Imaging and 3D elemental characterization of intact bacterial spores by high-resolution secondary ion mass spectrometry

We present a quantitative, imaging technique based on nanometer-scale secondary ion mass spectrometry for mapping the 3D elemental distribution present in an individual micrometer-sized Bacillus spore. We use depth profile analysis to access the 3D compositional information of an intact spore without the additional sample preparation steps (fixation, embedding, and sectioning) typically used to access substructural information in biological samples. The method is designed to ensure sample integrity for forensic characterization of Bacillus spores. The minimal sample preparation/alteration required in this methodology helps to preserve sample integrity. Furthermore, the technique affords elemental distribution information at the individual spore level with nanometer-scale spatial resolution and high (microg/g) analytical sensitivity. We use the technique to map the 3D elemental distribution present within Bacillus thuringiensis israelensis spores.     

Tandem mass spectrometry for sequencing proanthocyanidins

Proanthocyanidins (PAs) are a group of bioflavonoids consisting of oligomers based on catechin monomeric units. These polyphenolic compounds are widely distributed in higher plants and are an integral part of the human diet. A sensitive LC-tandem mass spectrometric (LC/ESI-MS(n)) method in the positive ion mode for sequencing these ubiquitous and highly beneficial antioxidants is described. The hydroxylation patterns and interflavanoid linkage for A- and B-type PAs were determined by fragment ions derived from a retro-Diels-Alder (RDA) fission, heterocyclic ring fission (HRF), a novel benzofuran-forming (BFF) fission described here for the first time, and a quinone methide (QM) fission. The subunit sequence of the PAs was determined by diagnostic ions derived from HRF/RDA fission, HRF/BFF fission, and RDA/HRF fission together with QM fission. A total of 26 PAs were reliably sequenced by the newly established tandem mass spectrometric protocol. It is shown that the protocol based on a combination of these different fragmentation patterns allows for uniquely identifying PA oligomers.     

Processing complex mixtures of intact proteins for direct analysis by mass spectrometry

For analysis of intact proteins by mass spectrometry (MS), a new twist to a two-dimensional approach to proteome fractionation employs an acid-labile detergent instead of sodium dodecyl sulfate during continuous-elution gel electrophoresis. Use of this acid-labile surfactant (ALS) facilitates subsequent reversed-phase liquid chromatography (RPLC) for a net two-dimensional fractionation illustrated by transforming thousands of intact proteins from Saccharomyces cerevisiae to mixtures of 5-20 components (all within approximately 5 kDa of one another) for presentation via electrospray ionization (ESI) to a Fourier transform MS (FTMS). Between 3 and 13 proteins have been detected directly using ESI-FTMS (or MALDI-TOF), and the fractionation showed a peak capacity of approximately 400 between 0 and 70 kDa. A probability-based identification was made automatically from raw MS/MS data (obtained using a quadrupole-FTMS hybrid instrument) for one protein that differed from that predicted in a yeast database of approximately 19,000 protein forms. This ALS-PAGE/RPLC approach to proteome processing ameliorates the "front end" problem that accompanies direct analysis of whole proteins and assists the future realization of protein identification with 100% sequence coverage in a high-throughput format.     

Tandem parallel fragmentation of peptides for mass spectrometry

Parallel fragmentations of peptides in the source region and in the collision cell of tandem mass spectrometers are sequentially combined to develop parallel collision-induced-dissociation mass spectrometry (p2CID MS). Compared to MS/MS spectra, the p2CID mass spectra show increased signal intensities (2-400-fold) and number of sequence ions. This improvement is attributed to the fact that p2CID MS virtually samples all the ions generated by electrospray ionization, including intact and fragment ions of different charge states from a peptide. We implement the method using a quadrupole time-of-flight tandem mass spectrometer. The instrument is operated in TOF-MS mode that allows the ions from source region broadband-passing the first mass analyzer to enter the collision cell. Cone voltage and collision energy are investigated to optimize the outcome of the two parallel CID processes. In the in-source parallel CID, elevated cone voltage produces singly charged intact peptide ions and large fragment ions, as well as decreases the charge-state distribution of peptide ions mainly to double and single charges. The in-collision-cell parallel CID is optimized to dissociate the ions from the source region to produce small and medium fragment ions. The method of p2CID MS is especially useful for sequencing of large peptides with labile amide bonds and peptides with C-terminal arginine. It has unique potential for de novo sequencing of peptides and proteome analysis, especially for affinity-enriched subproteomes.     

