Identification of biomarkers of whole Coxiella burnetii phase I by MALDI-TOF mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) specific biomarkers have been shown to be an effective tool for identifying microorganisms. In this study, we demonstrate the feasibility of using this technique to detect the obligate intracellular bacterium Coxiella burnetii, a category B bioterrorism agent. Specific biomarkers were detected in C. burnetii Nine Mile phase I (NMI) strain purified from embryonated egg yolk sac preparations. Whole organisms were applied directly to the MALDI target. MALDI-TOF MS analysis of C. burnetii NMI grown and purified at different times and places revealed a group of unique, characteristic, and reproducible spectral markers in the mass range of 1000-25000 Da. Statistical analysis of the averaged centroided masses uncovered at least 24 peptides or biomarkers. Three biomarkers observed in the MALDI-TOF MS spectrum consistently matched proteins that had been previously described in C. burnetii, one of them being the small cell variant protein A. MALDI-TOF MS analysis of whole organisms represents a sensitive and specific option for characterizing C. burnetii isolates, especially when coupled with antigen capture techniques. The method also has potential for several applications in basic microbial research, including regulation of gene expression.     

Liquid chromatographic analysis and mass spectrometric identification of farnesylated peptides

Farnesylation involves the post-translational attachment of a 15 carbon unit to the C-terminus of proteins, thus allowing them to incorporate into membranes. The farnesylation reaction requires farnesyldiphosphate as the farnesyl group donor and is catalyzed by the farnesyltransferase. Some of the most familiar farnesylated proteins belong to the Ras protein superfamily, well-known oncoproteins. As Ras proteins require the membrane localization for the transduction of extracellular signals, farnesyltransferase inhibitors are discussed as chemotherapeutic agents. Despite the importance of this post-translational modification, farnesylated peptides have been investigated rarely by means of high-pressure liquid chromatography in combination with mass spectrometry. In this study, we examined the liquid chromatographic separation of farnesylated peptides with the help of the multidimensional protein identification technology. The peptides were further ionized by electrospray ionization and subsequently analyzed by tandem mass spectrometry. We demonstrated that farnesylated peptides are more strongly retained by reversed phase than nonfarnesylated peptides. This allowed for the identification of farnesylated peptides, if spiked into complex peptide samples. In some cases the farnesyl group was apparently split off from the peptide during the ionization process, and tandem mass spectra often revealed a neutral loss of the farnesyl moiety.     

Efficient identification of phosphorylation by mass spectrometric phosphopeptide fingerprinting

We describe a rapid and efficient method for the identification of phosphopeptides, which we term mass spectrometric (MS) phosphopeptide fingerprinting. The method involves quantitative comparison of proteolytic peptides from native versus completely dephosphorylated proteins. Dephosphorylation of serine, threonine, and tyrosine residues is achieved by in-gel treatment of the separated proteins with hydrogen fluoride (HF). This chemical dephosphorylation results in enrichment of those unmodified peptides that correspond to previously phosphorylated peptides. Quantitative comparison of the signal-to-noise ratios of peaks in the treated versus untreated samples are used to identify phosphopeptides, which can be confirmed and further studied by tandem mass spectrometry (MS/MS). We have applied this method to identify eight known phosphorylation sites of Xenopus Aurora A kinase, as well as several novel sites in the Xenopus chromosome passenger complex (CPC).     

Isotopic analyses based on the mass spectrum of carbon dioxide

Measurements of carbon and oxygen isotopic abundances are commonly based on the mass spectrum of carbon dioxide, but analysis of that spectrum is not trivial because three isotope ratios (17O/16O, 18O/16O, and 13C/12C) must be determined from only two readily observable ion-current ratios (45/44 and 46/44). Here, approaches to the problem are reassessed in the light of new information regarding the distribution of oxygen isotopes in natural samples. It is shown that methods of calculation conventionally employed can lead to systematic errors in the computed abundance of 13C and that these errors may be related to incorrect assessment of the absolute abundance of 17O. Further, problems arising during the analysis of samples enriched by admixture of 18O-labeled materials are discussed, and it is shown (i) that serious inaccuracies arise in the computed abundance of 17O and 13C if methods of calculation conventionally employed in the analysis of natural materials are applied to material labeled with 18O but (ii) that computed fractional abundances of 18O are always within 0.4% of the correct result. Methods for exact calculation of two isotope ratios when the third is known are presented and discussed, and a more exact approach to the computation of all three isotope ratios in natural materials is given.     

