Liquid chromatographic determination of 2-thioxothiazolidine-4-carboxylic acid isolated from urine by affinity chromatography on organomercurial agarose gel

Urinary 2-thioxothiazolidine-4-carboxylic acid is a useful indicator to assess the degree of occupational exposure to carbon disulfide. A new procedure is described for the isolation of this compound from urine prior to reversed-phase high-performance liquid chromatography. It is based on liquid-liquid extraction with methyl tert-butyl ether, followed by affinity chromatography on organomercurial agarose gel. 5-Carboxythiouracil is used as internal standard. The superior selectivity of affinity chromatography for the isolation of 2-thioxothiazolidine-4-carboxylic acid from urine permits an isocratic high-performance liquid chromatographic analysis. The total recovery of 2.5 mg of 2-thioxothiazolidine-4-carboxylic acid/g of creatinine in spiked urine by liquid-liquid extraction combined with affinity chromatography was 48.0% (SD 2.0%, n = 8). Within-run and within-day relative standard deviations averaged 4.0% (means = 2.48 mg/g of urinary creatinine, n = 9) and 6.5% (means = 1.19 mg/g of urinary creatinine, n = 15), respectively. The detection limit of the method was estimated at 0.05 mg of 2-thioxothiazolidine-4-carboxylic acid/g of urinary creatinine. The identity and spectral purity of 2-thioxothiazolidine-4-carboxylic acid detected in the urine extracts were confirmed by diode-array UV-vis detection. Typical 2-thioxothiazolidine-4-carboxylic acid levels in individual workers exposed to carbon disulfide ranged from nondetectable to 11 mg/g of urinary creatinine, several of which exceeded the generally accepted biological exposure index of 5 mg/g of urinary creatinine.     

Enrichment and identification of cysteine-containing peptides from tryptic digests of performic oxidized proteins by strong cation exchange LC and MALDI-TOF/TOF MS

The extreme complexity of sample and uninformative fragmentation of peptides in MS/MS experiments are two of several real challenges faced by proteomics. In this work, a strategy aimed at tackling these two problems is presented. Briefly, proteins were first oxidized by performic acid to cleave the disulfide bonds and simultaneously convert cysteine residue into its sulfonic form. Then the resultant sulfonic peptides were enriched by SCX chromatography, exploiting the negative solution charge of sulfonic group. The sulfonic peptide could be easily detected by MALDI-MS in negative mode and showed both enhanced fragmentation efficiency and a simplified spectrum in MALDI-MS/MS experiment in positive mode. The strength of the strategy was demonstrated by applying it to bovine serum albumin. Potential use of the strategy in proteomics was also discussed.     

Enrichment of cysteine-containing peptides from tryptic digests using a quaternary amine tag

A new strategy for specifically targeting cysteine-containing peptides in a tryptic digest is described. The method is based on quantitatively derivatizing cysteine residues with a quaternary amine tag (QAT). Tags were introduced into proteins following reduction of disulfide bonds through derivatization of cysteine residues with (3-acrylamidopropyl)trimethylammonium chloride. After trypsin digestion, derivatized cysteine-containing peptides were enriched by strong cation exchange chromatography. The method was validated using model peptides and a protein. The QAT strategy has several advantages over other methods for the selection of cysteine-containing peptides. One is that it increases the ionization efficiency of cysteine-containing peptides. The other is that chromatographic selection is achieved with simple, robust cation exchange chromatography columns. As a result, this new strategy provides a simple way to facilitate enrichment of cysteine-containing peptides, thereby reducing sample complexity in bottom-up proteomics.     

