Matrix-dependent cationization in MALDI mass spectrometry

The matrix dependence in cationization processes, the competition between cationization and protonation and the question of whether gas-phase cation transfer or attachment of free cations dominates in matrix-assisted laser desorption/ionization mass spectrometry were studied. Two different sample preparation methods were employed, the dried-droplet sample preparation and a mixture of solid matrix, analyte and salt. The latter ensures that the formation of cation adducts takes place in the gas phase. By monitoring the suppression of matrix signals for different matrices, it was found that matrices with high gas-phase metal ion binding energies require high analyte concentrations for matrix suppression to occur. By comparing the mass spectra obtained using sinapinic acid or sinapinic methyl ester as a matrix, a correlation between cationization and deprotonation of matrix molecules was found. It is also demonstrated that attachment of free gas-phase cations, rather than cation transfer from the cationized matrix, is the predominant process in cationization.     

Application of nonpolar matrices for the analysis of low molecular weight nonpolar synthetic polymers by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The application of nonpolar matrices for the analysis of low molecular weight nonpolar synthetic polymers using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is demonstrated. Anthracene, pyrene, and acenaphthene were utilized as nonpolar matrices for the analysis of polybutadiene, polyisoprene, and polystyrene samples of various average molecular weights ranging from about 700 to 5000. The standard MALDI-MS approach for the analysis of these types of polymers involves the use of conventional acidic matrices, such asall-trans-retinoic acid, with an additional cationization reagent. The nonpolar matrices used in this study are shown to be as equally effective as the conventional matrices. The uniform mixing of the nonpolar matrices and the nonpolar analytes enhances the MALDI-MS spectral reproducibility. Silver salts were found to be the best cationization reagents for all of the cases studied. Copper salts worked well for polystyrene, poorly for polyisoprene, and not at all for polybutadiene samples. These matrices should be useful for the characterization of hydrocarbon polymers and other analytes, such as modified polymers, which may potentially be sensitive to acidic matrices.     

The cationization of synthetic polymers in matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry: Investigations of the salt‐to‐analyte ratio

Matrix‐assisted laser desorption/ionization (MALDI) is a soft ionization technique that when used to analyze synthetic polymer analytes often requires the addition of a metal cationization agent (herein termed the “salt”). The choice of both the matrix and the cationization agent needs to be taken into account when considering the polymer under study; different polymers have shown different affinities toward different cationization agents, and their selectivity can change as the matrix changes. Salt‐to‐analyte ratio (S/A) plots are used in this work to investigate the effect of the quantity of cationization agent employed in the analysis of a poly (methylmethacrylate) (PMMA) analyte with different MALDI matrices. The point at which analyte signal stops increasing with the added cationization agent is termed the “cation saturation point,” and it was found to occur around a S/A of 1. When the analyte signal after this point remains constant, it is termed an “ideal case.” The “non‐ideal case” occurs when the analyte signal decreases after the cation saturation point. The amount of matrix present (measured as the matrix‐to‐analyte molar ratio, M/A) and the use of different counterions for the salt are also found to affect the intensity of the analyte signal. In non‐ideal cases, changes in the counterion or an increase in the M/A are found to increase the analyte signal, often converting an initially observed non‐ideal case into an ideal case. Several experiments attempting to uncover the reason for observation of the non‐ideal S/A behavior are also described.     

Effect of metal cationization on the tandem mass spectra of glycosyl dithioacetals

The effect of metal cationization on the tandem mass spectra of glycosyl dithioacetals of glucose, mannose, galactose, rhamnose, arabinose and xylose was studied by electrospray ionization mass spectrometry under ammonium and metal (Li, Na, Ag and Cu) ion cationization conditions. The ammonium-cationized glycosyl dithioacetals fragment by loss of ammonia followed by either two molecules of EtSH or one molecule of EtSH and one molecule of H2O. Lithium cationization leads to additional eliminations such as EtSEt and EtSSEt and C-C cleavages. Elimination of EtSH is not observed under sodium cationization. Silver cationization, on the other hand, leads to additional fragmentations involving the elimination of silver as AgOH and AgSEt. Copper cationization results in adducts where copper has undergone a change of oxidation state from II to I. Li+, Ag+ and Cu+ cationization seem to favour cyclization resulting in elimination of EtSH. However, the mechanisms seem to be differently affected by different metal ions. Li+ and Ag+ cationization appear to be non-specific and favour cyclization involving C2-, C4- and C5-hydroxyl hydrogens, whereas Cu+ cationization seems to favour cyclization involving C4-hydroxyl hydrogen.     

