Analysis of biopolymers by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry is an analytical technique enabling the mass analysis of biopolymers with masses up to at least 300,000 Da. Incorporation of analyte in a matrix consisting of small highly absorbing organic molecules and excitation with short pulses of intense laser light enables the production of intact molecule ions to be analyzed in a time-of-flight mass spectrometer. Mass accuracies of up to 0.01% can be achieved from sample amounts of 1 pmol or less. Proteins, glycoproteins, oligonucleotides and oligosaccharides have been analyzed. The short analysis time of several minutes makes the method well suited for combination with other biochemical methods.     

Matrix-assisted laser desorption/ionization (MALDI) mechanism revisited

Although matrix-assisted laser desorption/ionization (MALDI) was developed more than a decade ago and broad applications have been successfully demonstrated, detailed mechanism of MALDI is still not well understood. Two major models; namely photochemical ionization (PI) and cluster ionization (CI) mechanisms have been proposed to explain many of experimental results. With the photochemical ionization model, analyte ions are considered to be produced from a protonation or deprotonation process involving an analyte molecule colliding with a matrix ion in the gas phase. With the cluster ionization model, charged particles are desorbed with a strong photoabsorption by matrix molecules. Analyte ions are subsequently produced by desolvation of matrix from cluster ions. Nevertheless, many observations still cannot be explained by these two models. In this work, we consider a pseudo proton transfer process during crystallization as a primary mechanism for producing analyte ions in MALDI. We propose an energy transfer induced disproportionation (ETID) model to explain the observation of an equal amount of positive and negative ions produced in MALDI for large biomolecules. Some experimental results are used for comparisons of various models.     

Protein desolvation in UV matrix-assisted laser desorption/ionization (MALDI)

We describe experiments in MALDI-TOF and MALDI-TOF-TOF showing that the ejection of protein-matrix cluster ions and their partial decay in the source occur in MALDI. The use of radial beam deflection and small size detector in linear mode allows detection of ions with higher time-of-flight and kinetic energy deficit. MALDI-TOF-TOF experiments were carried out by selecting chemical noise ions at m/z higher than that of a free peptide ion. Whatever the selected m/z (up to m/z 300) the molecular peptide ion appeared as the main fragment. The production of protein-matrix clusters and their partial decay in the source was found to increase with the size of the protein (MW from 1000 to 150,000 u), although it decreases with increasing charge state. These effects were observed for different matrices (HCCA and SA) and in a large laser fluence range. Experimental results and calculation highlight that a continuous decay of protein-matrix cluster ions occurs in the source. This decay-desolvation process can account for the high-mass tailing and peak shifting as well as the strong noise/background in the mass spectra of proteins.     

Ionic matrices for matrix-assisted laser desorption/ionization time-of-flight detection of DNA oligomers

Salts with low melting points, also termed room-temperature ionic liquids, can be used as matrices in matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF). They have great vacuum stability, and can dissolve polar and apolar solutes including carbohydrates, biological oligomers and proteins. The ionic liquids give much more homogeneous sample solutions compared with solid matrices. We demonstrate the usefulness of using ionic matrices to determine the molecular weight of DNA oligomers by direct TOF mass spectrometric analysis. Three oligonucleotides were tested, (d(pT)(10), d(pC)(11), and d(pC)(12)), with several ionic matrices synthesized from different bases associated to two acids (3-hydroxypicolinic acid and 2,5-dihydroxybenzoic acid). The results obtained show that the best ionic matrices enhance the ion peak intensity of the oligonucleotides with respect to conventional molecular matrices under our experimental conditions. In one case, an ionic matrix provided a signal-to-noise ratio ten times higher than the corresponding molecular matrix. Several of the tested ionic matrices were liquids. However, all working ionic matrices were solids.     

