Matrix-assisted Laser Desorption/ionization Time-of-flight Spectra Research Articles

Application of ionic liquid matrices in spectral analysis of poly(lactide) - solid state NMRspectroscopyversus matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry

The work presented here shows the complementarity of Solid State NMR (SS NMR) spectroscopy and Matrix-Assisted Laser Desorption/Ionization-time-of-flight (MALDI-TOF) mass spectrometry in spectral analysis of poly(L-lactide) (PLLA) using second-generation ionic liquid matrices (ILM II) prepared from N,N-diisopropylethylamine (DEA) and DHB (2,5-dihydroxybenzoic acid), IAA (3-indoleacrylic acid), and HABA (2-(4-hydroxyphenylazo) benzoic acid). The 13C cross-polarization (CP) magic angle spinning (CP/MAS) SS NMR technique was used to study the structure of ionic liquid matrices, their thermal stability, and the influence of ILM on the morphology of polymer. A comparison of MALDI-TOF spectra for samples prepared employing the dried droplet (DD) and the solvent free (SF) mode showed that the former approach gave better results (signal to noise ratio) very likely due to intimate contact between analyte and matrix domains. This hypothesis was verified by analysis of CP build-up curves for DHB–DEA–PLLA samples prepared employing both methods. We also showed that an alternative method of sample preparation based on the melting of ILM II together with a suspended polymer in the liquid matrix is unsatisfactory, particularly for those matrices which can undergo isomerization at higher temperatures (e.g., HABA–DEA and IAA–DEA).

Proline Isomerase Pin1 Represses Terminal Differentiation and Myocyte Enhancer Factor 2C Function in Skeletal Muscle Cells

Reversible proline-directed phosphorylation at Ser/Thr-Pro motifs has an essential role in myogenesis, a multistep process strictly regulated by several signaling pathways that impinge on two families of myogenic effectors, the basic helix-loop-helix myogenic transcription factors and the MEF2 (myocyte enhancer factor 2) proteins. The question of how these signals are deciphered by the myogenic effectors remains largely unaddressed. In this study, we show that the peptidyl-prolyl isomerase Pin1, which catalyzes the isomerization of phosphorylated Ser/Thr-Pro peptide bonds to induce conformational changes of its target proteins, acts as an inhibitor of muscle differentiation because its knockdown in myoblasts promotes myotube formation. With the aim of clarifying the mechanism of Pin1 function in skeletal myogenesis, we investigated whether MEF2C, a critical regulator of the myogenic program that is the end point of several signaling pathways, might serve as a/the target for the inhibitory effects of Pin1 on muscle differentiation. We show that Pin1 interacts selectively with phosphorylated MEF2C in skeletal muscle cells, both in vitro and in vivo. The interaction with Pin1 requires two novel critical phospho-Ser/Thr-Pro motifs in MEF2C, Ser98 and Ser110, which are phosphorylated in vivo. Overexpression of Pin1 decreases MEF2C stability and activity and its ability to cooperate with MyoD to activate myogenic conversion. Collectively, these findings reveal a novel role for Pin1 as a regulator of muscle terminal differentiation and suggest that Pin1-mediated repression of MEF2C function could contribute to this function.

Matrix Assisted Laser Desorption Ionization-Time of Flight Mass Spectral Analysis of Marine Lipopeptides with Potential Therapeutic Implications

Marine Bacillus circulans DMS-2 (MTCC 8281) was found to produce lipopeptides, which reduced the surface tension of the medium from 69 to 28 mNm−1. The methanol extracts of the lipopeptides were resolved in RP-HPLC, which resulted in the presence of two major surface active fractions corresponding to the peaks E and F were eluted at the retention times of 16.8 and 18 min, respectively. Fourier transform infrared spectroscopy and matrix assisted laser desorption ionization time of flight (MALDI-ToF) mass spectral analysis showed that the produced lipopeptides belonged to surfactin family. In the MALDI-ToF spectrum, the major molecular mass of the HPLC purified isoforms were identified at m/z 1,044 and 1,058 Da, respectively. In addition, this marine strain also produced new variants of surfactins with molecular weights ranging from m/z 1,066 to 1,098 Da, which have not been reported earlier. Both surface-active fractions were found to have potent antimicrobial activity against the Gram-positive and Gram-negative bacterial strains.

