Low-resolution Mass Spectrometry Research Articles

Reliable Detection of Deamidated Peptides from Lens Crystallin Proteins Using Changes in Reversed-Phase Elution Times and Parent Ion Masses

Identifying deamidated peptides using low-resolution mass spectrometry is difficult because traditional database search programs cannot accurately detect modified peptides when the mass differences are only 0.984 Da. In this study, we utilized differential reversed-phase elution behavior of deamidated and corresponding unmodified peptide forms to significantly improve deamidation detection on a low-resolution LCQ ion trap instrument. We also improved the mass measurements of unmodified and deamidated peptide forms by averaging survey scans across each chromatogram peak. Tryptic digests of a series of normal (3-day old, 2-year old, 18-year old, 35-year old, and 70-year old) and cataractous (93-year old) human lens samples were used to produce large numbers of potentially deamidated peptides. The complex peptide mixtures were separated by strong cation exchange (SCX) chromatography followed by reversed-phase (RP) chromatography. Synthetic peptides were used to show that unmodified and deamidated peptides coeluted during the SCX separation and were completely resolved with the RP conditions used. Retention time shifts (RTS) and mass differences (DeltaM) of deamidated lens peptides and their corresponding unmodified forms were manually determined for the 70-year old lens sample. These values were used to assign correct or incorrect deamidation identifications from SEQUEST searches where deamidation was specified as a variable modification. Manual validation of SEQUEST identifications from synthetic peptides, 3-day old, and 70-year old samples had an overall 42% deamidation detection accuracy. Filtering SEQUEST identifications using RTS and DeltaM constraints resulted in >93% deamidation detection accuracy. An algorithm was developed to automate this method, and 72 Crystallin deamidation sites, 18 of which were not previously reported in human lens tissue, were detected.

Improved model-based, platform-independent feature extraction for mass spectrometry

Mass spectrometry (MS) is increasingly being used for biomedical research. The typical analysis of MS data consists of several steps. Feature extraction is a crucial step since subsequent analyses are performed only on the detected features. Current methodologies applied to low-resolution MS, in which features are peaks or wavelet functions, are parameter-sensitive and inaccurate in the sense that peaks and wavelet functions do not directly correspond to the underlying molecules under observation. In high-resolution MS, the model-based approach is more appealing as it can provide a better representation of the MS signals by incorporating information about peak shapes and isotopic distributions. Current model-based techniques are computationally expensive; various algorithms have been proposed to improve the computational efficiency of this paradigm. However, these methods cannot deal well with overlapping features, especially when they are merged to create one broad peak. In addition, no method has been proven to perform well across different MS platforms. We suggest a new model-based approach to feature extraction in which spectra are decomposed into a mixture of distributions derived from peptide models. By incorporating kernel-based smoothing and perceptual similarity for matching distributions, our statistical framework improves existing methodologies in terms of computational efficiency and the accuracy of the results. Our model is parameterized by physical properties and is therefore applicable to different MS instruments and settings. We validate our approach on simulated data, and show that the performance is higher than commonly used tools on real high- and low-resolution MS, and MS/MS data sets.

Experimental study on low temperature thermal treatment of polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) in fly ash

The effects of temperature and time on polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/Fs) distribution in fly ash of a municipal solid waste incinerator (MSWI) were studied in a tubular oven under nitrogen atmosphere. The PCDD/Fs in the gas phase and solid phase were detected by high-resolution gas chromatography coupled with low resolution mass spectrometry (HRGC/LRMS) separately. The experimental results showed that the major congener was octa-chlorinated dibenzo-p-dioxin (OCDD) in the gas phase and the low chlorinated congeners were the major products in the solid phase. There were high levels of OCDD in the gas phase in several experimental conditions although the PCDD/Fs in the solid phase could be decomposed. The optimum condition for PCDD/Fs decomposition in fly ash was a heating time of 60 min at 400 °C under nitrogen atmosphere.

Analysis of a Parallel Branch in the Mitomycin Biosynthetic Pathway Involving the mitN-Encoded Aziridine N-Methyltransferase

Mitomycin C is a natural product with potent alkylating activity, and it is an important anticancer drug and antibiotic. mitN, one of three genes with high similarity to methyltransferases, is located within the mitomycin biosynthetic gene cluster. An inframe deletion in mitN of the mitomycin biosynthetic pathway was generated in Streptomyces lavendulae to produce the DHS5373 mutant strain. Investigation of DHS5373 revealed continued production of mitomycin A and mitomycin C in addition to the accumulation of a new mitomycin analog, 9-epi-mitomycin C. The mitN gene was overexpressed in Escherichia coli, and the histidine-tagged protein (MitN) was purified to homogeneity. Reaction of 9-epi-mitomycin C with MitN in the presence of S-adenosylmethionine yielded mitomycin E showing that the enzyme functions as an aziridine N-methyltransferase. Likewise, MitN was also shown to convert mitomycin A to mitomycin F under the same reaction conditions. We conclude that MitN plays an important role in a parallel biosynthetic pathway leading to the subclass of mitomycins with 9alpha-stereochemistry but is not involved directly in the biosynthesis of mitomycins A and C.