Influence of Solvent Additive Composition on Chromatographic Separation and Sodium Adduct Formation of Peptides in HPLC–ESI MS

Mobile phase additives have a strong influence on sensitivity and spectral quality when HPLC is coupled to mass spectrometry. They may cause ion suppression, as frequently observed with trifluoroacetic acid, or may lead to increased adduct formation, complicating data analysis and interfering with automated LC–MS–MS analysis. When reversed-phase HPLC is performed under neutral conditions, ammonium acetate is the additive of choice because of its volatility. Because of the limited solubility of ammonium acetate in commonly employed organic solvents like acetonitrile usually a small amount of water is added for dissolution, and in addition, acetic acid can be added to obtain a buffered system. We compared six different acetonitrile/ammonium acetate blends, differing in their water and acetic acid content and evaluated their performance for HPLC–MS analysis of model peptides (bradykinins) under neutral conditions. The results demonstrate that mobile phase preparation strongly influences chromatographic separation and adduct formation in electrospray ionization MS, as shown for a series of bradykinin peptides. By varying the solvent composition only slightly to influence the pH curves during gradient elution, three nearly coeluting peptides could be baseline separated. At the same time, the formation of sodiated instead of protonated peptides was found to vary by up to a factor of four, depending on the preparation of the solvent blends.     

Effect of surface cationization on the conformal deposition of polyelectrolytes over cotton fibers

The effect of surface cationization on the conformal deposition of alternating nanolayers of poly(sodium styrene sulfonate) (PSS) [Kleinfeld E, Ferguson G (1996) Chem Mater 8:1575–1578] and poly(allylamine hydrochloride) (PAH) over cotton fibers is reported. Three different levels of cotton cationization were evaluated. Variations in the cationization degree were achieved by manipulating the ratio of 3-chloro-2-hydroxy propyl trimethyl ammonium to NaOH. Experimental results obtained via Carbon–Hydrogen–Nitrogen–Sulfur (CHNS) elemental analysis and X-ray Photoelectron Spectroscopy (XPS) indicated that the deposition process was not significantly influenced by the degree of cotton cationization. The build up of further polyelectrolyte layers was found to be less sensitive to variations in the cationic character of the substrates once a critical number of alternating layers was deposited.     

Laser desorption/ionization mass spectrometry fingerprinting of complex hydrocarbon mixtures: application to crude oils using data mining techniques

Crude oil fingerprints were obtained from four crude oils by laser desorption/ionization mass spectrometry (LDI-MS) using a silver nitrate cationization reagent. Replicate analyses produced spectral data with a large number of features for each sample (>11,000 m/z values) which were statistically analyzed to extract useful information for their differentiation. Individual characteristic features from the data set were identified by a false discovery rate based feature selection procedure based on the analysis of variance models. The selected features were, in turn, evaluated using classification models. A substantially reduced set of 23 features was obtained through this procedure. One oil sample containing a high ratio of saturated/aromatic hydrocarbon content was easily distinguished from the others using this reduced set. The other three samples were more difficult to distinguish by LDI-MS using a silver cationization reagent; however, a minimal number of significant features were still identified for this purpose. Focus is placed on presenting this multivariate statistical method as a rapid and simple analytical procedure for classifying and distinguishing complex mixtures.     

A study of cationic glucomannan as a paper strength agent

In this paper, konjac glucomannan was cationized by 3-chloro-2-hydroxypropyl trimethyl ammonium chloride (a kind of cationic reagent) as a paper strength agent. The effects of dosage of cationic reagent, cationization time, cationization temperature and the pH of papermaking on paper properties were analyzed. The results showed that when 1% cationic glucomannan (the cationization temperature was 70°C, dosage of cationic reagent was 20% and cationization time was 5 h) was added, tensile index, burst index and folding endurance of the paper were improved by 11.0%, 13.2%, 72.0%, respectively, compared with the control. The results of SEM analysis, FTIR analysis, and elemental analysis illustrated that the addition of paper strengthening agent make fibers combined more closely, and further lead to the increase of paper strength properties.     