Sensitive detection of Bacillus anthracis spores by immunocapture and liquid chromatography-tandem mass spectrometry

Bacillus anthracis is one of the most dangerous agents of the bioterrorism threat. We present here a sensitive immuno-liquid chromatography-tandem mass spectrometry (immuno-LC-MS/MS) approach to spore detection in complex environmental samples. It is based on the combined specificity and sensitivity of two techniques: immunocapture and targeted mass spectrometry. The immunocapture step, realized directly on the intact spores, is essential for their selective isolation and concentration from complex environmental samples. After parallel trypsin and Glu-C digestions, proteotypic peptides corresponding to small acid-soluble spore protein-B (SASP-B) are specifically monitored in the multiple reaction monitoring (MRM) mass spectrometry mode. Peptide ratio is carefully monitored and provides an additional level of specificity, which is shown to be highly useful for distinguishing closely related samples and avoiding false-positive/negative results. Sensitivity at the level of the infectious dose is demonstrated, with limits of detection of 7 × 10(3) spores/mL of milk or 10 mg of soil. This mass spectrometry approach is thus complementary to polymerase chain reaction (PCR) techniques.     

Comprehensive assignment of mass spectral signatures from individual Bacillus atrophaeus spores in matrix-free laser desorption/ionization bioaerosol mass spectrometry

We have fully characterized the mass spectral signatures of individual Bacillus atrophaeus spores obtained using matrix-free laser desorption/ionization bioaerosol mass spectrometry (BAMS). Mass spectra of spores grown in unlabeled, 13C-labeled, and 15N-labeled growth media were used to determine the number of carbon and nitrogen atoms associated with each mass peak observed in mass spectra from positive and negative ions. To determine the parent ion structure associated with fragment ion peaks, the fragmentation patterns of several chemical standards were independently determined. Our results confirm prior assignments of dipicolinic acid, amino acids, and calcium complex ions made in the spore mass spectra. The identities of several previously unidentified mass peaks, key to the recognition of Bacillus spores by BAMS, have also been revealed. Specifically, a set of fragment peaks in the negative polarity is shown to be consistent with the fragmentation pattern of purine nucleobase-containing compounds. The identity of m/z = +74, a marker peak that helps discriminate B. atrophaeus from Bacillus thuringiensis spores grown in rich media is [N1C4H12]+. A probable precursor molecule for the [N1C4H12]+ ion observed in spore spectra is trimethylglycine (+N(CH3)3CH2COOH), which produces a m/z = +74 peak when ionized in the presence of dipicolinic acid. A clear assignment of all the mass peaks in the spectra from bacterial spores, as presented in this work, establishes their relationship to the spore chemical composition and facilitates the evaluation of the robustness of "marker" peaks. This is especially relevant for peaks that have been used to discriminate Bacillus spore species, B. thuringiensis and B. atrophaeus, in our previous studies.     

Detection of anthrax simulants with microcalorimetric spectroscopy: Bacillus subtilis and Bacillus cereus spores