Comparison of the reliability of the identification with diode array detector and mass spectrometry

The reliability of compound identification by low resolution mass spectrometry (MS) was quantitatively compared with that for diode array detection (DAD). The quantity of the information obtained with one parent ion and two characteristic daughter ions at low resolution MS–MS was compared with that obtained by UV–vis spectra with large wavelength range from DAD. It was shown that if the UV–vis spectra are reproducible, i.e. with low RSD values for selected wavelengths and relative absorption, the reliability of the identification with DAD is comparable with the one for low resolution MS–MS. Because the sensitivity of the DAD is one or two orders of magnitude lower than that of mass spectrometry, on-column focusing was applied and reliable identification was achieved at 1 ppb concentration. The surprisingly high reliability of identification obtained with contemporary DADs should increase the analysts’ confidence in this method of detection/confirmation.

Evaluation of GC-ion trap-MS/MS methodology for monitoring PCDD/Fs in infant formulas

The application of high resolution gas chromatography in combination with low resolution mass spectrometry with electron ionization and MS/MS detection (HRGC-MS/MS) is tested for its use in the analysis of PCDD/Fs in infant formulas. Development of the analytical method was based upon EPA directrices and international recommendations. Calibration linearity was tested and average relative response for any native and labelled compound over the five-point calibration range below 14% was found. The precision and accuracy of the proposed analytical procedure are also presented. Results obtained are in agreement with EPA criteria. The method is applied to the analysis of a number of initial and follow-on milk based infant formulas. In general, HRGC-MS/MS constitutes an interesting method for the analysis of dioxins in such matrices.

POPs and organic polysufides in sediments of Lake Ladoga

The study included one station close to a pollution source (depth 59 m) and another far from polluted areas (depth 40 m). Samples were analysed for organic chlorine, bromine and sulfur compounds. Samples taken with a corer were sliced to the layers of 0-1, 1-4, 4-7 cm etc. down to the depth of 34 cm. The dating was made with two independent methods, the 210Po method and with the soot particle counting method. The analyses were made with a multiresidue method. Gas chromatography was connected to low resolution mass spectrometry (LRMS) or to high resolution mass spectrometry (HRMS). A different extraction was applied to screen the possible occurrence of polysulfides. Typical chlorophenol and chlorohydrocarbon traces from chlorobleaching of pulp were non-detectable. Also PCDDs, PCDFs, PCBs and various organochloride pesticides, fire retardant residues PBDEs, PBDDs and PBDFs and polychlorinated phenyl salicylates were not detected. Instead, methylated dibenzothiophenes Me-DBTs and Di-Me-DBTs, typical traces of oil based defoamers used in e.g. paper mills were found in the surface layers of the polluted site. Polysulfides were analyzed from layers representing years 1955-1970 from the polluted site. The structures of five congeners were according to GC/MS dimethyl trisulfide, 1,2,3,4-tetrathiepane, pentathiane, 1,2,3,4-tetrathiane and 1,2,4,5-tetrathiepane. Many of the analyzed pollutants have not been reported earlier from Lake Ladoga. The only positive observations were from oil-based defoamers used in paper mills, and from polysulfides, which are either of natural origin or indicate a discharge in the 1950s and 1960s. Owing to the large spreadout and dilution, the concentrations are low even at moderate distances from the pollution centers.

Analytical methods for PCBs and organochlorine pesticides in environmental monitoring and surveillance: a critical appraisal.

Analytical methods for the analysis of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) are widely available and are the result of a vast amount of environmental analytical method development and research on persistent organic pollutants (POPs) over the past 30–40 years. This review summarizes procedures and examines new approaches for extraction, isolation, identification and quantification of individual congeners/isomers of the PCBs and OCPs. Critical to the successful application of this methodology is the collection, preparation, and storage of samples, as well as specific quality control and reporting criteria, and therefore these are also discussed. With the signing of the Stockholm convention on POPs and the development of global monitoring programs, there is an increased need for laboratories in developing countries to determine PCBs and OCPs. Thus, while this review attempts to summarize the current best practices for analysis of PCBs and OCPs, a major focus is the need for low-cost methods that can be easily implemented in developing countries. A “performance based” process is described whereby individual laboratories can adapt methods best suited to their situations. Access to modern capillary gas chromatography (GC) equipment with either electron capture or low-resolution mass spectrometry (MS) detection to separate and quantify OCP/PCBs is essential. However, screening of samples, especially in areas of known use of OCPs or PCBs, could be accomplished with bioanalytical methods such as specific commercially available enzyme-linked immunoabsorbent assays and thus this topic is also reviewed. New analytical techniques such two-dimensional GC (2D-GC) and “fast GC” using GC–ECD may be well-suited for broader use in routine PCB/OCP analysis in the near future given their relatively low costs and ability to provide high-resolution separations of PCB/OCPs. Procedures with low environmental impact (SPME, microscale, low solvent use, etc.) are increasingly being used and may be particularly suited to developing countries.Electronic supplementary materialSupplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00216-006-0765-y and is accessible for authorized users.

