Direct quantitative analysis of peptides using matrix assisted laser desorption ionization

The protocol and various matrices were examined for quantification of biomolecules in both the low ca. 1200 amu and mid mass 6000-12000 amu ranges using an internal standard. Comparative studies of different matrices of MALDI quantitative analysis showed that the best accuracy and standard curve linearity were obtained for two matrices: (a) 2,5-dihydroxybenzoic acid (DHB) combined with a comatrix of fucose and 5-methoxysalicylic acid (MSA) and (b) ferulic acid/fucose. In the low mass range, the quantitative limit was in the 30 fmol range and in the mid mass range the quantitative limit was in the 250 fmol range. Linear response was observed over 2-3 decades of analyte concentration. The relative error of the standard curve slope was 1.3-1.8% with correlation coefficients of 0.996-0.998.The main problem for quantitative measurement was suppression of the signal of the less concentrated component (analyte or internal standard) by the more concentrated component. The effect was identified with saturation of the matrix by the analyte. The threshold of matrix saturation was found to be in the range of 1/(3000-5000) analyte/matrix molar ratio. To avoid matrix saturation the (analyte+internal standard) to matrix molar ratio should be below this threshold. Thus the internal standard concentration should be as low as possible.DHB/MSA/fucose and ferulic acid/fucose matrices demonstrated good accuracy and linearity for standard curves even when the internal standard had chemical properties different from the analyte. However, use of an internal standard with different chemical properties requires highly stable instrumental parameters as well as constant (analyte+internal standard)/matrix molar ratio for all samples.     

Direct quantitative analysis of peptides using matrix assisted laser desorption ionization

The protocol and various matrices were examined for quantification of biomolecules in both the low ca. 1200 amu and mid mass 6000–12000 amu ranges using an internal standard. Comparative studies of different matrices of MALDI quantitative analysis showed that the best accuracy and standard curve linearity were obtained for two matrices: (a) 2,5-dihydroxybenzoic acid (DHB) combined with a comatrix of fucose and 5-methoxysalicylic acid (MSA) and (b) ferulic acid/fucose. In the low mass range, the quantitative limit was in the 30 fmol range and in the mid mass range the quantitative limit was in the 250 fmol range. Linear response was observed over 2–3 decades of analyte concentration. The relative error of the standard curve slope was 1.3–1.8% with correlation coefficients of 0.996–0.998.The main problem for quantitative measurement was suppression of the signal of the less concentrated component (analyte or internal standard) by the more concentrated component. The effect was identified with saturation of the matrix by the analyte. The threshold of matrix saturation was found to be in the range of 1/(3000–5000) analyte/matrix molar ratio. To avoid matrix saturation the (analyte+internal standard) to matrix molar ratio should be below this threshold. Thus the internal standard concentration should be as low as possible.DHB/MSA/fucose and ferulic acid/fucose matrices demonstrated good accuracy and linearity for standard curves even when the internal standard had chemical properties different from the analyte. However, use of an internal standard with different chemical properties requires highly stable instrumental parameters as well as constant (analyte+internal standard)/matrix molar ratio for all samples.     

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...     

Analysis of quaternary protein ensembles by matrix assisted laser desorption/ionization mass spectrometry

The intact noncovalent structure of the homo-oligomeric complexes of streptavidin (52 kDa), alcohol dehydrogenase (150 kDa), and beef liver catalase (240 kDa) have been observed using the matrix 2,6-dihydroxyacetophenone in an organic solvent. Intact streptavidin tetramers could also be observed with ferulic acid and other hydroxyacetophenone derivatives. Intact complexes are observed only for the first shot at a given position, which may be due to physical segregation or precipitation of the noncovalent complexes at the crystal surface. This effect is independent of the macroscopic crystal structure or the type of substrate (hydrophobic versus hydrophilic). Observation of intact complexes is not affected by addition of less than 10 mM salts or buffers, and appears to be independent of the pH stability range of the protein samples investigated.     

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.     

Aggregation-induced emission compounds as new assisted matrices for laser desorption/ionization time-of-flight mass spectrometry

Here, N,N′-bis(4-hydroxylsalicylidene)-p-phenylenediamine (BSPD-OH), N,N′-bis(4-methoxylsalicylidene)-p-phenylenediamine (BSPD-OMe) and N,N′-bis(salicylidene)-p-phenylenediamine (BSPD), which belong to the same category of aggregation-induced emission (AIE) compounds based on Schiff base reactions, were synthesized and applied as new matrices in the analysis of small molecules by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). This type of AIE compounds can be good MALDI matrices. Conventional organic matrices often produce large amounts of matrix ions, hindering the analysis of low molecular weight (LMW) compounds. However, these AIE compounds generate few matrix ions and less background interference because their presence as aggregates decreases the generation of matrix interference. The sensitivity of the AIE matrix is high because the aggregates can improve the absorption of the applied laser emissions. We can regulate the ionization efficiency of the AIE matrix by changing its aggregation state. During this study, BSPD-OH exhibited better ionization efficiency than the other two AIE matrices because it has more phenolic hydroxyl groups. BSPD-OH was successfully applied to the analysis of various LMW compounds including amino acids, organic amine compounds, isoquinoline compounds and fluoroquinolones compounds. This material also can be employed during the qualitative and quantitative analysis of LMW metabolites in human urine without requiring complicated separation processes.     