Recent advances in the development of ultrasensitive micromechnical thermal detectors have led to the advent of novel subfemtojoule microcalorimetric spectoscopy (CalSpec). On the basis of principles of photothermal IR spectroscopy combined with efficient thermomechanical transduction, CalSpec provides acquisition of vibrational spectra of microscopic samples and absorbates. We use CalSpec as a method of identifying nanogram quantities of biological micro-organisms. Our studies focus on Bacillus subtilis and Bacillus cereus spores as simulants for Bacillus anthracis spores. Using CalSpec, we measured IR spectra of B. subtilis and B. cereus spores present on surfaces in nanogram quantities (approximately 100-1,000 spores). The spectra acquired in the wavelength range of 690-4000 cm(-1) (2.5-14.5 microm) contain information-rich vibrational signatures that reflect the different ratios of biochemical makeup of the micro-organisms. The distinctive features in the spectra obtained for the two types of microorganism can be used to distinguish between the spores of the Bacillus family. As compared with conventional IR and Fourier-transform IR microscopic spectroscopy techniques, the advantages of the present technique include significantly improved sensitivity (at least a full order of magnitude), absence of expensive IR detectors, and excellent potential for miniaturization.     

Stable isotope labeling of entire Bacillus atrophaeus spores and vegetative cells using bioaerosol mass spectrometry

Single vegetative cells and spores of Bacillus atrophaeus, formerly Bacillus subtilis var. niger, were analyzed using bioaerosol mass spectrometry. Key biomarkers were identified from organisms grown in 13C and 15N isotopically enriched media. Spore spectra contain peaks from dicipolinate and amino acids. The results indicate that compounds observed in the spectra correspond to material from the spore's core and not the exosporium. Standard compounds and mixtures were analyzed for comparison. The biomarkers for vegetative cells were clearly different from those of the spores, consisting mainly of phosphate clusters and amino acid fragments.     

Top-down approaches for measuring expression ratios of intact yeast proteins using Fourier transform mass spectrometry

The extension of quantitation methods for small peptides to ions above 5 kDa, and eventually to global quantitative proteomics of intact proteins, will require extensive refinement of current analytical approaches. Here we evaluate postgrowth Cys-labeling and 14N/15N metabolic labeling strategies for determination of relative protein expression levels and their posttranslational modifications using top-down mass spectrometry (MS). We show that intact proteins that are differentially alkylated with acrylamide (+71 Da) versus iodoacetamide (+57 Da) have substantial chromatographic shifts during reversed-phase liquid chromatography separation (particularly in peak tails), indicating a requirement for stable isotopes in alkylation tags for top-down MS. In the 14N/15N metabolic labeling strategy, we achieve 98% 15N incorporation in yeast grown 10 generations under aerobic conditions and determine 50 expression ratios using Fourier transform ion cyclotron resonance MS in comparing these cells to anaerobically grown control (14N) cells. We devise quantitative methods for top-down analyses, including a correction factor for accurate protein ratio determination based upon the signal-to-noise ratio. Using a database of 200 yeast protein forms identified previously by top-down MS, we verify the intact mass tag concept for protein identification without tandem MS. Overall, we find that top-down MS promises work flows capable of large-scale proteome profiling using stable isotope labeling and the determination of >5 protein ratios per spectrum.     

Identification of unusual bacterial glycosylation by tandem mass spectrometry analyses of intact proteins

The characterization of protein glycosylation can be a complex and time-consuming procedure, especially for prokaryote O-linked glycoproteins, which often comprise unusual oligosaccharide structures with no known glycosylation motif. In this report, we describe a "top-down" approach that provides information on the extent of glycosylation, the molecular masses, and the structure of oligosaccharide residues on bacterial flagella, important structural proteins involved in the motility of pathogenic bacteria. Flagella from four bacterial pathogens, namely, Campylobacter jejuni, Helicobacter pylori, Aeromonas caviae, and Listeria monocytogenes, were analyzed by this top-down mass spectrometry approach. The approach needs minimal sample preparation and can be performed within a few minutes compared to the tedious and often time-consuming "bottom-up" approach involving proteolytic digestion and LC-MS-MS analyses of the suspected glycopeptides. Multiply protonated protein precursor ions subjected to low-energy collisional activation in a quadrupole time-of-flight instrument showed extensive and specific gas-phase deglycosylation resulting in the formation of abundant oxonium ions with very few fragment ions from peptidic bond cleavages. Structural information on individual carbohydrate residues is obtained using a second-generation product ion scan of oxonium ions formed by collisional activation of the intact protein ions in the source region. The four bacterial flagella examined differed not only by the extent of glycosylation but also by the nature of carbohydrate substituents. For example, the flagellin from the Gram-positive bacterium, L. monocytogenes showed O-linked GlcNAc residues at up to 6 sites/protein monomer. In contrast, the three Gram-negative bacterial pathogens C. jejuni, H. pylori and A. caviae displayed up to 19 Ser/Thr O-linked sites modified with residues structurally related to N-acetylpseudaminic acid (Pse5Ac7Ac) and in the case of Campylobacter include a novel N-acetylglutamine substituent on Pse5Am7Ac.     