A statistical basis for testing the significance of mass spectrometric protein identification results

A method for testing the significance of mass spectrometric (MS) protein identification results is presented. MS proteolytic peptide mapping and genome database searching provide a rapid, sensitive, and potentially accurate means for identifying proteins. Database search algorithms detect the matching between proteolytic peptide masses from an MS peptide map and theoretical proteolytic peptide masses of the proteins in a genome database. The number of masses that matches is used to compute a score, S, for each protein, and the protein that yields the best score is assumed as the identification result. There is a risk of obtaining a false result, because masses determined by MS are not unique; i.e., each mass in a peptide map can match randomly one or several proteins in a genome database. A false result is obtained when the score, S, due to random matching cannot be discerned from the score due to matching with a real protein in the sample. We therefore introduce the frequency function, f(S), for false (random) identification results as a basis for testing at what significance level, alpha, one can reject a null hypothesis, H0: "the result is false". The significance is tested by comparing an experimental score, S(E), with a critical score, S(C), required for a significant result at the level alpha. If S(E) > or = S(C), H0 is rejected. f(S) and S(C) were obtained by simulations utilizing random tryptic peptide maps generated from a genome database. The critical score, S(C), was studied as a function of the number of masses in the peptide map, the mass accuracy, the degree of incomplete enzymatic cleavage, the protein mass range, and the size of the genome. With S(C) known for a variety of experimental constraints, significance testing can be fully automated and integrated with database searching software used for protein identification.     

High-affinity capture of proteins by diamond nanoparticles for mass spectrometric analysis

Carboxylated/oxidized diamond nanoparticles (nominal size 100 nm) exhibit exceptionally high affinity for proteins through both hydrophilic and hydrophobic forces. The affinity is so high that proteins in dilute solution can be easily captured by diamonds, simply separated by centrifugation, and directly analyzed by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry (MS). No preseparation of the adsorbed molecules from diamonds is required for the mass spectrometric analysis. Compared to conventional MALDI-TOF-MS, an enhancement in detection sensitivity by more than 2 orders of magnitude is achieved for dilute solution containing cytochrome c, myoglobin, and albumin because of preconcentration of the probed molecules. The lowest concentration detectable is 100 pM for a 1-mL solution. Aside from the enhanced sensitivity, the overall performance of this technique does not show any sign of deterioration for highly contaminated protein solutions, and furthermore, no significant peak broadening and band shift were observed in the mass spectra. The promise of this new method for clinical proteomics research is demonstrated with an application to human blood serum.     

Selective enrichment and mass spectrometric identification of nitrated peptides using fluorinated carbon tags

Protein tyrosine nitration (PTN) is a post-translational modification that is related to several acute or chronic diseases. PTN introduces a nitro group in the ortho position of the phenolic hydroxyl group of tyrosine residues. PTN has been shown to be involved in the pathogenesis of inflammatory responses, cancers, and neurodegenerative and age-related disorders. Furthermore, it has been proposed that PTN regulates signal cascades related to nitric oxide (NO·) production and NO-mediated processes. Although nitrated proteins as markers of oxidative stress are confirmed by immunological assays in various affected cells or tissues, it is not known how many different types of proteins in living cells are nitrated. Since protein nitration is a low-abundance post-translational modification, development of an effective enrichment method for nitrated proteins is needed to detect nitrated peptides or proteins from the limited amount of pathophysiological samples. In the present study, we developed an enrichment method using specific chemical tagging. Nitroproteome profiling using chemical tagging and mass spectrometry was validated by model proteins. Furthermore, we successfully identified numerous nitrated proteins from the Huh7 human hepatoma cell line.     