Peptide and protein identification by matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay time-of-flight mass spectrometry

The potential of matrix-assisted laser desorption ionization (MALDI) and MALDI-post-source decay (PSD) time-of-flight mass spectrometry for the characterization of peptides and proteins is discussed. Recent instrumental developments provide for levels of sensitivity and accuracy that make these techniques major analytical tools for proteome analysis. New software developments employing protein database searches have greatly enhanced the fields of application of MALDI-PSD. Peptides and proteins can be easily identified even if only a partial sequence information is determined. Derivatization procedures have been optimized for MALDI-PSD to increase the structural information and to obtain a complete peptide sequence even in critical cases. They are fast, simple and can be performed on target. MALDI-PSD is also a very powerful tool to characterize or elucidate post-translational or chemically induced modifications. In association with database searches, proteins issued from electrophoretic gels can be identified after specific enzymatic cleavages and peptide mapping.     

A new method for analysis of disulfide-containing proteins by matrix-assisted laser desorption ionization (MALDI) mass spectrometry

A simple and high-throughput method for the identification of disulfide-containing peptides utilizing peptide-matrix adducts is described. Some commonly used matrices in MALDI mass spectrometry were found to specifically react with sulfhydryl groups within peptide, thus allowing the observation of the peptide-matrix adduct ion [M+n+n′ matrix+H]⁺ or [M+n+n′ matrix+Na]⁺ (n = the number of cysteine residues, n′=1, 2,…, n) in MALDI mass spectra after chemical reduction of disulfide-linked peptides. Among several matrices tested, α-cyano-4-hydroxycinnamic acid (CHCA, molecular mass 189 Da) and α-cyano-3-hydroxycinnamic acid (3-HCCA) were found to be more effective for MALDI analysis of disulfide-containing peptides/proteins. Two reduced cysteines involved in a disulfide bridge resulted in a mass shift of 189 Da per cysteine, so the number of disulfide bonds could then be determined, while for the other matrices (sinapinic acid, ferulic acid, and caffeic acid), a similar addition reaction could not occur unless the reaction was carried out under alkaline conditions. The underlying mechanism of the reaction of the matrix addition at sulfhydryl groups is proposed, and several factors that might affect the formation of the peptide-matrix adducts were investigated. In general, this method is fast, effective, and robust to identify disulfide bonds in proteins/peptides.     

Characteristics of photodissociation at 193 nm of singly protonated peptides generated by matrix-assisted laser desorption ionization (MALDI)

Photodissociation (PD) at 193 nm of various singly protonated peptides was investigated. These include peptides with an arginine residue at the C-terminus, N-terminus, at both termini, inside the chain, and those without an arginine residue. Monoisotopomeric selection was made for the precursor ions. Interference from the post-source decay (PSD) product signals was reduced as much as possible by using the deflection system (reported previously) and subtracting the remaining signals from the laser-on signals. The presence of an arginine residue and its position inside the peptide were found to significantly affect the PD spectra, as reported previously. Presence of a proline, aspartic acid, or glutamic acid residue hardly affected the PD spectral patterns. By comparing the PD spectra obtained at a few different wavelengths, it is concluded that the dissociation of the photoexcited ions occurs in their ground electronic states. Tentative explanations for the observed spectral correlations based on the statistical picture for the reactions are also presented.     

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.     

Characterization of polyethylenimine by electrospray ionization and matrix-assisted laser desorption/ionization

Branched polyethylenimines (PEIs) with lower average molecular weights (600, 1200 and 1800 Da) have been studied by Electrospray Ionization (ESI) and Matrix‐Assisted Laser Desorption/Ionization (MALDI) mass spectrometry. In both, ESI and MALDI mass spectra, the main distribution arises from protonated PEI oligomers with NH₂ end groups, [PEI + H]⁺, which are observed at m/z 43n + 18. A trace of sodium contamination in the PEI samples results in the presence of a series that appears at m/z 43n + 40 [PEI + Na]⁺. However, only the MALDI mass spectra show a [PEI + K]⁺ series at m/z 43n + 56, because of matrix contamination with potassium, and a series generated by condensation of the matrix with PEI at m/z 43n + 30. Collisionally activated dissociation tandem mass spectrometry (CAD (MS/MS)) of protonated PEI oligomers is shown to yield three fragment ion series b_n, and K_n. The experiments have demonstrated the capabilities of these mass spectrometry techniques, along with CAD MS/MS to detect and characterize such polar synthetic polymers. Copyright © 2011 John Wiley & Sons, Ltd.     

