Potassium cation affinities of matrix assisted laser desorption ionization matrices determined by threshold collision-induced dissociation: application to benzoic acid derivatives

Threshold collision-induced dissociation of K+(xBA) complexes with xenon is studied using guided ion beam mass spectrometry. The xBA ligands studied include benzoic acid and all of the mono- and dihydroxy-substituted benzoic acids: 2-, 3-, and 4-hydroxybenzoic acid and 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dihydroxybenzoic acid. In all cases, the primary product corresponds to endothermic loss of the intact xBA ligand. The cross section thresholds are interpreted to yield 0 and 298 K bond dissociation energies (BDEs) for K+-xBA after accounting for the effects of multiple ion-neutral collisions, the kinetic and internal energy distributions of the reactants, and dissociation lifetimes. Density functional theory calculations at the B3LYP/6-31G* level of theory are used to determine the structures of the xBA ligands and their complexes with K+. Theoretical BDEs are determined from single-point energy calculations at the B3LYP/6-311+G(2d,2p) and MP2(full)/6-311+G(2d,2p) levels using B3LYP/6-31G* optimized geometries. Four favorable binding modes for the K+(xBA) complexes are found. In all complexes to an xBA ligand that does not have a 2-hydroxyl substituent, the most favorable binding mode corresponds to a single interaction with the carbonyl oxygen atom. Formation of a 4-membered ring via chelation interactions with both oxygen atoms of the carboxylic acid group is found to be the most favorable binding mode for all of the 2-hydroxy-substituted systems except K+(2,3-dihydroxybenzoic acid). In these complexes, a hydrogen-bonding interaction between the hydrogen atom of the carboxylic acid moiety and the oxygen atom of the 2-hydroxy substituent provides additional stabilization. Formation of a 5-membered chelation ring via interaction of K+ with the oxygen atoms of adjacent hydroxyl substituents is also favorable and corresponds to the ground-state geometry for the K+(23DHBA) complex. Formation of a 6-membered chelation ring via interaction of K+ with the carbonyl and 2-hydroxyl oxygen atoms is also quite favorable but does not correspond to the ground-state geometry for any of the systems examined here. The experimental BDEs determined here are in very good agreement with the calculated values.     

Mass spectrometry--not just a structural tool: the use of guided ion beam tandem mass spectrometry to determine thermochemistry

Guided ion beam tandem mass spectrometry has proved to be a robust tool for the measurement of thermodynamic information. Over the past twenty years, we have elucidated a number of factors necessary to make such thermochemistry accurate. Careful attention must be paid to the reduction of the raw data, ion intensities versus laboratory ion energies, to a more useful form, reaction cross sections versus relative kinetic energy. Analysis of the kinetic energy dependence of cross sections for endothermic reactions can then reveal thermodynamic data for both bimolecular and collision-induced dissociation (CID) processes. Such analyses need to include consideration of the explicit kinetic and internal energy distributions of the reactants, the effects of multiple collisions, the identity of the collision partner in CID processes, the kinetics of the reaction being studied, and competition between parallel reactions. This work provides examples illustrating the need to consider this multitude of effects along with details of the procedures developed in our group for handling each of them.     

Electrospray ionization tandem mass spectrometry determined regioisomeric structures of disubstituted beta-cyclodextrin derivatives

Electrospray ionization tandem mass spectrometry was applied to the analysis of regiospecifically disubstituted beta-cyclodextrins. Each disubstituted regioisomer showed unique fragmentation, distinguishing each of them unambiguously. These results demonstrate that mass spectrometry is the most straightforward method among those reported in the assignment of regioisomeric structures of these oligosaccharides.     

Reactive MALDI mass spectrometry: application to high mass alkanes and polyethylene

A molecular solid of fullerene (C(60)) intercalated with cobalt cyclopentadienyl dicarbonyl (CoCp(CO)(2)) was shown to be an effective matrix for matrix-assisted laser desorption/ionization mass spectrometry (MALDI) of large alkanes (demonstrated up to C(94)H(190)) and polyethylenes that otherwise cannot be produced as intact ions in the gas phase.     

