure 2), whatever the Group 1 matrix used (i.e., CNA, IAA, HABA, DAN, DIT, DCTB and CHCA). This m/z 2627 fragment appears in variable relative intensity and could possibly include a contribution from product degradation occurring at the moment of target preparation, instead of being produced during the MALDI process. Among the matrix-dependent fragments observed in the m/z 2500–2850 range, the highest m/z detected fragment ion (m/z 2860) corresponding to [POMc12-O + H]– was observed only with DAN and CHCA as matrix.
The second type of fragment ions corresponds to those that are detected below m/z 2000 (Figure 2). These fragments, observed especially at elevated laser powers, are produced through prompt in-source dissociations, also known as in-source decay (ISD). ISD has been mainly reported for peptides and proteins and ISD experiments on peptides mainly display c and z in-source fragments . Such sequencing fragment ions were produced through radical fragmentation processes as demonstrated by hydrogen/deuterium transfer . Similarly, dissociations of benzyl-pyridines used as thermometer molecules were explained through two fragmentation mechanisms, one similar to peptide fragmentation  and the other by a thermal dissociation route . For the investigation of glycans, ISD produced hydrogen abstraction (still as H•) . ISD experiments are often successful at producing abundant fragments when DHB, SA or DAN are employed [30, 31]. These matrixes exhibit a propensity to readily provide H• during the MALDI process and this possibility can induce extensive in-source fragmentations. Thus, matrix selection is an important parameter for ISD experiments, and the tendency of a given molecule toward fragmentation is clearly matrix-dependent .
In examining the matrixes of the first group characterized by abundant in-source fragment ions in our experiments, the matrixes listed (except for DAN ) are not usually considered to be efficient in generating ISD on more commonly encountered analytes (e.g., peptides). For example, the widely used CHCA matrix that offered particularly extensive in-source fragmentations in our MALDI experiments is usually considered to be an inefficient matrix for promoting ISD [30, 34, 35, 36]. However, the CHCA matrix found an appreciation as the most efficient matrix to provide metastable dissociations of peptides or proteins during so-called post-source decay (PSD) experiments . The kinetics of PSD and ISD processes are quite different . ISD is a prompt process leading to direct fragmentation in the ion source, as opposed to PSD, which leads to the production of metastable fragment ions that are produced/appear only after leaving the ion source (some tens of microseconds following the laser shot). ISD fragments are formed promptly during the first nanoseconds in the MALDI plume or even earlier during laser irradiation, while analyte molecules are still in the crystalline solid . Such prompt fragmentations found a coherent explanation in the proposed radical origin of these processes, which can also rationalize the matrix dependence of the decomposition behavior. Hydrogen radical donating abilities of various matrixes have been measured  and results show that DHB or DAN matrixes are capable of providing H• much more readily than CHCA. Conversely, PSD is a much slower dissociation process induced by vibrational energy uptake of the analytes, similar to the energy uptake occurring in collision induced decomposition (CID) processes .
The fragmentation mechanism of POMc12 with its oxometallic core coordination structure cannot be compared with peptides that have a linear primary structure. The origin of the observed facile fragmentations in POMs is thus likely to be different from the radical-induced process that operates for peptides. We thus consider that hydrogen radical transfer is not a major factor in ISD of POMc12 because most of the matrixes observed to induce in-source fragmentations in our experiments are not efficient providers of H• (e.g., CHCA, CNA, DCTB).
Electron emission is a well-documented occurrence in MALDI, and the matrix itself can be a supplier of electrons, as can be the MALDI plate surface . In the case of CNA, the only matrix ions observed in the mass spectrum are CNA•–/CNA•+ (see Supplementary Materials, Figure S2). In an attempt to clarify the role of electrons in fragmentation processes, an electrically insulated polymer plate was used as the MALDI support. Surprisingly, the level of fragmentation and the presence of fragments containing an extra electron were even higher when using the insulated plate (see Supplementary Materials, Figure S3). We conclude that the ISD fragmentation process is not being driven by electronic emission from the plate.
Calculation of ISD Fragment Yields and Ranking of Matrix Propensity Toward Fragmentation
Number of peaks in a given isotopic cluster used to calculate the in-source fragmentation yield
Number of W in the ion
Number of peaks integrated to reach 50% of total signal’s area
Fragmentation yield and peak resolution in the region of the molecular adduct ion at 1.5 × threshold laser power. Mixtures consisted of matrix at 10 g/L in ternary solvent with 5 mM AlCl3
Peak resolution on [POM + Al]–
Peak resolution on [SiW5O17Al]–
The second group of matrixes (i.e., DHB, HPA, SA and FA) required higher laser powers to give optimal signals, and we speculate that this is due to an increased difficulty to desolvate the molecular adduct ion upon desorption. This second group of matrixes showed no in-source fragmentations whatsoever, and only a variety of low-level signals were observable above the molecular mass, indicating that the expected specific adduct ions were less promoted or disfavored.