Characterization of microorganisms and biomarker development from global ESI-MS/MS analyses of cell lysates

The capability for sensitive and accurate identification of microorganisms has potential applications that include the monitoring of industrial bioprocessing operations, food safety analyses, disease diagnosis, and detection of potential biological hazards. Efforts based upon matrix-assisted laser desorption/ionization mass spectrometry to detect and identify specific microorganisms have been actively pursued for several years. We report a new method being developed to select useful biomarkers for the identification of microorganisms based upon electrospray ionization (ESI)-ion trap mass spectrometry. Crude cell lysates are processed using a recently developed dualmicrodialysis device and then directly infused into an ion trap MS. The low ESI flow rate and precursor ion accumulation capability of the ion trap MS enables high-sensitivity MS/MS analyses. Precursor ions are automatically selected and analyzed using tandem MS (MS/MS) to produce "global" MS/MS surveys and processed to yield two-dimensional MS/MS spectral displays. Such global MS/MS surveys are demonstrated for Escherichia coli lysates. The distinctive MS/MS spectral patterns can be used to identify mass spectrometric-detected species useful as biomarkers, which then provide a basis for confident microorganism identification. The results presented demonstrate the application of this method for the identification of microorganisms, as well as for detection of bacteriophage MS2 in the presence of a large excess of E. coli.     

InsPecT: identification of posttranslationally modified peptides from tandem mass spectra

Reliable identification of posttranslational modifications is key to understanding various cellular regulatory processes. We describe a tool, InsPecT, to identify posttranslational modifications using tandem mass spectrometry data. InsPecT constructs database filters that proved to be very successful in genomics searches. Given an MS/MS spectrum S and a database D, a database filter selects a small fraction of database D that is guaranteed (with high probability) to contain a peptide that produced S. InsPecT uses peptide sequence tags as efficient filters that reduce the size of the database by a few orders of magnitude while retaining the correct peptide with very high probability. In addition to filtering, InsPecT also uses novel algorithms for scoring and validating in the presence of modifications, without explicit enumeration of all variants. InsPecT identifies modified peptides with better or equivalent accuracy than other database search tools while being 2 orders of magnitude faster than SEQUEST, and substantially faster than X!TANDEM on complex mixtures. The tool was used to identify a number of novel modifications in different data sets, including many phosphopeptides in data provided by Alliance for Cellular Signaling that were missed by other tools.     

Facile identification of phosphorylation sites in peptides by radical directed dissociation

Identification of phosphorylation sites is of interest due to their importance in protein regulation; however, the identification of the exact sites of this modification is not always easily obtained due to the dynamic nature of phosphorylation and the challenges faced during mass spectrometric analysis. Herein we elaborate on our previous communication (Diedrich, J. K.; Julian, R. R. J. Am. Chem. Soc. 2008, 130, 12212-12213) describing a novel technique for assignment of phosphorylation in a site-specific and facile manner. Phosphorylation sites are selectively modified through β elimination followed by Michael addition chemistry to install a photolabile group. Photodissociation with 266 nm light yields homolytic cleavage at the modification site, generating a β radical which is poised to fragment the peptide backbone. Dissociation primarily yields d-type ions at the previously phosphorylated residue, allowing facile identification. Radical directed fragmentation also occurs in smaller abundances at neighboring residues. The mechanisms behind this selective radical fragmentation are presented and the utility is discussed. Fragmentation is shown to be independent of charge state allowing analysis of a wide variety of peptide sequences including peptides with multiple phosphorylation sites. A comparison of this technique is made with collision induced dissociation (CID) and electron capture dissociation (ECD) for representative peptides.     

Enrichment of carbonylated peptides using Girard P reagent and strong cation exchange chromatography