Silver cluster interferences in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of nonpolar polymers

Potential difficulties associated with background silver salt clusters during matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of nonpolar polymers are reported. Silver salt cluster ions were observed from m/z 1500 to 7000 when acidic, polar matrices, such as 2,5-dihydroxybenzoic acid (DHB), all-trans-retinoic acid (RTA) or 2-(4-hydroxyphenylazo)benzoic acid (HABA), were used for the analysis of nonpolar polymers. These background signals could be greatly reduced or eliminated by the use of nonpolar matrices such as anthracene or pyrene. Representative examples of these background interferences are demonstrated during the analysis of low molecular weight nonpolar polymers including polybutadiene and polystyrene. Nonpolar polymers analyzed with acidic, polar matrices (e.g., RTA) and silver cationization reagents can yield lower quality mass spectral results when interferences due to silver clusters are present. Replacing the polar matrices with nonpolar matrices or the silver salts with copper salts substantially improved the quality of the analytical results. In addition, it was found that silver contamination cannot be completely removed from standard stainless steel sample plates, although the presence of silver contamination was greatly reduced after thorough cleaning of the sample plate with aluminum oxide grit. Carry-over silver may cationize polymer samples and complicate the interpretation of data obtained using nonpolar matrices in the absence of added cationization reagents.     

纳升级电喷雾离子源-四极杆-飞行时间质谱中磷酸化肽的离子抑制作用

目前磷酸化蛋白质组学研究中的主要技术是蛋白质酶解产生的磷酸化肽的质谱检测。但是实际样品中的磷酸化肽(特别是多磷酸化肽)很难被检测到。其原因普遍认为是由于质谱检测时,非磷酸化肽抑制磷酸化肽。但也有认为非磷酸化肽对磷酸化肽没有抑制作用。另外磷酸化肽之间是否存在离子抑制作用还没有报道。本文采用相同氨基酸序列的标准磷酸化肽和非磷酸化肽,将其单独和混合进行质谱检测,通过对比混合前后磷酸化肽的信号强度,证明了非磷酸化肽对磷酸化肽有离子抑制作用;单磷酸化肽对二磷酸化肽有一定的抑制作用,但不太显著;单磷酸化肽对三磷酸化肽、二磷酸化肽对三磷酸化肽均有显著的离子抑制作用。该研究为今后单磷酸化肽和多磷酸化肽的分段富集和检测提供了有力的证明。     

A study of gas-phase cationization in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A specially constructed split sample probe was used to unequivocally demonstrate that gas-phase cationization occurs within the desorption plume during a matrix-assisted laser desorption/ionization experiment. Two separate samples were prepared for analysis: on side A, a mixture of poly(ethylene glycol) (PEG) 1500 analyte and 2,5-dihydroxybenzoic acid (DHB) matrix, and on side B a mixture of DHB matrix and lithium hydroxide (LiOH), the cationization reagent. Analysis of the data showed that when the ionization laser was focused on the split (so that both sides were illuminated), Li(+)-cationized PEG peaks were observed. Since the PEG analyte did not come into contact with Li(+) in either the solution or solid phase, the only possibility for the observed cationization was a reaction in the gas phase. Due to the difficulty in completely removing the adventitious cations (Na(+) and K(+)) present in DHB and on sample surfaces, gas-phase cationization could not be demonstrated to be either the only or most important mechanism operating in the MALDI experiment.     

The cationization mechanism study of novel oligo (<Emphasis Type="Italic">p</Emphasis>-phenyleneethynylene)s

Two series of new oligo(p-phenyleneethynylene)s (OPEs) O1-O4 and O5-O8, which have been proven to be one of the chief classes of molecules mainly used as the wires and other potential backbones of molecular electronic devices, have been synthesized by stepwise synthetic approach. The characterization of these oligomers was performed on MALDI TOF MS. Different cationization salts have been applied to investigate the ionization processes of these series of oligomers under MALDI conditions. The experimental results show that these oligomers display a strong tendency to undergo radical cationization and varied ionization efficiency with different cationization agents attributable to their difference in cationic diameters. Furthermore, we found that these two series of oligomers differed in ionization properties because of their different end-groups even when the same cationization agent was used.     