Detection of cyclic lipopeptide biomarkers from Bacillus species using atmospheric pressure matrix-assisted laser desorption/ionization mass spectrometry

A novel approach to microbial detection using atmospheric pressure matrix-assisted laser desorption/ionization with an ion trap mass spectrometer to analyze whole cell bacteria is introduced. This new approach was tested with lyophilized spores and cultures of Bacillus globigii (BG) grown on agar media for 4 days or longer. At each stage of growth, it was found that biomarkers, identified as cyclic lipopeptides known as fengycin and surfactin, could be detected by pulsed ultraviolet laser irradiation of intact BG cells (approximately 5 mg) cocrystallized with alpha-cyano-4-hydroxycinnamic acid. Furthermore, definitive amino acid sequence information was obtained by performing tandem mass spectrometry on the precursor ions of the cyclic lipopeptides. The investigation was broadened to include the examination of aerosolized BG spores collected from the atmosphere and directly deposited onto double-sided tape. Subsequent analysis of the recovered spores resulted in the production of mass peaks consistent with fengycin. Other Bacillus species were analyzed for comparison and showed mass spectral peaks also identified as originating from various cyclic lipopeptides. Further studies were conducted using a pulsed infrared laser as the excitation source to analyze BG cells (approximately 5 mg) suspended in a matrix of 0.03 M ammonium citrate and glycerol resulting in the production of ions characteristic of fengycin and surfactin.     

On-line hollow-fiber flow field-flow fractionation-electrospray ionization/time-of-flight mass spectrometry of intact proteins

Capabilities of mass spectrometry for the analysis of intact proteins can be increased through separation methods. Flow field-flow fractionation (FlFFF) is characterized by the particularly "soft" separation mechanism, which is ideally suited to maintain the native structure of intact proteins. This work describes the original on-line coupling between hollow-fiber FlFFF (HF FlFFF), the microcolumn variant of FlFFF, and electrospray ionization/time-of-flight mass spectrometry (ESI/TOFMS) for the analysis and characterization of intact proteins. The results show that the native (or pseudonative) structure of horse heart myoglobin and horseradish peroxidase is maintained. Sample desalting is also observed for horse heart myoglobin. Correlation between the molar mass values independently measured by HF FlFFF retention and ESI/TOFMS allows us to confirm the protein aggregation features of bovine serum albumin and to indicate possible changes in the quaternary structure of human hemoglobin.     

Method of measuring Bacillus anthracis spores in the presence of copious amounts of Bacillus thuringiensis and Bacillus cereus