Electrospray and matrix-assisted laser desorption/ionization mass spectral characterization of linear single nylon-6 oligomers

Synthetic nylon-6 single molecular mass oligomers were studied by matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) mass spectrometry. These oligomers, considered as model compounds for the study of nylon-6 polymers, gave good mass spectrometric results using both MALDI and ESI. In spite of the gentle nature of both techniques, the MALDI and ESI spectra showed evidence of end-group cleavage from the oligomer chains. MALDI-MS was found to give similar fragmentation patterns for all of the oligomer samples. An increase in doubly charged ion signals with increasing oligomer mass was observed in the ESI mass spectra, as was end-group fragmentation. Signals from oligomer clusters were observed in ESI-MS for the dimer, tetramer and hexamer, most likely due to non-covalent bonding among the low-mass oligomer molecules.     

Chromophore effect in photodissociation at 266 nm of protonated peptides generated by matrix-assisted laser desorption ionization (MALDI)

Chromophore effect in the photodissociation of protonated peptides at 266 nm was investigated using synthetic peptides with the sequence RGGXGGGGGR where X was a phenylalanyl(F), tyrosyl(Y), cysteinyl(C), glycyl(G), seryl(S), or histidyl(H) residue. The peptides with an F or Y residue dissociated efficiently. Fragment ions due to cleavages at either end of the chromophore were especially prominent just as for the peptide with a tryptophanyl residue reported previously.1Photodissociation was observed even for the peptides without any noticeable chromophore at 266 nm. Here, dissociation at all the peptide bonds was almost equally prominent. Photodissociation of the protonated angiotensin I was investigated using the spectral correlation rules observed in the model systems. Role of the chromophores and the plausible mechanisms involved are discussed.     

Electrospray sample deposition for matrix-assisted laser desorption/ionization (MALDI) and atmospheric pressure MALDI mass spectrometry with attomole detection limits

Electrospray sample deposition was explored for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In this method, nanoliter volumes of matrix/analyte mixture were electrosprayed from a high voltage biased (1-2 kV) fused-silica capillary onto a grounded MALDI plate mounted 100-500 microm from the capillary outlet. Electrospray deposition with these conditions produced sample spots 200-300 microm in diameter thus matching the laser spot size. Varying spray voltage and distance resulted in different crystal sizes and volatilization rates for alpha-cyano-4-hydroxycinnamic acid matrix. Best results were obtained when the sample was deposited as wet droplets as opposed to deposition as dried solid. Under 'wet-spray' conditions, 2-4 microm diameter crystals were formed and detection limits for several neuropeptides were 0.7-25 amol. Samples could be pre-concentrated on the plate by spraying continuously and allowing sample to evaporate in a small spot. Sample volumes as large as 580 nL were deposited yielding a detection limit of 35 pM for neurotensin 1-11. Electrospray sample deposition yielded similar results when using atmospheric pressure-MALDI coupled with a quadrupole ion trap mass spectrometer, except that the sensitivity was approximately seven-fold worse.     

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.     

Analysis of plant phosphatidylcholines by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The use of matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS) for the quantitative determination of phospholipid (PL) molecular species has been problematic, due primarily to the formation of multiple signals (corresponding to the molecular ion and other adducts) for some classes of PL. For example, analysis of phosphatidylcholine (PC) yielded signals that corresponded to protonated and sodiated molecules in the MALDI spectrum. The resulting spectral overlap among various molecular species (e.g. [PC(16:0/18:2) + Na] and [PC(18:2/18:3)]) made it impossible to ascertain their relative amounts using this technique. Other spectral ambiguities existed among different structural isomers, such as PC(18:1/18:1) and PC(18:0/18:2). We determined that molecular species could be resolved by MALDI-TOFMS by first removing the polar head (e.g. phosphocholine) from the phospholipid to effect production of only the sodiated molecules of the corresponding diacylglycerols (DAGs). Analysis of the resulting spectrum allowed unequivocal determination of the molecular species profile of PC from potato tuber and soybean. Estimation of fatty acid composition based on the molecular species determined by MALDI-TOFMS analysis agreed with that from GC-FID analysis. Post-source decay (PSD) was used to resolve standard isomers of PC (e.g. 18:1/18:1 vs. 18:0/18:2). Our results indicated that PSD is a useful approach for resolving structural isomers of PL molecular species.     