Identification and characterization of a mosquito pupicidal metabolite of a Bacillus subtilis subsp. subtilis strain

The culture supernatant of a strain of Bacillus subtilis subsp. subtilis isolated from mangrove forests of Andaman and Nicobar islands, India was found to kill larval and pupal stages of mosquitoes. A chloroform extract of the culture supernatant of the bacterium showed pupicidal effects at an LC(50) dose of 1 microg/ml. The mosquitocidal metabolite(s) produced by this strain were purified by gel permeation chromatography. The purified fraction was subjected to Fourier transform infrared (FTIR) spectroscopy and Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry. The FTIR spectrum of active fraction/CHCl3 residue showed strong band characteristic of peptides. MALDI-TOF spectrum of the sample showed well-resolved group of peaks at m/z values 1,030.6, 1,046.7, 1,044.6, 1,060.5, 1,058.6, 1,058.7, and 1,074.6. The results indicated production of different isoforms of surfactin, ranging from C13-C15. Further, the sfp gene responsible for the production of surfactin was amplified and sequenced. In conclusion, this study showed that the mosquito pupicidal metabolite(s), produced by B. subtilis subsp. subtilis is the cyclic lipopeptide, surfactin. The mode of action of surfactin on pupae of mosquitoes is discussed. This is the first report on the mosquito pupicidal activity of surfactin produced by B. subtilis subsp. subtilis.

Identification of Mycobacterium tuberculosis Clinical Isolates with Altered Phagocytosis by Human Macrophages Due to a Truncated Lipoarabinomannan

Phenotypically distinct clinical isolates of Mycobacterium tuberculosis are capable of altering the balance that exists between the pathogen and human host and ultimately the outcome of infection. This study has identified two M. tuberculosis strains (i.e. HN885 and HN1554) among a bank of clinical isolates with a striking defect in phagocytosis by primary human macrophages when compared with strain Erdman, a commonly used laboratory strain for studies of pathogenesis. Mass spectrometry in conjunction with NMR studies unequivocally confirmed that both HN885 and HN1554 contain truncated and more branched forms of mannose-capped lipoarabinomannan (ManLAM) with a marked reduction of their linear arabinan (corresponding mainly to the inner Araf-alpha(1-->5)-Araf unit) and mannan (with fewer 6-Manp residues and more substitutions in the linear Manp-alpha(1-->6)-Manp unit) domains. The truncation in the ManLAM molecules produced by strains HN885 and HN1554 led to a significant reduction in their surface availability. In addition, there was a marked reduction of higher order phosphatidyl-myo-inositol mannosides and the presence of dimycocerosates, triglycerides, and phenolic glycolipid in their cell envelope. Less exposed ManLAM and reduced higher order phosphatidyl-myo-inositol mannosides in strains HN885 and HN1554 resulted in their low association with the macrophage mannose receptor. Despite reduced phagocytosis, ingested bacilli replicated at a fast rate following serum opsonization. Our results provide evidence that the clinical spectrum of tuberculosis may be dictated not only by the host but also by the amounts and ratios of surface exposed mycobacterial adherence factors defined by strain genotype.