Use of vapor-phase acid hydrolysis for mass spectrometric peptide mapping and protein identification

A method for mass spectrometric peptide mapping was developed, based on hydrolysis of a solid protein by acid vapor followed by mass spectrometric analysis of the cleavage products. The method is applicable to lyophilized samples as well as proteins present in gels after separation by SDS-PAGE. The cleavage specificity was established using a number of standard proteins. Three different types of cleavages were observed: specific internal backbone cleavages at Asp, Ser, Thr, and Gly and N- and C-terminal sequence ladders. On the basis of the observed cleavage characteristics, a strategy for protein identification based on the peptide mass maps was developed. The identification strategy utilizes the specific internal backbone cleavages as well as the partial sequence information, obtained from the sequence ladders.     

基于移动质量-频率曲线的结构损伤识别方法

针对土木工程中结构实测得到的模态信息少,结构损伤识别方法对噪声敏感且依赖高精确度有限元模型等问题,提出利用移动质量-频率曲线识别结构损伤的方法。首先,在结构上附加移动质量块,获得结构频率关于附加质量位置的关系曲线;然后,建立结构近似有限元模型,通过计算实际模型与近似有限元模型之间的质量-频率曲线的相关性,建立目标函数进行优化;最后,利用变截面简支梁结构进行数值模拟。结果表明:在模型存在较大误差情况下,该方法仍能够准确识别损伤位置,验证其具有较高的适用性。     

Mass spectrometric methods for generation of protein mass database used for bacterial identification

The availability of a suitable database is critical in a proteomic approach for bacterial identification by mass spectrometry (MS). The major limitation of the present public proteome database is the lack of extensive low-mass bacterial protein entries with masses experimentally verified for most bacteria. Here, we present a method based on mass spectrometry to create protein mass tables specifically tailored for bacterial identification. Several issues related to the detection of bacterial proteins for the purpose of database creation are addressed. Three species of bacteria, namely, Escherichia coli, Bacillus megaterium, and Citrobacter freundii, which can be found in the ambient environment, were chosen for this study. Direct matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis of each bacterial extract reveals 20-29 protein components in the mass range from 2000 to 20,000 Da. HPLC fractionation of bacterial extracts followed by off-line MALDI-TOF analysis of individual fractions detects 156-423 components. Analysis of the extracts by HPLC/electrospray ionization MS shows the number of detectable proteins in the range of 46-59. Although a number of components were common to the three detection schemes employed, some unique components were found using each of these techniques. In addition, for E. coli where a large proteome database exists in the public domain, a number of masses detected by the mass spectrometric methods do not match with the proteome database. Compared to the public proteome database, the mass tables generated in this work are demonstrated to be more useful for bacterial identification in an application where the bacteria of interest have limited protein entries in the public database. The implication of this work for future development of a comprehensive mass database is discussed.     

基于虚拟变形法的移动质量识别方法研究

在车-桥耦合系统的移动质量(荷载)识别反问题中,识别移动质量会面临重构系统、优化缓慢的问题;而若直接识别移动荷载常常会遇到病态问题且对噪音敏感。针对这些缺陷,根据虚拟变形法(VDM)的结构快速重分析思想,提出移动动态影响矩阵,实现利用较少的传感器即可快速而准确地识别移动质量(荷载)。以移动质量为优化变量,避免了识别荷载常遇到的病态问题,对噪音鲁棒性强;且需要传感器信息少。每步优化中,利用移动动态影响矩阵,无需时时重构车-桥耦合系统的时变系统参数矩阵,优化效率高。VDM方法的思想是将实际结构的响应计算转化为初始结构模型在相同外荷载作用下的响应,与在结构模型发生改变的位置施加相关的虚拟变形或虚拟力引起的响应的线性叠加。通过简支梁模型和框架梁模型验证了该方法的可行性和有效性,即使在5%的噪声影响下,利用一个传感器就可以很好地识别多个移动质量。     