Analysis of solid-supported oligonucleotides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This study presents matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) as a powerful tool to analyze and characterize oligonucleotides covalently linked to a solid support during their synthesis. The analysis of the fragment ions generated either in negative or positive mode allows direct and easy access to the nucleotide sequence and identification of the internucleosidic linkage. The mechanisms of the fragmentation of the solid-supported oligonucleotides induced by MALDI-TOFMS are discussed. Copyright 2000 John Wiley & Sons, Ltd.     

Analysis of hair components by nanoparticle-assisted laser desorption/ionization mass spectrometry imaging

Using a vertical hair-slice section, we compared the components of normal and damaged hair regions using two ionization methods, matrix-assisted laser desorption/ionization and nanoparticle-assisted laser desorption/ionization (Nano-PALDI) mass spectrometry. Nano-PALDI is useful for small-molecule and high spatial resolution (5 μm) analyses due to the lack of noise. Thus, clear images were obtained from thin hair samples. In Nano-PALDI mass spectrometry imaging, cystine and 18-methyleicosanoic acid as endogenous hair components localized in the cuticle and cortex and cuticle of normal hair, respectively. In contrast, both components were absent in damaged hair.     

Bimetallic silver-gold clusters by matrix-assisted laser desorption/ionization

Pure gold clusters (Aun+) were produced up to the cluster size of n = 100 by matrix-assisted laser desorption/ionization (MALDI). The mass spectrum of the resulting clusters showed alteration in the ion intensity at odd-even clusters size. On the other hand, intensity drops at cluster size predicted by the jellium model theory was also observed. Positively and negatively charged bimetallic silver-gold clusters were produced under MALDI conditions from the mixture of HAuCl4/silver trifluoroacetate and the 2-(4-hydroxyphenylazo)benzoic acid (HABA) matrix. A linear correlation was found between the intensity ratio of AunAgm+ to Au(n+1)Ag(m-1)+ cluster ions and the molar ratio of the gold to silver salt. It was observed that the composition and the distribution of the clusters can be varied with the molar ratio of the silver and gold salts. It was also found that the resulting cluster sizes obey the lognormal distribution.     

基质辅助激光解析电离-飞行时间质谱分析寡核苷酸G-四链体

我们发展了一种利用基质辅助激光解析电离飞行时间质谱技术(MALDI-TOF MS)分析对金属离子具有较高亲和力的寡核苷酸G-四链体的方法.考察了不同基质:3-羟基吡啶甲酸(3-HPA)与柠檬酸氢二铵(DHC)混合基质、3,4-二胺基苯基苯甲酮(DABP)及DABP/DHC混合基质,应用于G-四链体分析的效果.实验结果表...     

Parameterising matrix-assisted laser desorption/ionization (MALDI): effect of solvents and co-additives on analyte peak intensities

The peak intensities obtained when 2,5-dihydroxybenzoic acid (DHB) was used as a 'classic' matrix were measured using substance P (SP) and betacyclodextrin (BCD) as analytes. Enhancements in peak intensities were observed going from 1:1 MeOH/H2O to dimethylforamide (DMF) as matrix solvents. Also non-covalent interactions between SP and solvent and DHB were observed suggesting close interactions between matrix, solvent and analyte in the gas-phase. Peak enhancements were previously reported with 'superDHB' (DHB and 2-hydroxy-5-methoxybenzoic acid at 10% v/v). Co-addition of structural analogues and their respective absorption coefficients were determined. It was found that other analogues used as co-matrices could give analyte peak enhancement similar to reported for sDHB with the additional benefit that some analogues could act as matrices with DHB addition. No direct correlation was observed between absorption coefficient and the ability of the molecule to act as a 'good' UV MALDI matrix.