Capillary isoelectric focusing coupled offline to matrix assisted laser desorption/ionization mass spectrometry

This work presents several critical details for making cIEF-MALDI-MS a robust technique which will allow for more routine application and aid in automation. This includes emphasis on the hardware necessary for syringe pump mobilization and proper protocol for preventing disruption from gas bubbles. Following these guidelines, excellent elution time reproducibility is demonstrated for six pI markers (RSD <5%). Additionally, the pI markers are used to calibrate the pH gradient and determine experimental pIs of proteins detected offline by mass spectrometry. This was demonstrated using a standard protein mixture of myoglobin and two forms of β-lactoglobulin. Experimental determination of protein pIs and molecular weights were found to be in agreement with literature values. The technical details discussed provide a sound foundation for applying the offline coupling of MALDI-MS with cIEF.     

Analysis of the multimeric state of proteins by matrix assisted laser desorption/ionization mass spectrometry after cross-linking with glutaraldehyde

We have undertaken a systematic study on the suitability of matrix-assisted laser desorption/ionization mass spectrometry to analyze and determine the multimericity of several proteins after cross-linking with glutaraldehyde. Using both commercially available proteins and others of viral origin currently being characterized in our laboratory, we studied the range of concentrations of cross-linker and protein for optimal analysis. Under the conditions developed during this study, we confirmed the multimeric states of three phage PRD1 structural proteins with monomeric masses ranging from 13.5 to 63 kDa. In addition, we addressed the question of the general applicability of the method by using it successfully to confirm the stoichiometry of the heptameric chaperonin GroEL, a bacterial protein with a mass well over 450 kDa.     

Study of synthetic aliphatic copolyamides by time-of-flight matrix assisted laser desorption/ionization mass spectrometry

Synthetic copolyamides based on aliphatic diamines (1,3-propanediamine and 1,4-butanediamine) and dichlorides of aliphatic carboxylic acids (adipic and sebacic acid dichlorides) were investigated using time-of-flight matrix assisted laser desorption/ionization mass spectrometry. Their mass spectra showed peaks for cationized (Na⁺ and K⁺) and protonated (less intense peaks) oligomers with NH₂-NH₂, NH₂-COOH, or COOH-COOH end groups. No cyclic oligomers were detected in the samples. The compositions of oligomers were determined, and the relative reactivities of homologous comonomers in polycondensation were estimated. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1320–1324, July, 2007.     

Two-photon ionization thresholds of matrix-assisted laser desorption/ionization matrix clusters

Direct two-photon ionization of the matrix has been considered a likely primary ionization mechanism in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. This mechanism requires that the vertical ionization threshold of matrix materials be below twice the laser photon energy. Because dimers and larger aggregates may be numerous in the early stages of the MALDI plume expansion, their ionization thresholds are important as well. We have used two-color two-photon ionization to determine the ionization thresholds of jet cooled clusters of an important matrix, 2,5-dihydroxy benzoic acid (DHB), and mixed clusters with the thermal decomposition product of DHB, hydroquinone. The thresholds of the clusters were reduced by only a few tenths of an eV compared to the monomers, to an apparent limit of 7.82 eV for pure DHB clusters. None of the investigated clusters can be directly ionized by two nitrogen laser photons (7.36 eV), and the ionization efficiency at the thresholds is low.     

Controlling matrix suppression for matrix‐assisted laser desorption/ionization analysis of small molecules

The need for high‐throughput methodologies providing both qualitative and quantitative information has grown substantially in the pharmaceutical laboratory in recent years. Currently, tandem mass spectrometry (MS/MS) using quadrupole technology offers analysis in the minutes time scale. The use of matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) offers the advantage of speed and automation and enables analysis in the seconds time scale with accurate mass capabilities that are not typically found in quadrupole MS/MS. However, one of the limitations of MALDI for the analysis of small molecules is the abundance of interfering matrix peaks in the low molecular weight region of the mass spectrum. Described herein is an evaluation of a pre‐prepared MALDI target plate that has been coated with a thin layer of α‐cyano‐4‐hydroxycinnamic acid (CHCA) and nitrocellulose. This modified plate has been shown to suppress or eliminate CHCA matrix signals without any significant loss of analyte sensitivity when compared with analysis of the same sample using an unmodified target plate. Copyright © 2004 John Wiley & Sons, Ltd.     

Ionic liquids as matrices in microfluidic sample deposition for high-mass matrix- assisted laser desorption/ionization mass spectrometry

Sample preparation for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) via a microfluidic deposition device using ionic liquid matrices addresses several problems of standard protocols with crystalline matrices, such as the heterogeneity of sample spots due to the co-crystallization of sample and matrix and the limited capability for high-throughput analysis. Since ionic liquid matrices do not solidify during the measurement, the resulting sample spots are homogeneous. The use of these matrices is also beneficial for automated sample preparation, since crystallization of the matrix is avoided and, thus, no clogging of the spotting device can occur. The applicability of ionic liquids to the analysis of biomolecules with high molecular weights, up to ≈ 1 MDa is shown, as well as a good sensitivity (5 fmol) for recombinant human fibronectin, a protein with a molecular weight of 226 kDa. Microfluidic sample deposition of proteins with high molecular weights will, in the future, allow parallel sample preparation for MALDI-MS and for electron microscopy.     