Comparison of different mass spectrometric techniques combined with liquid chromatography for confirmation of pesticides in environmental water based on the use of identification points

Three mass spectrometric techniques have been used and compared for the confirmation of the presence of several pesticides that had been detected in environmental water samples by a previously reported SPE-LC-MS/MS screening method. The 2002/657/EC European Comission Decision establishes the need to obtain at least three identification points (IPs) in order to confirm organic residues and contaminants in live animals and animal products. In this paper, a similar approach has been applied for confirmation of pesticides in water samples, using triple quadrupole mass spectrometry (QqQ), time-of-flight mass spectrometry (TOF), and hybrid quadrupole time-of-flight mass spectrometry (QTOF) to achieve the required IPs. The number of IPs collected, the sensitivity, and the practical advantages and disadvantages of these techniques have been discussed. In summary, the QqQ instrument allowed the confirmation of detected pesticides even at very low concentrations (ng/L) achieving between four and five IPs when adding confirmatory transitions. The direct confirmation with a TOF instrument was only feasible for those compounds showing sufficient sensitivity, isotopic pattern, or easy in-source fragmentation. In other cases, the required IPs could be reached by adding IPs earned with this technique to those obtained from the MS/MS screening method. Finally, the use of a QTOF instrument allowed obtaining up to 20 IPs in a single run at relatively high concentrations (submicrograms per liter) as no "ion shopping" was required. Additionally, the application of TOF and QTOF techniques made it possible to detect some nontarget organic contaminants, which were not included in the screening method.     

A robust, detergent-friendly method for mass spectrometric analysis of integral membrane proteins

Recent breakthroughs in the high-resolution structural elucidation of ion channels and transporters are prompting a growing interest in methods for characterizing integral membrane proteins. These methods are proving extremely valuable in facilitating the production of X-ray diffraction-grade crystals. Here we present a robust and straightforward mass spectrometric procedure that utilizes matrix-assisted laser desorption/ionization to analyze integral membrane proteins in the presence of detergents. The utility of this method is illustrated with examples of high-quality mass spectral data obtained from membrane proteins for which atomic resolution structural studies are ongoing.     

Genetic identification by mass spectrometric analysis of single-nucleotide polymorphisms: ternary encoding of genotypes

An approach to genetic identification using biallelic single-nucleotide polymorphism (SNP) genetic markers is described in which the three possible genotypes, AA, Aa, or aa, where "A" and "a" represent the two SNP alleles, are assigned a ternary (base 3) digit of 0, 1, or 2, respectively. Genotyping an individual over a panel of separate SNP markers produces a composite ternary genetic code that can be converted to an easily stored, decimal (base 10) genetic identification number. The unambiguous identification of 11 individuals is demonstrated using ternary genetic codes generated from MALDI-TOF mass spectrometric genotyping data from 7 different SNP markers.     

Parallel detection of intrinsic fluorescence from peptides and proteins for quantification during mass spectrometric analysis

Direct mass spectrometric quantification of peptides and proteins is compromised by the wide variabilities in ionization efficiency which are hallmarks of both the MALDI and ESI ionization techniques. We describe here the implementation of a fluorescence detection system for measurement of the UV-excited intrinsic fluorescence (UV-IF) from peptides and proteins just prior to their exit and electrospray ionization from an ESI capillary. The fluorescence signal provides a quantifiable measure of the amount of protein or peptide present, while direct or tandem mass spectrometric analysis (MS/MS) on the ESI-generated ions provides information on identity. We fabricated an inexpensive, modular fluorescence excitation and detection device utilizing an ultraviolet light-emitting diode for excitation in a ～300 nL fluorescence detection cell integrated into the fused-silica separation column. The fluorescence signal is linear over 3 orders of magnitude with on-column limits of detection in the low femtomole range. Chromatographically separated intact proteins analyzed using UV-IF prior to top-down mass spectrometry demonstrated sensitive detection of proteins as large as 77 kDa.     

