Quantitative analysis of synthetic polymers using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Quantitative analyses of synthetic polymers were accomplished using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI TOF MS). Many factors have hindered the development of quantitative measurement of polymers via MALDI TOF MS, e.g., laser power, matrix, cation salt, and cocrystallization. By probing the optimal conditions, two sets of polymers were studied. Fair repeatability of the samples ensures acceptable results. In set 1, two poly(ethylene glycols) with different end groups showed equal desorption/ionization efficiencies. Two synthetic polymers in set 2 with different chemical properties resulted in different MALDI responses. Good linearity was achieved by plotting the relationship between the sample concentration ratio and the total signal intensity ratio in both sets.     

Gender identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

A rapid, simple, and reliable gender determination of human DNA samples was successfully obtained using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Detection sensitivity reached 0.01 ng or less for DNA samples.     

Identification of 4-hydroxy-2-nonenal-modified peptides within unfractionated digests using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The lipid peroxidation product 4-hydroxy-2-nonenal (HNE) is generated as a consequence of oxidative stress and can readily react with nucleophilic sites of proteins (e.g., histidine residues), mainly via a Michael addition. The formation of such lipid-protein conjugates can alter protein properties and biological functions, thus leading to highly deleterious effects. The present work describes a rapid (very limited sample preparation) and sensitive (low-femtomole range) procedure to identify HNE-modified peptides (Michael adducts) within unfractionated tryptic digests. The protocol involves the formation of dinitrophenylhydrazones of the Michael adducts, when using 2,4-dinitrophenylhydrazine as reactive matrix, followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The hydrazone derivatives present high desorption/ionization yield and can thus be preferentially detected compared to unmodified peptides. The MALDI mass spectrum obtained is therefore drastically different from the one obtained with the classical 4-hydroxy-alpha-cyanocinnamic acid matrix. Moreover, the presence of HNE, or more generally speaking carbonylated peptides, could be highlighted by 180 mass units differences (corresponding to the dinitrophenylhydrazone moiety) between these two MALDI mass spectra. Further information (e.g., localization/identification of the modified residues, peptide sequences) could be obtained by performing MALDI postsource decay (or electrospray) MS/MS experiments on the ions of interest.     

Chemical cleavage sequencing of DNA using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In this paper, we report for the first time use of laser desorption mass spectrometry for measurement of chemical cleavage sequencing products of DNA. In this method, the target DNA was labeled with biotin and subjected to chemical modification and cleavage according to the Maxam-Gilbert sequencing protocol. The biotin-containing fragments were captured by streptavidin-coated magnetic beads and separated from the other fragments. The captured fragments were released by hot ammonia treatment, and the released fragments were analyzed by mass spectrometry. Potential applications of this method in resolving sequence ambiguities and sequencing repeat sequences as well as in the analysis of DNA-protein interactions are discussed.     

End-group analysis of blue light-emitting polymers using matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry

An analytical method based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied to provide information on the structure of a copolymer, e.g., repeat unit and end group. Seven conjugated polymers, which have been demonstrated as the active component in blue light-emitting diodes, were synthesized through Suzuki polycondensation reaction in the presence of Pd(PPh3)4 catalyst. Their molecular weights were obtained using gel permeation chromatography analysis. MALDI-TOF MS was used to investigate the structure information in detail. The proposed end-group structures were confirmed by the identity between the observed and the simulated isotopic distribution of each polymer. The results demonstrate that these synthetic polymers possess various end groups and even contain macrocycles. The catalyst Pd(PPh3)4 was found to introduce phenyl end groups via aryl-aryl exchange between the catalytic palladium intermediate and the triphenylphosphine ligand. All these results are based on the analysis of the mass spectrum data, which suggests that MALDI-TOF MS is an extraordinarily strong tool in synthetic polymer structure analysis.     

Analysis of microbial mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Many different laboratories are currently developing mass-spectrometric techniques to analyze and identify microorganisms. However, minimal work has been done with mixtures of bacteria. To demonstrate that microbial mixtures could be analyzed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), mixed bacterial cultures were analyzed in a double-blind fashion. Nine different bacterial species currently in our MALDI-MS fingerprint library were used to generate 50 different simulated mixed bacterial cultures similar to that done for an initial blind study previously reported (Jarman, K. H.; Cebula, S. T.; Saenz, A. J.; Petersen, C. E.; Valentine, N. B.; Kingsley, M. T.; Wahl, K. L. Anal. Chem. 2000, 72, 1217-1223). The samples were analyzed by MALDI-MS with automated data extraction and analysis algorithms developed in our laboratory. The components present in the sample were identified correctly to the species level in all but one of the samples. However, correctly eliminating closely related organisms was challenging for the current algorithms, especially in differentiating Serratia marcescens, Escherichia coli, and Yersinia enterocolitica, which have some similarities in their MALDI-MS fingerprints. Efforts to improve the specificity of the algorithms are in progress.     