Analysis of acids and degradation products related to iron and sulfur in the Swedish warship Vasa

Abstract Aqueous wood extracts from the historic Swedish warship Vasa have been analyzed by 1H-NMR spectroscopy, ion chromatography, and matrix assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry as part of studies on the chemical degradation related to increased levels of iron and sulfur. The results show that low molecular organic acids have accumulated in the Vasa wood after the 1961 salvage. The increased acidity was found in a context of chemical degradation of the wood polymers and the conservation agent polyethylene glycol (PEG) in iron-rich parts of the timber. Formic, glycolic and oxalic acid are all possible end-products of oxidative degradation of wood polymers, whereas hydrolysis of acetyl groups in xylan may have contributed to increased concentrations of acetic acid. MALDI-TOF spectra of PEG displaced towards low-molecular PEG oligomers, as reported earlier, were accompanied by increased levels of formic acid, indicating oxidative degradation of PEG. PEG with a carboxylic acid end group (PEGC) was observed to a minor degree in the wood. However, analysis of stored conservation treatment solutions showed high concentrations of PEGC yielding significant contributions to the acidity during the 1960s conservation period. PEGC was probably formed as a result of microbial processes during the early conservation regime. Calculations using concentrations and well-established acidity constants show that oxalic and formic acid are the primary contributors to a low pH in the wood. The increased acidity in the interior of the wood was found in the absence of sulfur compounds but in a context of iron. The majority of the sub-samples with significant levels of sulfate in the surface region with a prospective sulfur oxidation, however, showed neither a decreased pH nor significant depolymerization. This indicates that oxidation pathways of organically bound sulfur do not necessarily produce strong acids, and thereby free protons, as the final product. These observations imply opposing effects of iron and reduced sulfur species, with iron acting as a initiator in oxidative reactions of Fenton type, whereas the reduced organic sulfur compounds may act as anti-oxidants.

Spectroscopic detection and identification of infected cells with herpes viruses

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and Fourier transform infrared (FTIR) microspectroscopy were previously applied for the identification of various biological samples. In the present study, normal cells in culture and cells infected with herpes simplex virus type 2 (HSV-2) or varicella-zoster virus (VZV) were analyzed by MALDI-TOF and FTIR microscopy. Specific spectral biomarkers for rapid and reliable monitoring and identification of infected cells and probably for the discrimination between these viruses were searched. The results show consistent spectral peaks in all examined normal uninfected human fibroblast cells both in MALDI-T0F and FTIR microscopy. In HSV-2- or VZV-infected cells, two unique peaks for each appeared at m/z 5397 and 5813 or at m/z 3501 and 4951, respectively, in MALDI-TOF spectra. In addition, several peaks that appeared in control uninfected cells at the region m/z 13,000-20,000 disappeared completely in all examined infected samples. When these infected cells were examined by FTIR microscopy, a band at 859 cm(-1) in control uninfected cells was significantly shifted to 854 cm(-1) in both HSV2- and VZV-infected cells. In addition, phosphate levels were considerably increased in all infected cells compared to normal uninfected cells. These parameters could be used as a basis for developing a spectral method for the detection and identification of cells infected with herpes viruses.

Empty Class II Major Histocompatibility Complex Created by Peptide Photolysis Establishes the Role of DM in Peptide Association

DM catalyzes the exchange of peptides bound to Class II major histocompatibility complex (MHC) molecules. Because the dissociation and association components of the overall reaction are difficult to separate, a detailed mechanism of DM catalysis has long resisted elucidation. UV irradiation of DR molecules loaded with a photocleavable peptide (caged Class II MHC molecules) enabled synchronous and verifiable evacuation of the peptide-binding groove and tracking of early binding events in real time by fluorescence polarization. Empty DR molecules generated by photocleavage rapidly bound peptide but quickly resolved into species with substantially slower binding kinetics. DM formed a complex with empty DR molecules that bound peptide with even faster kinetics than empty DR molecules just having lost their peptide cargo. Mathematical models demonstrate that the peptide association rate of DR molecules is substantially higher in the presence of DM. We therefore unequivocally establish that DM contributes directly to peptide association through formation of a peptide-loading complex between DM and empty Class II MHC. This complex rapidly acquires a peptide analogous to the MHC class I peptide-loading complex.