An optimized protocol for nano-LC-MALDI-TOF-MS coupling for the analysis of proteolytic digests of glycoproteins

Matrix-assissted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analyses of complete proteolytic digests are often hampered by contaminations and the complexity of the sample. This results in suppression effects and the formation of adducts which are difficult to assign, thus leading to low scores in database searches. In particular, signals of post-translationally modified peptides such as glycopeptides are often of low intensity or completely suppressed. Online liquid chromatography electrospray ionization mass spectrometry (ESI-MS) can, in part, overcome this problem, but the analytes are completely consumed during the run. Coupling of nano-flow HPLC (nano-LC), microfractionation and MALDI-TOF-MS combines separation and high-sensitivity UV detection with the possibility of collecting fractionated peptides and preserving the sample for detailed mass spectrometric analyses. Here we report on an optimized protocol for nano-LC-MALDI-TOF-MS analyses of glycoproteins. This protocol improves spectral quality, resulting in better protein identification scores in database searches. Furthermore, post-translationally modified peptides could be detected with higher sensitivity by changing the experimental conditions, allowing assignment, localization and characterization of the respective carbohydrate substituents.     

A study of phospholipids by ion mobility TOFMS

Combining matrix-assisted laser desorption/ionization (MALDI) mass spectrometry with ion mobility (IM) results in the fast sorting of biomolecules in complex mixtures along trend lines. In this two-dimensional (2D) analysis of biological families, lipids, peptides, and nucleotides are separated from each other by differences in their ion mobility drift times in a timescale of hundreds of microseconds. Molecular ions of similar chemical type fall along trend lines when plotted in 2D plots of ion mobility drift time as a function of m/z. In this study, MALDI-IM MS is used to analyze species from all of the major phospholipid classes. Complex samples, including tissue extracts and sections, were probed to demonstrate the effects that radyl chain length, degree of unsaturation, and class/head group have upon an ion’s cross section in the gas phase. We illustrate how these changes can be used to identify individual lipid species in complex mixtures, as well as the effects of cationization on ion cross section and ionization efficiency.     

The use of pencil lead as a matrix and calibrant for matrix-assisted laser desorption/ionisation

Pencil lead is shown to be an effective matrix and calibrant in matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry. Various groups of analytes, including peptides, polymers and actinide metals, can be readily ionised using MALDI when deposited onto a pencil lead matrix. The matrix is seen to have advantages in sample preparation relating to its hydrophobic properties and almost complete suppression of the matrix during analysis. Using pencil lead as a matrix is a quick and convenient method of qualitative analysis and has been shown to be quantitative for the isotope ratio analysis of actinide metals.     

Molecular mass determination of saturated hydrocarbons: reactivity of eta5-cyclopentadienylcobalt ion (CpCo*+) and linear alkanes up to C-30

The present study demonstrates the feasibility of the eta5-cyclopentadienylcobalt ion (CpCo*+) as a suitable cationization reagent for saturated hydrocarbon analysis by mass spectrometry. Ion/molecule reactions of CpCo*+ and three medium chain-length n-alkanes were examined using Fourier-transform ion cyclotron resonance mass spectrometry. Second-order rate constants and reaction efficiencies were determined for the reactions studied. Loss of two hydrogen molecules from the CpCo-alkane ion complex was found to dominate all reactions ( > or = 80%). Furthermore, this dehydrogenation reaction efficiency increases with increasing chain length. These preliminary results suggest that the CpCo*+ ion may be a promising cationization reagent of longer chain saturated hydrocarbons and polyolefins.     

Matrix-assisted laser desorption/ionization collision-induced dissociation of linear single oligomers of nylon-6.