Protein-metal ion interactions,stoichiometries and relative affinities determined by on-line size exclusion gel filtration mass spectrometry

The modulation of metal ions on protein function is well recognized and of paramount importance in protein biochemistry. To date, very few methods allow direct determination of protein-metal ion interactions, as well as exact stoichiometric binding ratios. In this work we demonstrate the usefulness of two on-line size exclusion gel filtration mass spectrometry approaches to directly detect protein-metal ion adducts, as well as determine exact protein-metal ion stoichiometries. We show that on-line size exclusion column chromatography (SEC) and rapid in-line desalting (RILED) coupled to microelectrospray mass spectrometry (microESI-MS) can be used for such analyses. The SEC approach can be effectively used to both separate proteins in a complex mixture and exchange buffers prior to the electrospray process. While RILED does not allow for protein separation, it provides a much faster high-throughput desalting procedure than the conventional SEC technique. Specifically, we show that SEC/microESI-MS and RILED/MS can be used to determine calcium ion binding stoichiometries to a high-affinity, metal ion binding protein, calbindin D(28K). Furthermore, the same approaches can also be used to determine metal ion binding stoichiometries of low-affinity metal-binding proteins such as Spo0F.     

Negative ion tandem mass spectrometry of prenylated fungal metabolites and their derivatives

Liquid chromatography negative ion electrospray ionisation tandem mass spectrometry has been used for characterisation of naturally occurring prenylated fungal metabolites and synthetic derivatives. The fragmentation studies allow an elucidation of the decomposition pathways for these compounds. It could be shown, that the prenyl side chain is degraded by successive radical losses of C₅ units. Both the benzoquinones and the phenolic derivatives display significant key ions comprising the aromatic ring. In some cases, the formation of significant oxygen-free key ions could be evidenced by high-resolution MS/MS measurements. Furthermore, the different types of basic skeletons, benzoquinones and phenol type as well as cyclic prenylated compounds, can be differentiated by their MS/MS behaviour.

Matrix‐enhanced secondary ion mass spectrometry: the influence of MALDI matrices on molecular ion yields of thin organic films

In this work the effect in secondary ion mass spectrometry (SIMS) of several frequently used matrix‐assisted laser desorption/ionisation (MALDI) matrices on the secondary ion intensities of low molecular weight (m/z 400–800) organic dyes and a pharmaceutical is tested. Matrix (10^?1 M) and analyte (10^?2 M) solutions were made in methanol. Mixtures with several concentration ratios were prepared from these solutions and spincoated on Si substrates prior to time‐of‐flight (TOF)‐SIMS analysis. In some cases the presence of the MALDI matrices caused a considerable increase in the positive secondary (protonated) molecular ion signals. Enhancements of a factor of 20 and more were recorded. Generally, of the matrices used, 2,5‐dihydroxybenzoic acid and 2,4,6‐trihydroxyacetophenone brought about the highest intensity increases. It was also shown that matrix‐enhanced (ME‐)SIMS is capable of lowering the detection limits for molecule ions. However, the enhancement effect is strongly influenced by the analyte/matrix combination and its concentration ratio. As a result, finding an optimal analyte/matrix mixture can be a very time‐consuming process. Mostly, the presence of the matrices causes changes in the relative ion intensities in the TOF‐S‐SIMS spectra. Compared to the spectra recorded from samples without matrices, only a few additional peaks, such as signals that originate directly from the applied matrix or adduct ions, are observed in the mass spectra. Sometimes molecule ions and some characteristic fragments at high m/z values, that cannot be recorded without matrix, do appear in the spectrum when a matrix is present. In the negative mode no enhancement effect is observed on applying the studied MALDI matrices. The results obtained from samples treated with MALDI matrices are also compared to SIMS results for the same samples after Ag and Au metallisation (MetA‐SIMS). For three of the four tested compounds Au MetA‐SIMS resulted in higher ion yields than ME‐SIMS. For both techniques possible mechanisms that can account for the enhancement effect are proposed. Copyright © 2005 John Wiley & Sons, Ltd.     