It has been shown that oxidatively modified forms of proteins accumulate during oxidative stress, aging, and in some age-related diseases. One of the unique features of protein oxidation by a wide variety of routes is the generation of carbonyl groups. Of major interest in the study of oxidative stress diseases is which proteins in a proteome are being oxidized and the site(s) of oxidation. Based on the fact that proteins are generally characterized through tryptic peptide fragments, this paper reports a method for the isolation of oxidized peptides, which involves (1) derivatization of oxidized proteins with Girard P reagent (GRP; 1-(2-hydrazino-2-oxoethyl)pyridinium chloride), (2) following proteolysis enrichment of the derivatized peptide using strong cation exchange (SCX) chromatography, and (3) identification of oxidation sites using tandem mass spectrometry. Derivatization of aldehydes and ketones in oxidized proteins was accomplished by reacting protein carbonyls with the hydrazide of GRP. The resulting hydrazone bond was reduced by sodium cyanoborohydride to further stabilize the labeling. Derivatization time and concentrations of the derivatizing agent were optimized with model peptides. Oxidized transferrin was used as model protein to study derivatization efficiency at the protein level. Following metal-catalyzed oxidation of transferrin, the protein was derivatized with GRP and trypsin digested. Positively charged peptides were then selected from the digest with SCX chromatography at pH 6.0. Seven GRP-derivatized peptides were found to be selected from transferrin by MALDI-TOF-TOF analysis. Fourteen underivatized native peptides were also captured by the SCX column at pH 6.0. Mapping of the derivatized peptides onto the primary structure of transferrin indicated that the oxidation sites were all on solvent-accessible regions at the protein surface. Efficiency of the method was further demonstrated in the identification of oxidized proteins from yeast.     

Detection and quantification of lanthanide complexes in cell lysates by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Gadolinium (III) complexes are under intense scrutiny as contrast agents for magnetic resonance imaging. Although currently used mainly as extracellular agents, there is a growing interest to exploit their contrast enhancing ability in the intracellular environment. To ascertain the preservation of their chemical integrity upon the intracellular entrapment, it is necessary to have a method for their dosage in the cell lysates. Herein, a mass spectrometric method for detection and quantification of gadolinium complexes in cell lysates is reported. The detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was carried out by using a non-acidic matrix (2,4,6-trihydroxyacetophenone), which does not allow any leakage of gadolinium from the complex. Quantification has been possible by using as an internal standard an ytterbium complex with the same ligand of the analyte. Ytterbium was chosen because, among the lanthanides, it is the one with the isotopic distribution pattern the most similar to that of gadolinium. Sensitivity was enough to detect low micromolar quantities of a cationic complex and high micromolar quantities of a neutral complex in cell lysates of rat hepatoma cells. In the case of anionic complexes, sensitivity was too low for quantitative analysis. To the best of our knowledge, this is the first report concerning the quantification of metal complexes by MALDI-TOF-MS.     

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.     

Characterization of agarose microparticles prepared by water-in-water emulsification

Microparticles have a wide range of applications in various areas. In this study, agarose microparticles (AGM) were successfully prepared by using a water-in-water (w/w) emulsification (WWEM) technique to eliminate the use of a surfactant. An aqueous agarose solution was employed as the dispersed phase, and polyethylene glycol (PEG) was used as the continuous phase. We evaluated how the characteristics of the microparticles were affected by different processing factors, including temperature, agitation speed, stirring time, concentration of agarose, and proportions of the two phases. The agarose microparticles obtained were nearly perfect spheres, and their particle sizes decreased with an increase in the agitation speed. Physicochemical characterization suggested that agarose microparticles, due to their ubiquitous stability, could be applied in most mild environments.     

Selective enrichment of low-abundance peptides in complex mixtures by elution-modified displacement chromatography and their identification by electrospray ionization mass spectrometry

Trace components were selectively enriched and detected in the tryptic digest of recombinant human growth hormone using elution-modified displacement chromatography, a hybrid technique combining features of elution and displacement chromatography. Based on the retention behavior of sample components in the elution mode, rapid and selective trace enrichment and high-resolution separation was achieved in a single step by utilizing appropriate combinations of an eluent such as aqueous acetonitrile with the displacer. Mass spectral and chromatographic analysis of displacement zones revealed up to 400-fold enhancement of the concentration of some low-abundance sample components. Potential application of this technique in proteomics to augment the sensitivity of LC-MS and 2-D gel electrophoretic approaches for the detection of biologically important low-abundance species is discussed.     

