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.     

Detection and quantification of neurotensin in human brain tissue by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method was developed for mass spectrometric detection of neurotensin (NT)-like immunoreactivity and quantification of NT in human brain tissue. The method is based on immunoprecipitation followed by analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The identity of the major component of the immunoprecipitates as neurotensin was confirmed by fragment ion analysis on an electrospray ionization quadrupole time-of-flight instrument. MALDI-TOF-MS quantification of NT was achieved using stable-isotope-labeled NT as the internal standard, yielding an error of less than 5%. The method allowed detection of low-femtomole amounts of NT, staring from low-milligram amounts of lyophilized brain tissue. In addition to NT, several other peptides were detected in the purified samples, most of which, according to their molecular masses, corresponded to fragments of NT. The method is demonstrated with quantification of NT from human hypothalamus tissue, and a comparison is made with results obtained from competitive radioimmunoassay.     

Analysis of gangliosides directly from thin-layer chromatography plates by infrared matrix-assisted laser desorption/ionization orthogonal time-of-flight mass spectrometry with a glycerol matrix

A novel method is presented for direct coupling of high-performance thin-layer chromatography (HPTLC) with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for the analysis of biomolecules. A first key feature is the use of a liquid matrix (glycerol), which provides a homogeneous wetting of the silica gel and a simple and fast MALDI preparation protocol. A second is the use of an Er:YAG infrared laser, which ablates layers of approximately 10-microm thickness of analyte-loaded silica gel and provides a soft desorption/ionization of even very labile analyte molecules. The orthogonal time-of-flight mass spectrometer employed in this study, finally provides a high accuracy of the mass determination, which is independent of any irregularity of the silica gel surface. The analytical potential of the method is demonstrated by the compositional mapping of a native GM3 (II(3)-alpha-Neu5Ac-LacCer) ganglioside mixture from cultured Chinese hamster ovary cells. The analysis is characterized by a high relative sensitivity, allowing the simultaneous detection of various major and minor GM3 species directly from individual HPTLC analyte bands. The lateral resolution of the direct HPTLC-MALDI-MS analysis is defined by the laser focus diameter of currently approximately 200 microm. This allows one to determine mobility profiles of individual species with a higher resolution than by reading off the chromatogram by optical absorption. The fluorescent dye primuline was, furthermore, successfully tested as a nondestructive, MALDI-compatible staining agent.     

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.     

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.     

Elemental imaging via laser ionization orthogonal time-of-flight mass spectrometry

An elemental imaging method using a laser ionization orthogonal time-of-flight mass spectrometer system was developed for the simultaneous detection of all metal and nonmetal elements. The instrument control and data processing were realized by self-developed programs. This system is capable of simultaneous detection of metal and nonmetal elements, with a spatial resolution of 50 μm, the lowest detection limits of 3 × 10(-7) g/g (Li), and a dynamic range of 7 orders of magnitude. Moreover, this technique does not require standards for quantitative analysis and can be a powerful and versatile tool for elemental imaging.     

Molecular analysis of native tissue and whole oils by infrared laser mass spectrometry

We have employed infrared laser desorption ionization orthogonal time-of-flight mass spectrometry (IR-LDI-o-TOF-MS) to generate molecular ion profiles directly from native tissue and from whole oils. The method requires little sample preparation besides an eventual dissection of the areas of interest and drying of particularly water-rich samples. The lateral resolution of the analysis is on the order of the laser focal diameter, and in the third dimension, defined by the depth of material ejection, a few to 10 microm per laser pulse. Various types of small molecules are readily detected from minute volumes of sample. Among these are carbohydrates, phospholipids, triglycerides, and flavonoids. Substantially different molecular profiles were recorded from different areas of a single strawberry seed.     

Discrimination between bacterial spore types using time-of-flight mass spectrometry and matrix-free infrared laser desorption and ionization

We demonstrate that molecular ions with mass-to-charge ratios (m/z) ranging from a few hundred to 19 050 can be desorbed from whole bacterial spores using infrared laser desorption and no chemical matrix. We have measured the mass of these ions using time-of-flight mass spectrometry and we observe that different ions are desorbed from spores of Bacillus cereus, Bacillus thuringiensis, Bacillus subtilis, and Bacillus niger. Our results raise the possibility of identifying microorganisms using mass spectrometry without conventional sample preparation techniques such as the addition of a matrix. We have measured the dependence of the ion yield from B. subtilis on desorption wavelength over the range 3.05-3.8 microm, and we observe the best results at 3.05 microm. We have also generated mass spectra from whole spores using 337-nm ultraviolet laser desorption, and we find that these spectra are inferior to spectra generated with infrared desorption. Since aerosol analysis is a natural application for matrix-free desorption, we have measured mass spectra from materials such as ragweed pollen and road dust that are likely to form a background to microbial aerosols. We find that these materials are readily differentiated from bacterial spores.     