Charge assisted laser desorption/ionization mass spectrometry of droplets

We propose and evaluate a new mechanism to account for analyte ion signal enhancement in ultraviolet-laser desorption mass spectrometry of droplets in the presence of corona ions. Our new insights are based on timing control of corona ion production, laser desorption, and peptide ion extraction achieved by a novel pulsed corona apparatus. We demonstrate that droplet charging rather than gas-phase ion-neutral reactions is the major contributor to analyte ion generation from an electrically isolated droplet. Implications of the new mechanism, termed charge assisted laser desorption/ionization (CALDI), are discussed and contrasted with those of the laser desorption atmospheric pressure chemical ionization method (LD-APCI). It is also demonstrated that analyte ion generation in CALDI occurs with external electric fields about one order of magnitude lower than those needed for atmospheric pressure matrix assisted laser desorption/ionization or electrospray ionization of droplets.     

Synthesis and application of ionic liquid matrices (ILMs) for effective pathogenic bacteria analysis in matrix assisted laser desorption/ionization (MALDI-MS)

Application of two new series of ionic liquid matrices (ILMs) based on the two most predominantly used conventional organic matrices (Sinapinic acid and 2,5-DHB) in conjugation with various bases (aniline (ANI), dimethyl aniline (DMANI), diethylamine (DEA), dicyclohexylamine (DCHA), pyridine (Pyr), 2-picoline(2-P), 3-picoline(3-P)) for bacterial analysis in matrix assisted laser desorption/ionisation mass spectrometry (MALDI-MS) are reported. The results reveal that ionic liquid matrices could significantly enhance the protein signals, reduce spot-to-spot variation and increase spot homogeneity. More importantly, these novel matrices would not produce any interference during MALDI-MS analysis. Among these ILMs, 2,5-DHB/ANI, 2,5-DHB/DMANI and 2,5-DHB/Pyr can be successfully applied to intact bacterial studies compared with other ILMs. Base molecules when added to conventional matrix can promote proton transfer between the bacterial lysate and the matrices. Due to the enhanced proton transfer efficiency by the ionic liquid matrices, almost all the biomolecules of the intact bacterial cells can be ionized and detected in the MALDI-MS. All synthesized ILMs were characterized using ESI (+)/MS and UV-spectroscopy.     

Electric field assisted thermal desorption ionization using an infrared laser

A new technique has been developed for the formation of gas-phase electrons and ions by electric field assisted thermal desorption ionization at atmospheric pressure. Experiments were carried out using a sharp tungsten wire tip coated with a thin solid sample film which was irradiated by a 10.6 μm infrared laser. By applying a strong electric field on the laser-irradiated tungsten wire tip, abundant ions such as alkali ions, halide ions, and also multiply charged negative ions S(2)O(6)(2-) and S(2)O(8)(2-), were formed. Copyright 1999 John Wiley & Sons, Ltd.     

Size-selected (2-10 nm) gold nanoparticles for matrix assisted laser desorption ionization of peptides

In this report, first use of size-selected gold nanoparticles (AuNPs) as matrixes for matrix assisted laser desorption/ionization (MALDI) is described for peptides and proteins. In comparison with conventional organic acid MALDI matrixes, the optimum matrix-to-analyte ratio with AuNP matrixes is reduced by 10-14 orders of magnitude. Significant differences in the relative abundances of the ions observed in positive and negative mode MALDI-time-of-flight mass spectrometry (TOFMS) are revealed as the AuNP size distribution is decreased from 10 to 2 nm, whereby 2-nm AuNPs exhibit quantum confinement effects prevalent in quantum dots. AuNP matrixes allow for selective analyte ionization, as demonstrated in the selective MALDI-TOFMS of phosphotyrosine in a background of phosphoserine and phosphothreonine peptides.     

Quantitative analysis of technical polymer mixtures by matrix assisted laser desorption/ionization time of flight mass spectrometry

We report quantitative MALDI-TOF measurements for polydimethylsiloxane (PDMS) of two different molecular weights using the relative ratio of the signal intensities of integrated oligomer distributions for these two molecular weight distributions. By reporting the ratio of intensities of the integrals of two oligomer distributions, we assume that the ionization and desorption efficiencies, crystallization conditions and other factors affecting intensity are similar. Poly(methyl methacrylate) (PMMA-33,000) was mixed with PDMS samples to show whether the presence of another material might affect the desorption efficiency. Quantitative values for the number-average molecular weight (Mn), weight-average molecular weight (Mw) and polydispersities (D) were calculated using the oligomer distributions. The results show a linear relationship between the analyte concentrations and the signal intensities in the range from 1,000 Da to 10,000 Da, and the desorption efficiency of these two PDMS materials was the same even in the presence of PMMA.