Alkali-hydroxide-doped matrices for structural characterization of neutral underivatized oligosaccharides by MALDI time-of-flight mass spectrometry

We report new approaches using alkali-hydroxide-doped matrices to facilitate structural characterization of neutral underivatized oligosaccharides by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS. The approaches involved pretreatment of the analytes with NaOH or LiOH in aqueous solution, followed by mixing them with MALDI matrices prior to MS analysis. It was found that for open-ended neutral underivatized oligosaccharides partial alkaline degradation occurred upon laser desorption and ionization of the hydroxide-pretreated analytes in 2,5-dihydroxybenzoic acid (DHBA). The effect intensified when nonacidic compounds such as 2,4,6-trihydroxyacetophenone (THAP) and 5-amino-2-mercapto-1,3,4-thiadiazole (AMT) were used as matrix. The degradation allowed facile identification of the reducing end residue of the analyte and facilitated its structural characterization by postsource decay TOF-MS. Applying the same technique using matrices composed of LiOH and THAP or AMT led to the production of singly as well as multiple lithiated ions of oligosaccharides containing hexoses with free 3-OH groups. Extensive lithiation through multiple hydrogen-lithium exchanges up to 6 Li atoms was observed for maltoheptaose, beta-cyclodextrin, and dextran 1500. Such a 'lithium tagging' technique makes it possible to differentiate positional isomers of milk-neutral oligosaccharides, lacto-N-difucohexaose I and II (LNDFH-I and LNDFH-II), without the need of chemical derivatization or tandem MS analysis.     

Rapid comprehensive amino acid analysis by liquid chromatography/tandem mass spectrometry: comparison to cation exchange with post-column ninhydrin detection

Ion-exchange chromatography with ninhydrin detection remains the gold standard for detecting inborn errors of amino acid catabolism and transport. Disadvantages of such analysis include long chromatography times and interference from other ninhydrin-positive compounds. The aim of this project was to develop a more rapid and specific technique using liquid chromatography/tandem mass spectrometry (LC/MS/MS). Optimal fragmentation patterns for 32 amino acids were determined on a triple quadrupole mass spectrometer following butylation. Chromatographic characteristics of each of the amino acids were determined using C8 reversed-phase chromatography with 20% acetonitrile/0.1% formic acid as isocratic mobile phase. Quantitation using eleven deuterated internal standards was compared to cation exchange and ninhydrin detection on a Beckman 7300 system. Following methanol extraction and butylation, determination of 32 amino acids required 20 min. The dynamic range of each amino acid was generally 1-1000 micromol/L. Imprecision ranged from 7 to 23% (CV) over 6 months and recovery ranged from 88-125%. Deming regression with the Beckman 7300 yielded slopes from 0.4-1.2, intercepts from -21 to 65 micromol/L, correlation coefficients from 0.84-0.99 and Syx from 2-125 micromol/L. Isobaric amino acids were separated by chromatography (e.g. leucine, isoleucine) or by unique fragmentation (e.g., alanine, beta-alanine). LC/MS/MS is comparable to traditional LC-ninhydrin detection. Mass spectral detection shortens analysis times and reduces potential for interference in detecting inborn metabolic errors.     

离子色谱-串联质谱法检测茶叶中的高氯酸盐

建立了离子色谱-串联质谱检测茶叶中高氯酸盐的分析方法。选用高容量、强亲水性的IonPac AS20阴离子交换柱（2 mm）进行分离,以淋洗液自动发生器在线产生的70 mmol/L氢氧化钾为淋洗液等浓度淋洗,TSQ Quantiva三重四极杆质谱仪作检测器,采用电喷雾电离源负离子（ESI^-）模式、多反应监测（MRM）模式进行分析,内标法定量。结果表明,高氯酸盐在0.02~10.0 μg/L范围内线性关系良好,相关系数（r）为0.9991,定量限为2 μg/kg。运用该方法测定5种茶叶中的高氯酸盐,加标回收率为87.3%~112.2%。该方法具有操作简单、专属性强、灵敏度高等优点,可满足茶叶中高氯酸盐的检测要求。     