Among all of the matrixes of the first group, follow-up studies focused on the use of CNA. This matrix allows the observation of highly resolved peaks for intact molecular adduct ions and fragments ions at high S/N values compared with spectra obtained using other matrixes under identical instrumental conditions. The CNA matrix produced mainly a single high-abundance molecular adduct ion at just above the threshold laser power value. Compared with DAN or CHCA, which display higher fragmentation yield values, CNA preferentially induces the production of low mass fragments possessing less than 8 tungsten atoms (see Table 2). CNA induced higher in-source fragmentations than DIT, HABA, DCTB and IAA at laser power values of 1.5 times the laser threshold. As shown in Table 2, use of the CNA matrix resulted in the highest resolution spectra either for the molecular adduct ion, or for fragment ion signals, regardless of the laser power used.
Competition for Cation Attachment: Evaluation of POMc12's Affinity for Various Cations
Even if most trivalent cations (i.e., Fe3+, Al3+, Ga3+) display abundant singly charged [POM + cation]– signals, as might be expected for such quadruply negatively charged POM species, the trend cannot be explained simply on the basis of cation charge because another trivalent cation (i.e. Cr3+) shows a very poor propensity for POM attachment. In evaluating the alkali metals and their affinities for the polyanionic POM, Li+ gave very satisfactory results producing an abundant [POM + 3Li]– signal, whereas Na+ and K+ do not result in the detection of analogous resolved species in the molecular ion region. When two salts were introduced simultaneously, the molecular adduct ion signal generally revealed a preferential interaction of POMc12 for one of the two cations. For instance, the mixture of Ga3+ and Al3+ led only to the observation of the singly charged [POMc12 + Al]–. In a direct comparison of Al3+ versus Fe3+, exclusive formation of a specific adduct species was demonstrated by placing 10 times more Al3+ than Fe3+ in the sample preparation (matrix/Al3+/Fe3+/POMc12 ratio 1500/150/15/1); despite the disadvantage in terms of concentration, ionic molecular species containing uniquely Fe(III) were observed. However, mixtures of divalent and monovalent cations can provide both mixed and single cation-containing adduct species. For example, the mixture of Co2+ + Li+ produced two main species by MALDI: [POMc12 + 3Li]– and [POMc12 + Li + Co]– (see Figure 4); however, it is possible to rank these two cations (Li+ and Cu2+) by performing a comparison with a third cation, Ga3+. Gallium has a higher affinity for POMc12 than Cu2+ but lower than Li+.
Variation in the signal intensity of the adduct species as a function of the particular cation participating in adduction has been previously reported in MALDI-MS . In assessing POMc12 interactions with cations, no overwhelming trend can be drawn from the MALDI mass spectra concerning a preferred valence of the cation. Nevertheless, it was noticed that cations characterized by smaller radii do exhibit an increased tendency to form abundant molecular adduct species. The radii of cations such as Al3+ (54 pm), Li+ (76 pm), or Cu2+ (73 pm)  that efficiently form adducts with POMc12 and exhibit large signals in mass spectra are much smaller than those of the other alkali metals (e.g., Na+, K+, whose radii exceed 100 pm) that provide poor MALDI results, or even TMA+, which was never observed as an adduct on POMc12 during our MALDI experiments. To test whether the cation affinity trend was more general in nature, ESI experiments were undertaken to ascertain whether those cations exhibiting strong affinities for POMc12 in MALDI would perform equally well in electrospray ionization (ESI). Somewhat surprisingly, the use of ESI did not result in the observation of any species entailing lithium or aluminum adduction to the model POM. However, this result is in line with that of Deery et al. , who also observed divergent behaviors for MALDI-MS and ESI-MS analyses of polystyrene cationic adducts. In addition, the little NMR data pertaining to characterization of POMc12 in solution  establishes the proximity of counter-ions TMA+ or K+ to POMc12 in the liquid phase, but no NMR characterization has been performed in the presence of cations such as Fe3+ or Li+. Notably, results derived from in-solution conditions may not be applicable to a solid matrix such as that which constitutes the MALDI target.
Presence of Cations in Fragment Ions of POMc12
Abundant ions observed from a mixture of [SiW10O36(POC12H25)2]4– and various cations as a function of the number of tungsten atoms (#W) contained in a fragment. Results for addition of Li+, Al3+, and Co2+ cations are presented, along with those for the POM without any added salt (Ø). Underlined fragments represent species that have undergone a one-electron reduction. Fragments observed with zero cations incorporated invariably represent low abundance signals
Molecular adduct ion
[POMc12 + 3Li]–
[POMc12 + Al]–
[POMc12 + CoH]–
[POMc12 + 2Co]–
For POMc12, when triply charged cations, such as Al3+, Fe3+, Cr3+ and Ga3+ are employed, fragments observed contain an extra oxygen compared with the ones observed when adding singly charged cations [e.g., (SiW5O19Ga)– instead of (SiW5O18Li]–]. Despite the variety of fragment ion compositions, all of these species remain globally singly charged under MALDI conditions. For doubly charged cation addition (considering only Co and Cu because Mn and Mg did not display enough signal to be interpreted), however, two different outcomes are observed in MALDI mass spectra. Some POM fragments contain the divalent cation plus a proton (a “trivalent-like” cation), the elemental composition of which is similar to that observed in the presence of a triply charged cation (e.g., [SiW4O16CuH]– has the same core structure as [SiW4O16Fe]–). The second type of fragment ion has captured an electron, so the integrated divalent cation is included effectively as a monovalent cation (e.g., [SiW4O15Cu]– has the same core structure as [SiW4O15Li]–). Electron capture could be either considered as the reduction of the cation or the reduction of the core POM; this type of reduction has been commonly observed for Cu(II) , but it is more surprising to also observe this behavior for fragments containing Co(II).