Anionic adducts of oligosaccharides by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The formation and decomposition (postsource decay, PSD) of anionic adducts in negative ion matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry have been studied. Chloride, a small inorganic anion, has been found to form stable anionic adducts with a variety of neutral oligosaccharides that can survive the MALDI process to give readily identifiable signals (with characteristic isotope patterns) allowing subpicomole detection in the best cases. The matrixes that can aid the formation of chloride adducts of oligosaccharides have gas-phase acidities lower than or close to that of HCl (1373 kJ/mol). In PSD experiments, precursor chloride adducts of oligosaccharides yield fragment ions that retain the charge on the sugar molecule rather than solely forming Cl-, and these fragments can provide structurally informative product ions. In negative ion MALDI, highly acidic oligosaccharides do not form adducts with chloride anions, but mildly acidic saccharides (e.g., containing a carboxylic acid group) form both deprotonated molecules and chloride adducts, and each may provide structural information concerning the oligosaccharide upon decomposition.     

Proteomic profiling of intact mycobacteria by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Current methods for the identification of mycobacteria in culture are time-consuming, requiring as long as 12 weeks for positive identification. One potential approach to rapid mycobacterial identification is to utilize proteomic profiling of cultures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this report, we have applied MALDI-TOF MS to proteomic profiling of cultured microorganisms representing six species of the genus Mycobacterium. We find that analysis of acetonitrile/trifluoroacetic acid cellular extracts produces data similar to that of the analysis of deposited whole cells, while minimizing human contact with the microorganisms and rendering them nonviable. A matrix composition of alpha-cyano-4-hydroxycinnamic acid with fructose yields highly reproducible MALDI-TOF spectra. Statistical analysis of MALDI-TOF MS data allows differentiation of each individual mycobacterial species on the basis of unique mass fingerprints. The methodology allows identification of a number of unique (potentially diagnostic) biomarkers as targets for protein identification by MS/MS experiments. In addition, we observe a number of signals common to all mycobacterial species studied by MALDI-TOF MS, which may be genus-specific biomarkers. The potentially genus-specific biomarkers occur at low mass (<2 kDa) and are likely to be lipids and cell wall components such as mycolic acids. This study demonstrates the potential for mass spectrometry-based identification/classification of mycobacteria.     

Atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A novel ionization source for biological mass spectrometry is described that combines atmospheric pressure (AP) ionization and matrix-assisted laser desorption/ionization (MALDI). The transfer of the ions from the atmospheric pressure ionization region to the high vacuum is pneumatically assisted (PA) by a stream of nitrogen, hence the acronym PA-AP MALDI. PA-AP MALDI is readily interchangeable with electrospray ionization on an orthogonal acceleration time-of-flight (oaTOF) mass spectrometer. Sample preparation is identical to that for conventional vacuum MALDI and uses the same matrix compounds, such as alpha-cyano-4-hydroxycinnamic acid. The performance of this ion source on the oaTOF mass spectrometer is compared with that of conventional vacuum MALDI-TOF for the analysis of peptides. PA-AP MALDI can detect low femtomole amounts of peptides in mixtures with good signal-to-noise ratio and with less discrimination for the detection of individual peptides in a protein digest. Peptide ions produced by this method generally exhibit no metastable fragmentation, whereas an oligosaccharide ionized by PA-AP MALDI shows several structurally diagnostic fragment ions. Total sample consumption is higher for PA-AP MALDI than for vacuum MALDI, as the transfer of ions into the vacuum system is relatively inefficient. This ionization method is able to produce protonated molecular ions for small proteins such as insulin, but these tend to form clusters with the matrix material. Limitations of the oaTOF mass spectrometer for singly charged high-mass ions make it difficult to evaluate the ionization of larger proteins.     