Cinnamic acid derivatives as matrices for ultraviolet laser desorption mass spectrometry of proteins

The paper reports the discovery of three new matrices for the matrix-assisted laser desorption of proteins. These new matrices (sinapinic, ferulic and caffeic acids) are cinnamic acid derivatives that have several practical advantages over the nicotinic acid matrices previously used. These materials form much less intense photochemically generated adduct peaks in the protein quasimolecular ion signal and the adduct peaks that are present are easier to resolve. These matrices produce intense protonated-molecule ions from all of the proteins (over 50) so far examined. These new matrices are also very stable in a vacuum, allowing for their convenient use in very high vacuum applications (e.g., Fourier transform ion cyclotron resonance mass spectrometry).     

Analysis of polycyclic aromatic hydrocarbons by ion trap tandem mass spectrometry

An ion-trap mass spectrometer with a wave board and tandem mass spectrometry software was used to analyze gas chromatographically separated polycyclic aromatic hydrocarbons (PAHs) by using collision-induced dissociation (CID). The nonresonant (multiple collision) mode was used to determine the conditions for CID ionization of 18 PAHs. Unlike in electron impact (EI) analysis, the relative abundances of progeny ions of isomers were statistically different (using Student’s t-test) in CID analysis, thus making isomer identification by CID possible. For comparison, CID and EI were applied to the analysis of used motor oil. CID analysis was shown to be more sensitive than EI analysis of the used motor oil. Precision at the 10-ppb level for EI and CID showed relative standard deviations of 5. 2 and 7. 7%, respectively.     

Structural determination of cyclitol derivatives by fast-atom bombardment tandem mass spectrometry

Three cyclitol derivatives were isolated from the marine sponge Sarcotragus sp. by reversed-phase high-performance liquid chromatography and analyzed by fast-atom bombardment mass spectrometry (FAB-MS). Their structural elucidation was carried out with FAB tandem mass spectrometry (FAB-MS/MS). FAB-MS spectra produced a significant abundance of the sodium adducts [M+Na]+ and [M+2Na-H]+ from a mixture of m-NBA and NaI. In addition, trifluoroacetylation of the cyclitol derivatives was used for confirmation of the presence of the cyclitol ring. High abundance [M-5H+5CF3CO+Na]+ ions were observed in the FAB-MS spectra of the trifluoroacetyl-cyclitol derivatives. Collision-induced dissociation (CID) of the [M+Na]+ ions produced diverse product ions via a series of dissociative processes. Charge-remote fragmentation (CRF) patterns of [M+Na]+ ions were very useful for the identification of product ions which are characteristic for the cyclitol ring and long hydrocarbon chains substituted at the glycerol backbone. Moreover, the CID-MS/MS spectra of the [M+Na]+ ions yielded characteristic product ions at m/z 53, 83, 113, 155 and 171 for the cyclitol moiety, and at m/z 213, 229 and 245 for the glycerol backbone attached to the cyclitol ring.     

Fragmentation of phosphopeptides by atmospheric pressure MALDI and ESI/ion trap mass spectrometry

An investigation of phosphate loss from phosphopeptide ions was conducted, using both atmospheric pressure matrix-assisted laser desorption/ionization (AP MALDI) and electrospray ionization (ESI) coupled to an ion trap mass spectrometer (ITMS). These experiments were carried out on a number of phosphorylated peptides in order to investigate gas phase dephosphorylation patterns associated with phosphoserine, phosphothreonine, and phosphotyrosine residues. In particular, we explored the fragmentation patterns of phosphotyrosine containing peptides, which experience a loss of 98 Da under collision induced dissociation (CID) conditions in the ITMS. The loss of 98 Da is unexpected for phosphotyrosine, given the structure of its side chain. The fragmentation of phosphoserine and phosphothreonine containing peptides was also investigated. While phosphoserine and phosphothreonine residues undergo a loss of 98 Da under CID conditions regardless of peptide amino acid composition, phosphate loss from phosphotyrosine residues seems to be dependent on the presence of arginine or lysine residues in the peptide sequence.     