Analyses of Other POMs Using CNA as the MALDI Matrix
The optimized target preparation method was applied to other functionalized POMs with different charge states, such as species of the following molecular formulas: (PW11O39[GePhCCPhN3])4- or (PW11O39[SnPhI])4– or (SiW9O34[POC12H25]2)6– synthesized with TMA+, Na+, or K+ as counter ions . For these functionalized POMs, in-source fragments were observed in preference to molecular species. The most successful analyses, with respect to POMs of similar structure to POMc12, were performed for TMA3K(SiW10O36[POC10H21]2), TBA3K(SiW10O36[POC8H17]2), and TBA3K(SiW10O36[POH]2) that differ only by the length of the attached alkyl side chains (see Supplementary Materials, Figure S4). In addition, the non-functionalized POM K3[PW12O40] also gave useful results (see Supplementary Materials, Figure S5). In the presence of LiCl or NaCl, introduced in matrix solutions, the anions [PW12O40+2Li]– or [PW12O40+2Na]– were easily detected. This inorganic compound could be ionized by laser desorption ionization (LDI), i.e., without matrix addition, employing a high laser fluence. Yet, LDI spectra display, even in the presence of added LiCl, only PW12O39– ions (having undergone loss of one oxygen atom). The fact that POMs are not “electron sensitive” (i.e., they can stock and release electrons without further decompositions ), could be an important factor in promoting their survival in the plume, rich in matrix radical anions and cations.
Polyanionic polyoxometalates (POMs) have been successfully analyzed using MALDI-MS in the negative ion mode. Sample matrix optimization established that the matrix 9-cyanoanthracene (CNA), in the presence of an excess of cations, provided efficient analyses (intense and highly resolved peaks) either for singly charged intact POMc12 anions, or for in-source fragments when the laser power was largely increased. Comparing a first group of matrixes: CNA, IAA, HABA, DAN, DIT, DCTB, and CHCA to a second group: DHB, SA, FA, THAP, and HPA, we observed that the first group offers both more abundant intact singly charged anionic adducts and prevalent in-source fragmentations at elevated laser powers that are absent when employing the second group. The ability to induce in-source fragmentations was found to vary as a function of the employed matrix, thereby allowing us to establish a ranking of the matrixes starting with those that exhibit facile fragmentation to those that offer limited or no ISD: DAN > CHCA >CNA> DIT > HABA > DCTB > IAA > [ DHB, SA, FA, THAP, HPA]. We postulate that the first group of matrixes enables a higher level of energy uptake to POM species, thereby permitting an initial higher level of desolvation and eventually access to endothermic decompositions that are not accessible when employing the second group of matrixes. The observed prompt fragments are proposed to originate from transferred energy derived from initial electronic excitation of the matrix, which rationalizes the increase in ISD with rising laser power. We postulate that this energy is rapidly converted to vibrational energy that leads to prompt decompositions in the MALDI source. POMs are known for their capability to sequester and release electrons, and this property is exhibited in negative ion mass spectra that reveal molecular ions that have obligatorily accepted an electron.
Experiments designed to assess the competition of various cations toward adduct formation with POMc12 have enabled the construction of the following affinity ranking of POMc12 towards cations: Fe3+ > Al3+ > Li+ > Ga3+ > Co2+ > Cr3+ > Cu2+ > [Mn2+, Mg2+] > [Na+, K+]. This experimental affinity ranking reflects, at least in part, a gas-phase competition for attachment to the polyanionic POM that occurs prior to any fragmentation process. The observed “high affinity” adduct species are produced during the early stages of plume expansion as demonstrated by the observation of high affinity complexes from initial mixtures comprised of two matrix crystal preparations: the first containing only low affinity complexes with POMc12, and the second containing only the higher affinity salt. We additionally observe that in the vast majority of cases, formed fragment ions contain the cations with the strongest affinities for the polyanionic POM. This finding corroborates our conclusion that Group 1 matrixes permit thermal desorption of POM-containing aggregates that contain sufficient energy to permit movement and attachment of preferred cations, and to allow complete desolvation of cation-bearing, singly charged anionic POMs. If sufficient energy is made available at augmented laser powers, prompt fragmentation of these adducts can ensue.
The authors especially thank our colleagues Drs. Vincent Jallet and Geoffrey Guillemot and Pr. Anna Proust from the E-POM group of the IPCM laboratory for the POM products that they graciously provided. The authors also thank Dr. Joëlle Vinh for allowing them to perform experiments on the MALDI-Orbitrap at the ESPCI.