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.     

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.     

Matrix-implanted laser desorption/ionization mass spectrometry

The implantation of low-velocity massive gold clusters is shown to be a method of choice for homogeneous incorporation of a metallic matrix into the near-surface region of a solid biopolymer for subsequent laser desorption/ionization (LDI) MS analysis. Matrix implanted (MI)LDI spectra from cluster-implanted pure test peptide or tissue exhibit molecular ion peaks similar to those observed by matrix-assisted LDI. Moreover, the ion emission is very reproducible from any spot on the surface of these test samples. MILDI promises to be a powerful technique for mass spectrometric analysis of native biological samples as demonstrated by the first results on rat brain tissues.     

In vivo analysis and spatial profiling of phytochemicals in herbal tissue by matrix-assisted laser desorption/ionization mass spectrometry

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was developed for spatial profiling of phytochemicals and secondary metabolites in integrated herbal tissue without solvent extraction. Abundant alkaloid ions, including (+)-menisperine (m/z 356), magnoflorine (m/z 342), stepharanine (m/z 324), protonated sinomenine (m/z 330), protonated sinomendine (m/z 338), and a metabolite at m/z 314, could be directly desorbed from alpha-cyano-4-hydroxycinnamic acid- (CHCA-) coated stem tissue of Sinomenium acutum upon N2 laser (337 nm) ablation, while the ion signals desorbed from sinapinic acid- (SA-) coated and 2,5-dihydroxybenzoic acid- (DHB-) coated stem tissue were at least 10 times weaker. Solvent composition in the matrix solution could have significant effects on the ion intensity of the metabolites. Under optimized conditions that maximize the ion intensity and form homogeneous matrix crystals on the tissue surface, spatial distributions of the metabolites localized in different tissue regions, including cortex, phloem, xylem, rim, and pith, and their relative abundances could be semiquantitatively determined. The three metabolites detected at m/z 356, 342, and 314 showed specific distributions in the herbal samples collected from different growing areas, while others were not. By applying principal component analysis (PCA), the characteristic metabolites in specific tissue regions could be easily determined, allowing unambiguous differentiation of the herbal samples from different geographic locations.     

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.     

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.     

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.     

Thermal vaporization-vacuum ultraviolet laser ionization time-of-flight mass spectrometry of single aerosol particles

Single aerosol particles of ethylene glycol and oleic acid are vaporized on a heater at temperatures between 500 and 700 K, and the resulting vapor plume is ionized by a 10.5-eV vacuum ultraviolet (VUV) laser. The mass spectra are compared to those obtained by CO2 laser vaporization followed by VUV laser ionization. The relative intensities of the parent and fragment ion peaks are remarkably similar for the two modes of vaporization. A Maxwell-Boltzmann distribution of speeds accurately describes the dependence of the signal as a function of the VUV laser pulse timing. The signal levels obtained with this design are sufficient to obtain good-quality mass spectra.     

Ambient molecular imaging and depth profiling of live tissue by infrared laser ablation electrospray ionization mass spectrometry

Mass spectrometry in conjunction with atmospheric pressure ionization methods enables the in vivo investigation of biochemical changes with high specificity and sensitivity. Laser ablation electrospray ionization (LAESI) is a recently introduced ambient ionization method suited for the analysis of biological samples with sufficient water content. With LAESI mass spectrometric analysis of chimeric Aphelandra squarrosa leaf tissue, we identify the metabolites characteristic for the green and yellow sectors of variegation. Significant parts of the related biosynthetic pathways (e.g., kaempferol biosynthesis) are ascertained from the detected metabolites and metabolomic databases. Scanning electron microscopy of the ablated areas indicates the feasibility of both two-dimensional imaging and depth profiling with a approximately 350 microm lateral and approximately 50 microm depth resolution. Molecular distributions of some endogenous metabolites show chemical contrast between the sectors of variegation and quantitative changes as the ablation reaches the epidermal and mesophyll layers. Our results demonstrate that LAESI mass spectrometry opens a new way for ambient molecular imaging and depth profiling of metabolites in biological tissues and live organisms.     

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.     

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.