Serum Biomarkers to Differentiate Benign and Malignant Mammographic Lesions

Mammographic screening has increased detection of earlier-stage breast cancers and has decreased mortality from breast cancer. A Breast Imaging-Reporting and Data System (BIRADS) 4 classification prompts a biopsy that most often reveals benign disease. Our objective was to determine if serum protein-expression profiles could be used to differentiate between benign and malignant mammographic lesions. After IRB approval, women undergoing an image-guided biopsy for a BIRADS category 4 lesion were recruited. Serum was collected prebiopsy and labeled retrospectively after final pathology was reviewed. Serum was incubated with weak cation exchange magnetic beads and assayed in duplicate for analysis on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) instrumentation (Bruker Daltonics). Spectra were analyzed using ClinProTools 2.0 software (Bruker Daltonics), and classifications determined using a genetic-clustering algorithm. In a 14-month period, 260 subjects were recruited into this study. Sera from 92 subjects were randomly selected to form benign (n = 46) and cancer (n = 46) cohorts. The MALDI-TOF spectra analysis yielded 273 peaks, with 14 peaks expressed differentially (p < 0.05) between the cancer and benign cohorts. A genetic algorithm model was generated, yielding a sensitivity of 88.3% and specificity of 85.8%. MALDI-TOF protein-expression profiles generated from BIRADS 4 sera could be used to distinguish between benign and malignant mammographic lesions.

Peak selection from MALDI-TOF mass spectra using ant colony optimization

Due to the large number of peaks in mass spectra of low-molecular-weight (LMW) enriched sera, a systematic method is needed to select a parsimonious set of peaks to facilitate biomarker identification. We present computational methods for matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) spectral data preprocessing and peak selection. In particular, we propose a novel method that combines ant colony optimization (ACO) with support vector machines (SVM) to select a small set of useful peaks. The proposed hybrid ACO-SVM algorithm selected a panel of eight peaks out of 228 candidate peaks from MALDI-TOF spectra of LMW enriched sera. An SVM classifier built with these peaks achieved 94% sensitivity and 100% specificity in distinguishing hepatocellular carcinoma from cirrhosis in a blind validation set of 69 samples. Area under the receiver operating characteristic (ROC) curve was 0.996. The classification capability of these peaks is compared with those selected by the SVM-recursive feature elimination method. Supplementary material and MATLAB scripts to implement the methods described in this article are available at http://microarray.georgetown.edu/web/files/bioinf.htm. Supplementary data are available at Bioinformatics online.

The Crucial Role of Trehalose and Structurally Related Oligosaccharides in the Biosynthesis and Transfer of Mycolic Acids in Corynebacterineae

Trehalose (alpha-D-glucopyranosyl-alpha'-D-glucopyranoside) is essential for the growth of the human pathogen Mycobacterium tuberculosis but not for the viability of the phylogenetically related corynebacteria. To determine the role of trehalose in the physiology of these bacteria, the so-called Corynebacterineae, mutant strains of Corynebacterium glutamicum unable to synthesize trehalose due to the knock-out of the genes of the three pathways of trehalose biosynthesis, were biochemically analyzed. We demonstrated that the synthesis of trehalose under standard conditions is a prerequisite for the production of mycolates, major and structurally important constituents of the cell envelope of Corynebacterineae. Consistently, the trehalose-less cells also lack the cell wall fracture plane that typifies mycolate-containing bacteria. Importantly, however, the mutants were able to synthesize mycolates when grown on glucose, maltose, and maltotriose but not on other carbon sources known to be used for the production of internal glucose phosphate such as fructose, acetate, and pyruvate. The mycoloyl residues synthesized by the mutants grown on alpha-D-glucopyranosyl-containing oligosaccharides were transferred both onto the cell wall and free sugar acceptors. A combination of chemical analytical approaches showed that the newly synthesized glycolipids consisted of 1 mol of mycolate located on carbon 6 of the non reducing glucopyranosyl unit. Additionally, experiments with radioactively labeled trehalose showed that the transfer of mycoloyl residues onto sugars occurs outside the plasma membrane. Finally, and in contradiction to published data, we demonstrated that trehalose 6-phosphate has no impact on mycolate synthesis in vivo.