Furofuranic glycosylated lignans: a gas-phase ion chemistry investigation by tandem mass spectrometry

Alkali metal cation adducts, [M+Alk](+), and [M-H](-) ions of four known glycosylated furofuran lignans, (+)-pinoresinol 4-O-beta-D-glucopyranoside, (+)-phylliroside, (+)-8-hydroxypinoresinol 4-O-beta-D-glucopyranoside, and (+)-8-hydroxypinoresinol 8-O-beta-D-glucopyranoside, recently isolated from Carex distachya, were generated by electrospray ionization and allowed to undergo collisionally activated dissociation (CAD) in a quadrupole ion trap (QIT) and in a triple quadrupole (TQ) mass spectrometer. CAD mass spectra of [M+Na](+) and [M+Li](+) adducts revealed the presence of structurally diagnostic product ions. CAD mass spectra of deprotonated glycosylated furofuran lignans showed the typical neutral loss of 162 Da when the glucose residue was bound to a phenolic oxygen atom. When glycosylation occurred at an alcoholic oxygen, as for (+)-8-hydroxypinoresinol 8-O-beta-D-glucopyranoside, a neutral loss of 180 Da represented the main fragmentation pathway. Selective hydrogen/deuterium (H/D) exchange of all the acidic hydrogen atoms of furofuran glycosides, performed by introducing lignan glycosides in D(2)O/CH(3)OD solutions, were employed to obtain information on the nature of the product ions generated during TQ/CAD processes. Energy-resolved TQ/CAD mass spectra of deprotonated lignan glycosides and their deprotonated aglycones were used in a qualitative way to infer information on the integrated energetic picture of CAD fragmentations and to investigate the mechanism of the predominant dissociation/isomerization processes. On the basis of the hypothesized fragmentation mechanisms, gas-phase features of the furofuran ring were derived. The presence of an OH substituent in the C8 position decreased the electron density in the adjacent C8' position, modifying the fragmentation pathway.     

Characterization of N-palmitoylated human growth hormone by in situ liquid-liquid extraction and MALDI tandem mass spectrometry

Acylation is a common post-translational modification found in secreted proteins and membrane-associated proteins, including signal transducing and regulatory proteins. Acylation is also explored in the pharmaceutical and biotechnology industry to increase the stability and lifetime of protein-based products. The presence of acyl moieties in proteins and peptides affects the physico-chemical properties of these species, thereby modulating protein stability, function, localization and molecular interactions. Characterization of protein acylation is a challenging analytical task, which includes the precise definition of the acylation sites in proteins and determination of the identity and molecular heterogeneity of the acyl moiety at each individual site. In this study, we generated a chemically modified human growth hormone (hGH) by incorporation of a palmitoyl moiety on the N(epsilon) group of a lysine residue. Monoacylation of the hGH protein was confirmed by determination of the intact molecular weight by mass spectrometry. Detailed analysis of protein acylation was achieved by analysis of peptides derived from hGH by protease treatment. However, peptide mass mapping by MALDI MS using trypsin and AspN proteases and standard sample preparation methods did not reveal any palmitoylated peptides. In contrast, in situ liquid-liquid extraction (LLE) performed directly on the MALDI MS metal target enabled detection of acylated peptide candidates by MALDI MS and demonstrated that hGH was N-palmitoylated at multiple lysine residues. MALDI MS and MS/MS analysis of the modified peptides mapped the N-palmitoylation sites to Lys158, Lys172 and Lys140 or Lys145. This study demonstrates the utility of LLE/MALDI MS/MS for mapping and characterization of acylation sites in proteins and peptides and the importance of optimizing sample preparation methods for mass spectrometry-based determination of substoichiometric, multi-site protein modifications.     