Carbon nanotubes as assisted matrix for laser desorption/ionization time-of-flight mass spectrometry

Analysis of low molecular weight compounds with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) has been developed by using carbon nanotubes obtained from coal by arc discharge as the matrix. The carbon nanotube matrix functions as substrate to trap analytes of peptides, organic compounds, and beta-cyclodextrin deposited on its surface. It has been found that carbon nanotubes can transfer energy to the analyte under laser irradiation, which makes analytes well desorbed/ionized, and the interference of intrinsic matrix ions can be eliminated. At the same time, the fragmentation of the analyte can be avoided. A good sensitivity and excellent reproducibility of the spectrum signals are achieved. It is believed that this work not only will open a new field for applications of carbon nanotubes, but also will offer a new technique for high-speed analysis of low molecular weight compounds in areas such as metabolism research and characterization of natural products.     

High-throughput quantitative analysis of total N-glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Accurate and reproducible quantification of glycans from protein drugs has become an important issue for quality control of therapeutic proteins in biopharmaceutical and biotechnology industries. Mass spectrometry is a promising tool for both qualitative and quantitative analysis of glycans owing to mass accuracy, efficiency, and reproducibility, but it has been of limited success in quantitative analysis for sialylated glycans in a high-throughput manner. Here, we present a solid-phase permethylation-based total N-glycan quantitative method that includes N-glycan releasing, purification, and derivatization on a 96-well plate platform. The solid-phase neutralization enabled us to perform reliable absolute quantification of the acidic N-glycans as well as neutral N-glycans from model glycoproteins (i.e., chicken ovalbumin and porcine thyroglobulin) by only using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Furthermore, low-abundance sialylated N-glycans from human serum prostate specific antigen (PSA), an extremely valuable prostate cancer marker, were initially quantified, and their chemical compositions were proposed. Taken together, these results demonstrate that our all-inclusive glycan preparation method based on a 96-well plate platform may contribute to the precise and reliable qualitative and quantitative analysis of glycans.     

Single cell matrix-assisted laser desorption/ionization mass spectrometry imaging

Application of mass spectrometry imaging (MS imaging) analysis to single cells was so far restricted either by spatial resolution in the case of matrix-assisted laser desorption/ionization (MALDI) or by mass resolution/mass range in the case of secondary ion mass spectrometry (SIMS). In this study we demonstrate for the first time the combination of high spatial resolution (7 μm pixel), high mass accuracy (<3 ppm rms), and high mass resolution (R = 100?000 at m/z = 200) in the same MS imaging measurement of single cells. HeLa cells were grown directly on indium tin oxide (ITO) coated glass slides. A dedicated sample preparation protocol was developed including fixation with glutaraldehyde and matrix coating with a pneumatic spraying device. Mass spectrometry imaging measurements with 7 μm pixel size were performed with a high resolution atmospheric-pressure matrix-assisted laser desorption/ionization (AP-MALDI) imaging source attached to an Exactive Orbitrap mass spectrometer. Selected ion images were generated with a bin width of Δm/z = ±0.005. Selected ion images and optical fluorescence images of HeLa cells showed excellent correlation. Examples demonstrate that a lower mass resolution and a lower spatial resolution would result in a significant loss of information. High mass accuracy measurements of better than 3 ppm (root-mean-square) under imaging conditions provide confident identification of imaged compounds. Numerous compounds including small metabolites such as adenine, guanine, and cholesterol as well as different lipid classes such as phosphatidylcholine, sphingomyelin, diglycerides, and triglycerides were detected and identified based on a mass spectrum acquired from an individual spot of 7 μm in diameter. These measurements provide molecularly specific images of larger metabolites (phospholipids) in native single cells. The developed method can be used for a wide range of detailed investigations of metabolic changes in single cells.     

Identification of phosphorylation sites in N-linked glycans by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Glycan phosphorylation is a significant feature of complex carbohydrate chemistry and glycobiology. For example, N-linked glycans containing mannose-6-phosphate (Man-6-P) residues play a key role as targeting signals for the transport of proteins from the Golgi apparatus to lysosomes. Structural information on Man-6-P glycans involved in transport of proteins is usually obtained using nuclear magnetic resonance (NMR) spectroscopy. However, an alternative and simple method with comparable accuracy is desirable because large amounts of samples and special techniques are required for structural analysis using NMR. Recently, postsource decay (PSD) fragment spectra obtained by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) have provided critical information on complex carbohydrates. Since few Man-6-P-containing glycans are commercially available, very little information has been collected on the mass spectrometry of phosphorylated glycans. In this report, four kinds of phosphorylated glycans labeled with 2-aminopyridine (PA) were purified from yeast mannan, and their PSD spectra were measured in the positive ion mode. The phospho-6-O-mannose monoester linkages (PO3H-Man) in glycans are stable, although cleavage of the mannose-1-phosphate linkage (Man-alpha-1-PO3H) occurs readily. Fragment ions indicated the presence of the alpha-1,3-branching chain of an N-linked high-mannose-type glycan, and characteristic fragmentation patterns were observed for phosphorylated glycans. On the basis of the MALDI-PSD spectra, we deduced fragmentation rules for phosphorylated N-glycans that will be valuable for distinguishing the position of phosphorylation.     