Mass spectroscopic and IR spectroscopic evaluation of abnormal biological samples

Biomedical application of spectroscopic methods is on the rise in the last few years. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and microscopic Fourier transform infrared spectroscopy (FTIR) were applied for the analysis of human blood samples in order to detect spectral peaks which might serve as biomarkers for monitoring and identification of specific diseases. In the present study, samples of serum were obtained from healthy and patient persons suffering from diarrhea and analyzed by MALDI-TOF and advanced FTIR microscopy. The results show consistent spectral peaks in all examined serums obtained from healthy persons when tested in MALDI-TOF on the one hand, and in FTIR microscopy on the other hand. In all tested patient samples, two unique interesting peaks appeared at m/ z 11,553 and 11,710 in MALDI-TOF spectra. When these patients samples were examined by FTIR microscopy, a peak at area position near 1600 cm −1 completely disappeared in FTIR spectra. These parameters could be used as a basis for developing a spectral method for the detection and identification of specific human diseases and probably other disorders.

MP048 Mapping of the transcription start site for the hem operon

Biofilm formation, intra-osteoblastic persistence, small-colony variants (SCVs) and the dysregulation of agr, the major virulence regulon, are possibly involved in staphylococcal bone and joint infection (BJI) pathogenesis. We aimed to investigate the contributions of these mechanisms among a collection of 95 Staphylococcus aureus clinical isolates from 64 acute (67.4%) and 31 chronic (32.6%) first episodes of BJI. The included isolates were compared for internalization rate, cell damage and SCV intracellular emergence using an ex vivo model of human osteoblast infection. Biofilm formation was assessed in a microbead immobilization assay (BioFilm Ring test). Virulence gene profiles were assessed by DNA microarray. Seventeen different clonal complexes were identified among the screened collection. The staphylococcal internalization rate in osteoblasts was significantly higher for chronic than acute BJI isolates, regardless of the genetic background. Conversely, no differences regarding cytotoxicity, SCV emergence, biofilm formation and virulence gene distribution were observed. Additionally, agr dysfunction, detected by the lack of delta-toxin production using whole-cell matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis (n = 15; 15.8%), was significantly associated with BJI chronicity, osteoblast invasion and biofilm formation. These findings provide new insights into MSSA BJI pathogenesis, suggesting the correlation between chronicity and staphylococcal osteoblast invasion. This adaptive mechanism, along with biofilm formation, is associated with agr dysfunction, which can be routinely assessed by delta-toxin detection using MALDI-TOF spectrum analysis, possibly providing clinicians with a diagnostic marker of BJI chronicity at the time of diagnosis.

Laser-induced decomposition of 2,2-dimethoxy-2-phenylacetophenone and benzoin in methyl methacrylate homopolymerization studied via matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Pulsed laser polymerization (PLP) experiments were performed on the bulk polymerization of methyl methacrylate (MMA) at −34 °C. The aim of this study was to investigate the polymer end groups formed during the photoinitiation process of MMA monomer using 2,2-dimethoxy-2-phenylacetophenone (DMPA) and benzoin as initiators via matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Analysis of the MALDI-TOF spectra indicated that the two radical fragments generated upon pulsed laser irradiation show markedly different reactivity toward MMA: whereas the benzoyl fragment—common to both DMPA and benzoin—clearly participates in the initiation process, the acetal and benzyl alcohol fragments cannot be identified as end groups in the polymer. The complexity of the MALDI-TOF spectrum strongly increased with increasing laser intensity, this effect being more pronounced in the case of benzoin. This indicates that a cleaner initiation process is at work when DMPA is used as the photoinitiator. In addition, the MALDI-TOF spectra were analyzed to extract the propagation-rate coefficient, kp, of MMA at −34 °C. The obtained value of kp = 43.8 L mol−1 s−1 agrees well with corresponding numbers obtained via size exclusion chromatography (kp = 40.5 L mol−1 s−1). © 2002 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 40: 675–681, 2002; DOI 10.1002/pola.10150

Proteomic analysis of shrimp white spot syndrome viral proteins and characterization of a novel envelope protein VP466.