A sensitive and reliable method for the detection of Bacillus anthracis (BA; Sterne strain 7702) spores in presence of large amounts of Bacillus thuringiensis (BT) and Bacillus cereus (BC) is presented based on a novel PZT-anchored piezoelectric excited millimeter-sized cantilever (PAPEMC) sensor with a sensing area of 1.5 mm2. Antibody (anti-BA) specific to BA spores was immobilized on the sensing area and exposed to various samples of BA, BT, and BC containing the same concentration of BA at 333 spores/mL, and the concentration of BT + BC was varied in concentration ratios of (BA:BT + BC) 0:1, 1:0, 1:1, 1:10, 1:100, and 1:1000. In each case, the sensor responded with an exponential decrease in resonant frequency and the steady-state frequency changes reached were 14 +/- 31 (n = 11), 2742 +/- 38 (n = 3), 3053 +/- 19 (n = 2), 2777 +/- 26 (n = 2), 2953 +/- 24 (n = 2), and 3105 +/- 27 (n = 2) Hz, respectively, in 0, 27, 45, 63, 154, and 219 min. The bound BA spores were released in each experiment, and the sensor response was nearly identical to the frequency change during attachment. These results suggest that the transport of BA spores to the antibody immobilized surface was hindered by the presence of other Bacillus species. The observed binding rate constant, based on the Langmuir kinetic model, was determined to be 0.15 min-1. A hindrance factor (alpha) is defined to describe the reduced attachment rate in the presence of BT + BC and found to increase exponentially with BT and BC concentration. The hindrance factor increased from 3.52 at 333 BT + BC spores/mL to 11.04 at 3.33 x 105 BT + BC spores/mL, suggesting that alpha is a strong function of BT and BC concentration. The significance of these results is that anti-BA functionalized PEMC sensors are highly selective to Bacillus anthracis spores and the presence of other Bacillus species, in large amounts, does not prevent binding but impedes BA transport to the sensor.     

T3-sequencing: targeted characterization of the N- and C-termini of undigested proteins by mass spectrometry

A novel extension of the "top-down" approach is introduced for the selective characterization of protein termini that does not involve proteolytic digestion steps. N- and C-terminal peptides were generated from intact proteins in the mass spectrometer and further analyzed by MS/MS-an approach referred to as T(3)-sequencing. N-terminal and C-terminal fragment ion series were obtained by the pseudo-MS/MS technique in-source decay (ISD) on a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS). These ions provided near-terminal sequence tags from the undigested protein in the ISD spectrum acquired in reflector mode and allowed to screen for the proper processing state of the terminus with respect to a reference sequence. In the second step of T(3)-sequencing, the precursor ions, which have been generated by ISD and which included the N- or C-terminal sequence, were selected in the timed ion gate of a MALDI-TOF/TOF mass spectrometer for MS/MS analysis. These spectra allowed identification of the protein, the proper definition of both termini, and allowed confirmation of suspected terminal modifications. T(3)-Sequencing appears to be an alternative to classical Edman sequencing, which is fast and even permits the analysis of N-terminally blocked proteins and their C-terminus.     

Tandem mass spectrometry of sulfated heparin-like glycosaminoglycan oligosaccharides

The structural characterization of heparin-like glycosaminoglycans (HLGAGs) is a major challenge in glycobiology. These linear, sulfated oligosaccharides are expressed on animal cell surfaces, in extracellular matrixes, basement membranes, and mast cell granules and bind with varying degrees of specificity to families of proteases, growth factors, chemokines, and blood coagulation proteins. Cell surface HLGAGs bind growth factors and growth factor receptors and serve as coreceptors in these interactions. Understanding of the mechanism and regulation of growth factor-receptor binding requires efficient determination of cell surface HLGAG structures and the variations in their expression in response to the cellular environment. The solution to this problem entails rapid, sensitive structural analysis of these molecules. To date, HLGAG sequencing requires multistep processes that combine chemical and enzymatic degradation with gel-based or mass spectrometry-based detection systems. Although tandem mass spectrometry has revolutionized proteomics, the fragility of sulfate groups has limited its usefulness in the analysis of HLGAGs. This work demonstrates that tandem mass spectrometry can be effectively used to determine HLGAG structures while minimizing losses of SO3. First, collision-induced dissociation (CID) is shown to produce abundant backbone cleavage ions for HLGAG oligosaccharides, provided that most sulfate groups are deprotonated. Fragmentation of different precursor ion charge states produces complementary data on the structure of the HLGAG. Second, calcium ion complexation of HLGAGs stabilizes the sulfate groups, increases the relative abundances of backbone cleavage ions, and decreases the abundances of ions produced from SO3 losses.     