Identification of 2D-gel proteins: a comparison of MALDI/TOF peptide mass mapping to mu LC-ESI tandem mass spectrometry

A comparative analysis of protein identification for a total of 162 protein spots separated by two-dimensional gel electrophoresis from two fully sequenced archaea, Methanococcus jannaschii and Pyrococcus furiosus, using MALDI-TOF peptide mass mapping (PMM) and mu LC-MS/MS is presented. 100% of the gel spots analyzed were successfully matched to the predicted proteins in the two corresponding open reading frame databases by mu LC-MS/MS while 97% of them were identified by MALDI-TOF PMM. The high success rate from the PMM resulted from sample desalting/concentrating with ZipTip(C18) and optimization of several PMM search parameters including a 25 ppm average mass tolerance and the application of two different protein molecular weight search windows. By using this strategy, low-molecular weight (<23 kDa) proteins could be identified unambiguously with less than 5 peptide matches. Nine percent of spots were identified as containing multiple proteins. By using mu LC-MS/MS, 50% of the spots analyzed were identified as containing multiple proteins. mu LC-MS/MS demonstrated better protein sequence coverage than MALDI-TOF PMM over the entire mass range of proteins identified. MALDI-TOF and PMM produced unique peptide molecular weight matches that were not identified by mu LC-MS/MS. By incorporating amino acid sequence modifications into database searches, combined sequence coverage obtained from these two complimentary ionization methods exceeded 50% for approximately 70% of the 162 spots analyzed. This improved sequence coverage in combination with enzymatic digestions of different specificity is proposed as a method for analysis of post-translational modification from 2D-gel separated proteins.     

Quantitative analysis of crude plasma extracts by ion trap tandem mass spectrometry

The ion trap mass spectrometer is a tandem-in-time instrument that has promise as an extremely sensitive device for practical tandem mass spectrometry assays. An approach for the quantitative analysis of unknown drug levels in crude extracts, using combined capillary gas chromatography and the ion trap mass spectrometer in the tandem mode, is described. One-gram plasma samples were spiked with an anti-inflammatory drug at levels of 1–100 ng, and with 50 ng of a chemical analog internal standard. Crude extracts of the plasma samples are analyzed by using scan functions that utilize combined radiofrequency (rf) and de voltages. The need for combined rf- and de-voltage sequences for analysis of such extracts is demonstrated by comparison to attempted analyses using only rf voltages. Limitations of the method are: (1) the need for accurate calibration of ionization times to obtain linear calibration lines, and (2) the lack of automatic gain control for scans using combined rf and dc voltages to control and optimize parent ion populations and to allow a simpler analysis of “unknowns. ”     

Characterization of beam-induced reactions occurring in liquid secondary ion mass spectrometry/fast-atom bombardment by tandem mass spectrometry

Department of General and Physical Chemistry, University of Liege, Liege, Belgium A specific beam-induced secondary reaction involving the condensation of hydroxylic matrices with some organic groups (aldehydes, ketones, etc.) accompanied by the loss of a water molecule was investigated by liquid secondary ion mass spectrometry/fast-atom bombardment (LSIMS/FAB). A mechanistic scheme and a structure of the induced product are proposed. The features of this secondary reaction were studied and the influence of the types of solutes, acidic additives, and matrices analyzed. Rather than a drawback, LSIMS/FAB mass spectrometry can take advantage of this matrix effect to infer analytical information through tandem mass spectrometry experiments. Specific neutral loss scans can be conducted to highlight beam-induced reactive molecules, even when the detection of these species is prevented in normal scan spectra by other surface-active components.     

Synthesis of heptapeptides and analysis of sequence by tandem ion trap mass spectrometry

Two heptapeptides have been prepared by Fmoc methodology using Wang resin as solid support. For attachment of the first amino acid, several coupling systems were evaluated, and DIC/DMAP system could give yields of >99% and low levels of racemization. The selection of scavenger combination to deprotect side chains revealed that H₂O/p-cresol was good at scavenging trityl and 1,2-ethanedithiol was highly efficient for scavenging t-butyl. Through shortening the preactivation time to 5 min, the racemization which occurred during formation of amide bonds coupled by HBTU was minimized. The crude peptides were characterized by RP-HPLC and MS, and sequenced by MS/MS to acquire reliable amino acid sequence information.