Quantitative determination of heme for forensic characterization of bacillus spores using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A quantitative method was developed for the determination of heme (ferriprotoporphyrin IX) using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). The method was designed for forensic characterization of the use of blood agar in preparation of Bacillus spores. An alkali wash of 0.3 M ammonium hydroxide was used to solubilize heme from spore samples. The wash was concentrated and analyzed by MALDI-TOFMS. Experimental parameters were optimized to obtain the best signal intensity, maximize signal reproducibility, and improve day-to-day repeatability of the measurement. Sinapinic acid was found to be the best matrix. A sandwich sample preparation protocol was determined to increase the shot-to-shot and point-to-point reproducibility of the measurement. Cobalt(III) protoporphyrin was used as an internal standard and the analyte/internal standard ratio responses from solutions of known concentrations were used to construct a calibration curve (R(2) = 0.993). Limits of detection and quantitation for heme were calculated to be approximately 0.4 (200 fmol) and 0.8 microM (400 fmol), respectively. Spore samples prepared on blood agar and nonblood agar were analyzed using the method. Heme was detected at a concentration of approximately 0.3 ng/mg of spore on samples prepared on blood agar and purified by extensive washing. Heme was not detected on spore samples prepared without blood.     

Direct determination of the peptide content in microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A quantitative determination of peptides incorporated into poly(d,l-lactide-co-glycolide) microspheres by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was accomplished in a single step without pretreatment for extracting the peptide from the microsphere. The conventional extraction methods often underestimate the actual amount of peptide because of incomplete extraction from the microspheres or loss during the procedures. In this study, the microspheres dissolved in acetonitrile containing 0.1% trifluoroacetic acid were mixed with matrix solution containing the internal standard, and the peptide content was directly determined by MALDI-TOF MS. The drug content values determined by MALDI-TOF MS in both the leuprolide- and salmon calcitonin-incorporated microspheres were closer to the theoretical contents than those determined by the conventional extraction method. This method using MALDI-TOF MS could be a good alternative to time-consuming and less-accurate conventional methods.     

In-source and postsource decay in negative-ion matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of neutral oligosaccharides

Cross-ring cleavage ions produced by in-source decay (ISD), as well as deprotonated molecular ions [M - H]-, are invariably observed in negative-ion linear-mode matrix-assisted laser desorption/ionization time-of-flight mass spectrometry spectra of neutral oligosaccharides with 9H-pyrido[3,4-b]indole (norharman) as a matrix. The patterns of ISD ions depend on the oligosaccharide linkage type; thus, these ions are potentially useful in linkage analysis. In postsource decay (PSD) spectra from chlorinated molecular ions [M + Cl]-, all PSD ions are observed in the deprotonated form, although no deprotonated molecular ions are detected. In oligosaccharides having an alditol at the reducing end, deprotonated molecular ions [M - H]- are clearly seen in linear-mode mass spectra and survive in the PSD measurements. These results indicate that the deprotonation process drives ISD and PSD of oligosaccharides and that keto-enol tautomerization at the reducing terminal promotes ISD and PSD processes.     

Hollow-fiber flow field-flow fractionation for whole bacteria analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

This work proposes for the first time the use of hollow-fiber flow field-flow fractionation (HF FlFFF) for improved matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOFMS) of whole bacteria. HF FlFFF has proved to be able to prepurify or fractionate different species of whole bacteria. Sample preparation by HF FlFFF gives improved spectra quality because noncellular components possibly present in the sample can be separated from the cells. When a mixture of two bacteria (Bacillus subtilis and Escherichia coli) is fractionated through HF FlFFF, MALDI/TOFMS analysis of each separated bacterial species preserves the most characteristic ion signals of the species without the presence of characteristic signals of the other species. The main advantages of HF FlFFF for MALDI/TOFMS analysis of whole bacteria are miniaturization, simplicity, and low cost of the fractionator components. This low cost makes disposable usage of the fractionator possible, thus eliminating the risk of run-to-run contamination of spectra due to sample carryover. The low fractionator volume yields bacterial fractionation on the order of a few minutes, which is comparable to MALDI/TOFMS analysis time. The small fractionation volume makes sample dilution low enough so that additional sample concentration steps are not strictly required to preserve MALDI/TOFMS detection.     