Quantitative analysis of HIV-1 protease inhibitors in cell lysates using MALDI-FTICR mass spectrometry

In this report we explore the use of MALDI-FTICR mass spectrometry for the quantitative analysis of five HIV-1 protease inhibitors in cell lysates. 2,5-Dihydroxybenzoic acid (DHB) was used as the matrix. From a quantitative perspective, DHB is usually a poor matrix due to its poor shot-to-shot and poor spot-to-spot reproducibilities. We found that the quantitative precisions improved significantly when DMSO (dimethylsulfoxide) was added to the matrix solution. For lopinavir and ritonavir, currently the most frequently prescribed HIV-1 protease inhibitors, the signal-to-noise ratios improved significantly when potassium iodide was added to the matrix solution. The mean quantitative precisions, expressed as % relative standard deviation, were 6.4% for saquinavir, 7.3% for lopinavir, 8.5% for ritonavir, 11.1% for indinavir, and 7.2% for nelfinavir. The mean quantitative accuracies, expressed as % deviation, were 4.5% for saquinavir, 6.0% for lopinavir, 5.9% for ritonavir, 6.6% for indinavir, and 8.0% for nelfinavir. The concentrations measured for the individual quality control samples were all within 85-117% of the theoretical concentrations. The lower limits of quantification in cell lysates were 4 fmol/microL for saquinavir, 16 fmol/microL for lopinavir, 31 fmol/microL for ritonavir, and 100 fmol/microL for indinavir and nelfinavir. The mean mass accuracies for the protease inhibitors were 0.28 ppm using external calibration. Our results show that MALDI-FTICR mass spectrometry can be successfully used for precise, accurate, and selective quantitative analyses of HIV-1 protease inhibitors in cell lysates. In addition, the lower limits of quantification obtained allow clinical applications of the technique.     

High-resolution capillary isoelectric focusing of complex protein mixtures from lysates of microorganisms

High-resolution capillary isoelectric focusing separations of complex protein mixtures have been obtained for cellular lysates of Saccharomyces cerevisiae, Eschericia coli, and Deinococcus radiodurans. High quality separations are shown to be achievable for total protein concentrations of < 0.1 mg/mL. The separation reproducibility was examined, and the influence of the capillary inner wall coating on resolution investigated using fusedsilica capillaries coated with various hydrophilic polymers including hydroxypropyl cellulose, poly(vinyl alcohol), and linear polyacrylamide. Proteins having an isoelectric point (pI) difference of 0.004 are shown to be separated using a linear carrier ampholyte (linear pH gradient between two electrodes) of 3-10. Approximately 45 discrete peaks in the pI range of 5-7 were obtained for S. cerevisiae, approximately 80 peaks in the pI range of 4.5-8.5 for E. coli, and approximately 210 peaks in the pI range of 3-8.8 for D. radiodurans.     

一株酱醅中耐盐酵母Meyerozyma guilliermondii的分离鉴定及其挥发性香气成分分析

为筛选具有酱油生产应用潜能的微生物菌株,以推动传统产业与现代发酵的结合。从天然晒露法酿制酱油的第3年发酵酱醅中分离筛选到一株耐盐酵母3-J15,经鉴定为季也蒙毕赤酵母Meyerozyma guilliermondii。选用固相微萃取-气相色谱质谱联用(SPME-GC/MS)技术,对分离株在盐胁迫及非盐胁迫条件下代谢产生的挥发性香气成分进行分离测定。结果表明,从酵母3-J15发酵液中检测出包含醛、酮、醇、酸、酯在内的10种特征香气成分,其中主要的挥发性香气物质为3-甲基-1-丁醇和2-苯基乙醇。综合已有研究,这些呈香物质能够为酱油的特色风味带来正面影响,因此,酵母Meyerozyma guilliermondii具有进一步应用于酱油生产的开发潜力。     

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.     

固定化酵母菌填充柱对锶的吸附特性研究

锶为具有很强毒性的裂变产物.结合生物吸附和固定化技术的优点,用海藻酸钠-氯化钙包埋法制作固定酵母菌颗粒并填充成吸附柱,研究了固定化酵母菌颗拉的特性和在静态、动态吸附下的吸附率、吸附容量和柱层析参数.结果表明,酵母菌固定化颗粒具有较高的比表面积和较好的机械承受能力.静态吸附结果表明,填充柱具有较高的吸附容量,吸附平衡时平...     