Matrix-assisted laser desorption/ionization, collision induced-dissociation (MALDI-CID) has been used to obtain structural information for linear single oligomers of nylon-6. The effects of matrix and cationization agent in MALDI-CID analysis have been investigated. Fragmentation mechanisms are proposed for the series of ions that are observed in the MALDI-CID spectra of the hexamer, octamer and dodecamer. Fragmentation processes observed in the MALDI-CID spectra include cleavage of the end groups followed by dissociation of the m/z 113 unit. Cleavage of the oligamide chain occurs at the amide linkage, as well as at adjacent bonds. For the four matrices and three cationization agents investigated, 2,5-dihydroxybenzoic acid and sodium chloride showed the best performance for MALDI-CID analysis of the dodecamer. In addition, yields of the fragment ions in MALDI-CID spectra were found to be dependent on the chain length distribution.     

Natural Dyeing of Modified Cotton Fabric with Cochineal Dye

Natural dyes are not harmful to the environment owing to their biodegradability. For dye application to textiles, salts are necessary as mordant or electrolytes and make an environmental impact. In this paper, the influence of cationization during mercerization to the dyeing of cotton fabric with natural dye from Dactylopius coccus was researched. For this purpose, bleached cotton fabric as well as fabric cationized with Rewin OS was pre-mordanted using iron(II) sulfate heptahydrate (FeSO₄·7H₂O) and dyed with natural cochineal dye with and without electrolyte addition. For the characterization of surface changes after cationization, an electrokinetic analysis on SurPASS was performed and compared to pre-mordanting. For determination of dye exhaustion, the analysis of dye solution was performed on a UV/VIS spectrophotometer Cary 50 Solascreen. Spectrophotometric analysis was performed using a Datacolor 850 spectrophotometer, measuring remission ”until tolerance” and the whiteness degree, color parameters, color depth (K/S), and colorfastness of dyed fabric were calculated. Levelness was determined by visual assessment. Cationized cotton fabrics showed better absorption and colorfastness. Pre-mordanting and cationization showed synergism. The electrolytes improved the process of dye absorption. However, when natural dyeing was performed on cotton fabric cationized during mercerization, similar chromacity, uniform color, and colorfastness were achieved with and without electrolyte, resulting in pure purple hue of cochineal. For achieving a violet hue, pre-mordanting with Fe-salt was needed. Therefore, salt can be reduced or even unnecessary, which makes this process of natural dyeing more environmentally friendly.     

Diode array detection and simultaneous quantitation of the coeluting atenolol-related synthetic route impurities, PPA-Diol.

The diode array multichannel detector is used to acquire spectral information at specified intervals in the elution profile of atenolol and its related impurities for post-chromatographic data analysis. The applicability of a number of peak homogeneity testing methods, including spectral normalization, absorbance ratio, chromatographic derivatives, and spectral suppression (SS), are assessed for suitability in simultaneous determinations of the coeluting atenolol-related synthetic impurities, PPA-Diol. Spectral suppression displays a superior performance in comparison to all the other techniques in that both qualitative and quantitative information are acquired on the system.     

Factors affecting gas-phase deuterium scrambling in peptide ions and their implications for protein structure determination

In this report, we evaluate the validity of using hydrogen/deuterium exchange in combination with collision-induced dissociation mass spectrometry (CID MS) for the detailed structural and conformational investigation of peptides and proteins. This methodology, in which partly deuterated peptide ions are subjected to collision-induced dissociation in the vacuum of a mass spectrometer, has emerged as a useful tool in structural biology. It may potentially provide quantitatively the extent of deuterium incorporation per individual amino acid in peptides and proteins, thus providing detailed structural information related to protein structure and folding. We report that this general methodology has limitations caused by the fact that the incorporated deuterium atoms migrate prior or during the CID MS analysis. Our data are focused on a variety of transmembrane peptides, incorporated in a lipid bilayer, in which the near-terminal amino acids that anchor at the lipid-water interface are systematically varied. Our findings suggest that, under the experimental conditions we use, the extent of intramolecular hydrogen scrambling is strongly influenced by experimental factors, such as the exact amino acid sequence of the peptide, the nature of the charge carrier, and therefore most likely by the gas-phase structure of the peptide ion. Moreover, the observed scrambling seems to be independent of the nature of the peptide fragment ions (i.e., protonated B and Y' ' ions, and sodiated A and Y' ions). Our results strongly suggest that scrambling may be reduced by using alkali metal cationization instead of protonation in the ionization process.