An algorithm for automated bacterial identification using matrix-assisted laser desorption/ionization mass spectrometry

An algorithm for bacterial identification using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is being developed. This mass spectral fingerprint comparison algorithm is fully automated and statistically based, providing objective analysis of samples to be identified. Based on extraction of reference fingerprint ions from test spectra, this approach should lend itself well to real-world applications where samples are likely to be impure. This algorithm is illustrated using a blind study. In the study, MALDI-MS fingerprints for Bacillus atrophaeus ATCC 49337, Bacillus cereus ATCC 14579T, Escherichia coli ATCC 33694, Pantoea agglomerans ATCC 33243, and Pseudomonas putida F1 are collected and form a reference library. The identification of test samples containing one or more reference bacteria, potentially mixed with one species not in the library (Shewanella alga BrY), is performed by comparison to the reference library with a calculated degree of association. Out of 60 samples, no false positives are present, and the correct identification rate is 75%. Missed identifications are largely due to a weak B. cereus signal in the bacterial mixtures. Potential modifications to the algorithm are presented and result in a higher than 90% correct identification rate for the blind study data, suggesting that this approach has the potential for reliable and accurate automated data analysis of MALDI-MS.     

Capillary electrophoresis--matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using a vacuum deposition interface

An improved vacuum deposition interface for coupling capillary electrophoresis with MALDI-TOF MS has been developed. Liquid samples consisting of analyte and matrix were deposited on a moving tape in the evacuated source chamber of a TOF mass spectrometer, enabling 24 h of uninterrupted analysis. The vacuum deposition procedure was compared with the dried-droplet method, and it was found that vacuum deposition generated significantly more reproducible signal intensity, eliminating the need for "sweet spot" searching. A concentration detection limit in the low-nanomolar range has been achieved with a low-attomole amount of sample consumed per spectrum. In addition, ion suppression caused by hydrophobicity differences in the analytes was reduced. To minimize ion suppression further, separation prior to MALDI MS analysis was employed. The performance of capillary electrophoresis (CE)-MALDI-TOF MS using the vacuum deposition interface was evaluated with a peptide mixture injected at low-femtomole levels. All peptides were baseline resolved with separation efficiencies in the range of 250000-400000 plates/m (2-3-s band half-width), demonstrating the high separation efficiency of the CE-MALDI MS coupling. A fast (approximately 40 s) CE separation of a mixture of angiotensins was found to reduce significantly ion suppression and enable trace level detection. It was also shown, for the analysis of an enolase digest, that sequence coverage of 65% was obtained using CE separation compared to 52% using step-elution solid-phase extraction and 44% in the control experiment using an unseparated mixture.     

Two-step matrix application technique to improve ionization efficiency for matrix-assisted laser desorption/ionization in imaging mass spectrometry

A novel matrix application protocol for direct tissue mass spectrometry is presented. Matrix-assisted laser desorption/ionization is a popular ionization procedure for direct tissue analysis and imaging mass spectrometry. Usually, matrixes are applied by dispensing droplets through either pipettes or automated dispensing machines, or by airbrushing. These techniques are very simple, but it was difficult to obtain uniform matrix crystals on the tissue surface, and nonuniform crystals degrade the spectrum qualities. Here we report a new matrix application protocol, which is a combination of spraying and dispensing droplets, and we have succeeded in overcoming these problems in conventional matrix applications on tissue surfaces. We call our new technique the "spray-droplet method". In this technique, tiny matrix crystals formed by spraying act as seeds for crystal growth. Our technique leads to matrix spots that are filled homogeneously with minute crystals. Such matrix crystals dramatically improve peak intensity and signal-to-noise ratio. In an example on a rat brain section, the number of detectable peaks was increased and signal intensity of m/z 5440 in our method was approximately 30.6 times higher than that in conventional methods. We used this spray-droplet method with a chemical ink-jet technology for matrix deposition to succeed in MALDI imaging of signals, which were undetectable from the conventional matrix applications.