Highly sensitive immunoassay based on immunogold-silver amplification and inductively coupled plasma mass spectrometric detection

In this work, we demonstrated a highly sensitive inductively coupled plasma mass spectrometric (ICPMS) method for the determination of human carcinoembryonic antigen (CEA), which combined the inherent high sensitivity of elemental mass spectrometric measurement with the signal amplification of catalytic silver deposition on immunogold tags. The silver amplification procedure was easy to handle and required cheap reagents, and the sensitivity was greatly enhanced to 60-fold after a 15 min silver amplification procedure. The experimental conditions, including detection of gold and silver by ICPMS, immunoassay parameters, silver amplification parameters, analytical performance, and clinical serum samples analysis, were investigated. The ICPMS Ag signal intensity depends linearly on the logarithm of the concentration of human CEA over the range of 0.07-1000 ng mL(-1) with a limit of detection (LOD, 3σ) of 0.03 ng mL(-1) (i.e., 0.15 pM). The LOD of the proposed method is around 2 orders of magnitude lower than that by the widely used enzyme-linked immunosorbent assay (ELISA) and 1 order of magnitude lower than that by clinical routine chemiluminescence immunoassay (CLIA) or time-resolved fluoroimmunoassay (TRFIA) and conventional ICPMS immunoassay. The present strategy was applied to the determination of human CEA in clinical human serum samples, and the results were in good agreement with those obtained by chemiluminescence immunoassay.     

Ion-molecule reactions for mass spectrometric identification of functional groups in protonated oxygen-containing monofunctional compounds

Protonated oxygen-containing monofunctional compounds react with selected methoxyborane reagents by proton transfer followed by nucleophilic substitution of methanol at the boron atom in a Fourier transform ion cyclotron resonance mass spectrometer. The derivatized oxygen functionality can be identified by H/D exchange, collision-activated dissociation, or both. This information on the identity of the functionalities in the analyte, in conjunction with molecular formula information obtained from exact mass measurements on either the protonated or derivatized analyte, facilitates structure elucidation of unknown organic compounds in a mass spectrometer.     

Capillary high-performance liquid chromatography/mass spectrometric analysis of proteins from affinity-purified plasma membrane

Proteomics analysis of plasma membranes is a potentially powerful strategy for the discovery of proteins involved in membrane remodeling under diverse cellular environments and identification of disease-specific membrane markers. A key factor for successful analysis is the preparation of plasma membrane fractions with low contamination from subcellular organelles. Here we report the characterization of plasma membrane prepared by an affinity-purification method, which involves biotinylation of cell-surface proteins and subsequent affinity enrichment with strepavidin beads. Western blotting analysis showed this method was able to achieve a 1600-fold relative enrichment of plasma membrane versus mitochondria and a 400-fold relative enrichment versus endoplasmic reticulum, two major contaminants in plasma membrane fractions prepared by conventional ultracentrifugation methods. Capillary-HPLC/MS analysis of 30 microg of affinity-purified plasma membrane proteins led to the identification of 918 unique proteins, which include 16.4% integral plasma membrane proteins and 45.5% cytosol proteins (including 8.6% membrane-associated proteins). Notable among the identified membrane proteins include 30 members of ras superfamily, receptors (e.g., EGF receptor, integrins), and signaling molecules. The low number of endoplasmic reticulum and mitochondria proteins (approximately 3.3% of the total) suggests the plasma membrane preparation has minimum contamination from these organelles. Given the importance of integral membrane proteins for drug design and membrane-associated proteins in the regulation cellular behaviors, the described approach will help expedite the characterization of plasma membrane subproteomes, identify signaling molecules, and discover therapeutic membrane-protein targets in diseases.