Web and database software for identification of intact proteins using "top down" mass spectrometry

For the identification and characterization of proteins harboring posttranslational modifications (PTMs), a "top down" strategy using mass spectrometry has been forwarded recently but languishes without tailored software widely available. We describe a Web-based software and database suite called ProSight PTM constructed for large-scale proteome projects involving direct fragmentation of intact protein ions. Four main components of ProSight PTM are a database retrieval algorithm (Retriever), MySQL protein databases, a file/data manager, and a project tracker. Retriever performs probability-based identifications from absolute fragment ion masses, automatically compiled sequence tags, or a combination of the two, with graphical rendering and browsing of the results. The database structure allows known and putative protein forms to be searched, with prior or predicted PTM knowledge used during each search. Initial functionality is illustrated with a 36-kDa yeast protein identified from a processed cell extract after automated data acquisition using a quadrupole-FT hybrid mass spectrometer. A +142-Da delta(m) on glyceraldehyde-3-phosphate dehydrogenase was automatically localized between Asp90 and Asp192, consistent with its two cystine residues (149 and 153) alkylated by acrylamide (+71 Da each) during the gel-based sample preparation. ProSight PTM is the first search engine and Web environment for identification of intact proteins (https://prosightptm.scs.uiuc.edu/).     

Laser power dependence of mass spectral signatures from individual bacterial spores in bioaerosol mass spectrometry

Bioaerosol mass spectrometry is being developed to analyze and identify biological aerosols in real time. Characteristic mass spectra from individual bacterial endospores of Bacillus subtilis var. niger were obtained in a bipolar aerosol time-of-flight mass spectrometer using a pulsed 266-nm laser for molecular desorption and ionization. Spectra from single spores collected at an average fluence of approximately 0.1 J/cm2 frequently contain prominent peaks attributed to arginine, dipicolinic acid, and glutamic acid, but the shot-to-shot (spore-to-spore) variability in the data may make it difficult to consistently distinguish closely related Bacillus species with an automated routine. Fortunately, a study of the laser power dependence of the mass spectra reveals clear trends and a finite number of "spectral types" that span most of the variability. This, we will show, indicates that a significant fraction of the variability must be attributed to fluence variations in the profile of the laser beam.     

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.     

Bioinformatics and mass spectrometry for microorganism identification: proteome-wide post-translational modifications and database search algorithms for characterization of intact H. pylori

MALDI-TOF mass spectrometry has been coupled with Internet-based proteome database search algorithms in an approach for direct microorganism identification. This approach is applied here to characterize intact H. pylori (strain 26695) Gram-negative bacteria, the most ubiquitous human pathogen. A procedure for including a specific and common posttranslational modification, N-terminal Met cleavage, in the search algorithm is described. Accounting for posttranslational modifications in putative protein biomarkers improves the identification reliability by at least an order of magnitude. The influence of other factors, such as number of detected biomarker peaks, proteome size, spectral calibration, and mass accuracy, on the microorganism identification success rate is illustrated as well.     

Plasma electron capture dissociation for the characterization of large proteins by top down mass spectrometry

For the backbone dissociation of large (29 kDa) multiply charged protein ions in the gas phase by electron capture, the main experimental challenges are juxtaposition of the electron and ion for efficient capture, dissociation of tertiary noncovalent bonds that prevent product separation, and minimization of secondary electron capture that destroys larger product ions. A simple alternative methodology is described in which electrons (0.03-100 microA, 0.1-15 eV) are first impinged on a gas pulse in the ion cell of a Fourier transform mass spectrometer, followed by ion beam introduction. For carbonic anhydrase, the resulting plasma conditions produce 87% efficiency for electron capture; a single spectrum yields 512 product ions of 237 different masses from cleavage of 183 of the 258 interresidue bonds, while two spectra cleave 197 of these bonds. The problem of secondary dissociation of product ions is reduced by plasma conditions in which product ions are formed near electrons whose velocities are unfavorable and whose capture cross sections no longer have a square dependence on charge. One plasma ECD spectrum of ubiquitin provides its sequence de novo except for two residue pairs. ECD of casein identifies 126 of 208 interresidue cleavages, providing direct and specific characterization of all its 26 Ser/Thr/Tyr phosphorylation sites.