White spot syndrome virus (WSSV) is at present one of the major pathogens in shrimp culture worldwide. The complete genome of this virus has been sequenced recently. To identify the structural and functional proteins of WSSV, the purified virions were separated by SDS-PAGE. Twenty-four protein bands were excised, in-gel digested with trypsin, and subjected to matrix-assisted laser desorption ionization-time of flight mass spectrometry and electrospray ionization tandem mass spectrometry, respectively. Eighteen proteins matching the open reading frames of WSSV genome were identified. Except for three known structural proteins and collagen, the functions of the remaining 14 proteins were unknown. Temporal analysis revealed that all the genes were transcribed in the late stage of WSSV infection except for vp121. Of the newly identified proteins, VP466 (derived from band 16) was further characterized. The cDNA encoding VP466 was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein. Specific antibody was generated with the purified GST-VP466 fusion protein. Western blot showed that the mouse anti-GST-VP466 antibody bound specifically to a 51-kDa protein of WSSV. Immunogold labeling revealed that VP466 protein is a component of the viral envelope. Results in this investigation thus proved the effectiveness of proteomic approaches for discovering new proteins of WSSV.

Four Common Mutations of the Cystathionine β-Synthase Gene Detected by Multiplex PCR and Matrix-assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry

A deficiency of cystathionine beta-synthase (CBS) is the most frequent cause of homocystinuria. The effect of therapy is related to the underlying CBS genotype, which makes early diagnosis of this genetic defect important. Our aim was to develop a fast and reliable method based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the determination of common mutations of the CBS gene. We used MALDI-TOF mass spectrometry to detect four common CBS mutations (G307S, T272M, I278T, and V320A). The method is based on multiplex PCR of exons 7, 8, and 9, followed by single nucleotide extension in the presence of dideoxy NTPs of four primers targeted at the separate mutation sites. The extension products, as well as the 3-hydroxypicolinic acid matrix, were incubated with cation-exchange beads to remove disturbing salt contaminants. The above-mentioned mutations were determined in samples from 12 homocystinuria patients. The MALDI-TOF spectra allowed unambiguous discrimination between primers and extension products (>9 Da) in the mass range between 4500 and 7500 Da. No labeled primers or ddNTPs were required. The genotyping was verified by reference technique. Our results demonstrate fast, simple, and unambiguous multiplex genotyping of four common CBS mutations by MALDI-TOF mass spectrometry.

Novel Procedure for Molecular Weight Averages Measurement of Polydisperse Polymers Directly from Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectra

A new method for the determination of reliable molecular weight averages (MW) and polydispersity index (MWD) values for polydisperse polymers, directly analyzing their matrix-assisted laser/desorption ionization time-of-flight (MALDI-TOF) mass spectra, is reported. Our success is due to the concerted effect of an off-line procedure of MALDI-TOF spectrum processing that includes: (i) signal smoothing to improve the recorded traces of the spectra up to high m/z values; (ii) a subtraction of the misleading contribution to the signals introduced by the offset used in spectrum recording; (iii) examination of the spectra up to the highest possible m/z value for a better accuracy in estimating the MW values. To illustrate this method, three polymers with a wide polydispersity index, a polydimethylsiloxane, a polymethylmethacrylate, and a bisphenol-A copolyether, have been analyzed. Contrary to the general opinion that MW values can be measured directly by MALDI-TOF only for polymers with a very narrow mass distribution, the data reported here show that our off-line procedure is also able to measure reliable MW values for polydisperse materials.