Ultrasensitive identification of localization variants of modified peptides using ion mobility spectrometry

Localization of the modification sites on peptides is challenging, particularly when multiple modifications or mixtures of localization isomers (variants) are involved. Such variants commonly coelute in liquid chromatography and may be undistinguishable in tandem mass spectrometry (MS/MS) for lack of unique fragments. Here, we have resolved the variants of singly and doubly phosphorylated peptides employing drift tube ion mobility spectrometry (IMS) coupled to time-of-flight mass spectrometry. Even with a moderate IMS resolving power of ～80-100, substantial separation was achieved for both 2+ and 3+ ions normally generated by electrospray ionization, including for the variants indistinguishable by MS/MS. Variants often exhibit a distribution of 3-D conformers, which can be adjusted for optimum IMS separation by prior field heating of ions in a funnel trap. The peak assignments were confirmed using MS/MS after IMS separation, but known species could be identified using just the ion mobility "tag". Avoiding the MS/MS step lowers the detection limit of localization variants to <100 amol, an order of magnitude better than that provided by electron transfer dissociation in an Orbitrap MS.     

Separation and identification of peptides from gel-isolated membrane proteins using a microfabricated device for combined capillary electrophoresis/nanoelectrospray mass spectrometry

The coupling of microfabricated devices to nanoelectrospray mass spectrometers using both a triple quadrupole and a quadrupole time-of-flight mass spectrometer (QqTOF MS) is presented for the analysis of trace-level membrane proteins. Short disposable nanoelectrospray emitters were directly coupled to the chip device via a low dead volume connection. The analytical performance of this integrated device in terms of sensitivity and reproducibility was evaluated for standard peptide mixtures. A concentration detection limit ranging from 3.2 to 43.5 nM for different peptides was achieved in selected ion monitoring, thus representing a 10-fold improvement in sensitivity compared to that of microelectrospray using the same chip/mass spectrometer. Replicate injections indicated that reproducibility of migration time was typically less than 3.1% RSD whereas RSD values of 6-13% were observed on peak areas. Although complete resolution of individual components is not typically achieved for complex digests, the present chip capillary electrophoresis (chip-CE) device enabled proper sample cleanup and partial separation of multicomponent samples prior to mass spectral identification. Analyses of protein digests were typically achieved in less than 1.5 min with peak widths of 1.8-2.5 s (half-height definition) as indicated from individual reconstructed ion electropherograms. The application of this chip-CE/QqTOF MS system is further demonstrated for the identification of membrane proteins which form a subset of the Haemophilus influenzae proteome. Bands first separated by 1D-gel electrophoresis were excised and digested, and extracted tryptic peptides were loaded on the chip without any further sample cleanup or on-line adsorption preconcentration. Accurate molecular mass determination (< 5 ppm) in peptide-mapping experiments was obtained by introducing an internal standard via a postseparation channel. The analytical potential of this integrated device for the identification of trace-level proteins from different strains of H. influenzae is demonstrated using both peptide mass-fingerprint database searching and on-line tandem mass spectrometry.     

Fractionation of SWNT/nucleic acid complexes by agarose gel electrophoresis

We show that aqueous dispersions of single-walled carbon nanotubes (SWNTs), prepared with the aid of nucleic acids (NAs) such as RNA or DNA, can be separated into fractions using agarose gel electrophoresis. In a DC electric field, SWNT/NA complexes migrate in the gel in the direction of positive potential to form well-defined bands. Raman spectroscopy as a function of band position shows that nanotubes having different spectroscopic properties possess different electrophoretic mobilities. The migration patterns for SWNT/RNA and SWNT/DNA complexes differ. Parallel elution of the SWNT/NA complexes from the gel during electrophoresis and subsequent characterization by AFM reveals differences in nanotube diameter, length and curvature. The results suggest that fractionation of nanotubes can be achieved by this procedure. We discuss factors affecting the mobility of the nanotube complexes and propose analytical applications of this technique.