Alternative Sample Preparation Techniques in Gas Chromatographic-Mass Spectrometric Analysis of Urinary Androgenic Steroids

The following study describes the gas chromatography-mass spectrometry (GC-MS) based screening and confirmation analysis of urinary androgenic steroids. Four commercially available solid-phase extraction (SPE) cartridges, Serdolit PAD-1, Sep-pak <TEX>$C_{18}$</TEX>, amino-propyl, and Oasis HLB, and three different extractive organic solvents, diethyl ether, methyl tert-butyl ether (MTBE), and n-pentane, were tested for sample preparation. Overall, Oasis HLB combined with MTBE extraction provided the highest recoveries in 39 of 46 total androgenic steroids examined and it showed a good extraction yield (>82.1%) for polar steroids, such as metabolites of fluoxymesterone, oxandrolone, and stanozolol, which gave a poor recovery in both n-pentane (9.2-64.3%) and diethyl ether (22.2-73.6%) extractions. All SPE sorbents tested showed potential, because they were efficient in extraction for most or selective steroids. When applied to positive urine samples based on the results obtained, the present method allowed selective and sensitive analysis for detection of urinary androgenic steroids. The experiments showed that the high-resolution MS method is clearly more efficient than the low-resolution MS technique for the detection of many urinary steroids. However, comprehensive sample clean-up procedures also might be needed especially in confirmation analysis to increase detectability.

The synthesis of chromophore ended glycoles, part IV. Bis-coumarin derivatives ended polyglycols

The coumarin derivatives of bis-resorcin ended polyglycols were prepared in two ways: Bis-4-alkyl-7-oxycoumarin ended mono and diethyleneglycols were prepared starting from bis(3-hydroxyphenyl)glycols by coumarin condensation using relevant β-ketoesters. Accordingly, 4-methyl, 4-trifluoromethyl, 4-n-propyl and 4-phenyl derivatives of 7-hydroxycoumarins were prepared in good yields. They were then converted to bis-coumarin ended three and tetraethylenglycol derivatives by reacting with three and tetraethyleneglycols dichlorides in Na2CO3/DMF, respectively. The products were identified using IR, 1H nmr and low resolution mass spectrometry. The Li+, Na+ and Rb+ metal/Ligand selectivities of cation binding behaviour of products in acetonitrile were studied with steady state fluorescence spectroscopy.

Determination of C10-chloroalkane residues in fish matrices by short column gas chromatography/electron capture negative ion low resolution mass spectrometry applying single pure and representative synthesised chlorodecanes as standards

A new chlorodecane (CD) standard was developed consisting of five single compounds with 5–9 Cl-atoms, with which it was possible to determine chlorodecane residues in fish matrices from different countries using short column gas chromatography/electron capture negative ion low resolution mass spectrometry (SCGC/ECNI-LRMS). The concentrations found were between 4.8 and 30.2 ng/g fat. Pentachlorodecanes could not be detected in all samples. For an evaluation of the new CD-standard, the fish matrices were also quantified by several other polychlorinated decane (CP10) standards with different chlorine grade: 50, 55, 63.5, 65, and 70%. The concentrations found differed unsurprisingly considerable among the applied standards. Considering only these CP10:50–70% standards that showed the highest similarity in peak patterns with the fish samples, the differences in observed chlorodecane concentrations between these standards and the new CD-standard were low, varying only 1–16%. The CP10:50–70% standards were further quantified with the new CD-standard (chlorine content, 58.2%) with neglectable observed differences to the CP10:60, 63.5, and 65% standards. Highest differences were observed to the CP10:50, 55, and 70% standards. By this work, the quantification of eco-toxicologically relevant C10-chloroparaffins using the new CD-standard has led to reproducible and reliable results, which indicates further that these compounds are still a concerning class of substances in environmental fish samples.

Selection of IRS-P6 LISS-4 MO Mode Band for Producing Band-Sharpened Multispectral Imagery

The recently launched IRS-P6 satellite has a unique capability of acquiring simultaneously multispectral data at three different spatial resolutions from three independent optical sensors (LISS-4, LISS-3 and AWIFS). Of these, the LISS-4 sensor can be operated in two modes: (i) multispectral (MX) mode covering a swath of 23 km and (ii) monochromatic (MO) mode covering a 70-km swath, both at a spatial resolution of 5 m. One of the important uses of the LISS-4 MO data is in realizing a 5 m band-sharpened multispectral image by merging it with the low-resolution LISS-3 MS image. Operationally anyone of the three LISS-4 bands can be chosen for the MO mode data acquisition. The performance of each band for producing band-sharpened MS images is evaluated, and the choice of the band based on the spatial and spectral characteristics of the merged data is suggested. The LISS-4 Red-band is found to be optimal. It provides band-sharpened imagery with spatial and spectral qualities very similar to the LISS-4 MX data products.

Determination of polybrominated diphenyl ethers in indoor dust standard reference materials

Polybrominated diphenyl ethers (PBDEs) have been measured for the first time in three different indoor dust Standard Reference Materials (SRMs) prepared by the National Institute of Standards and Technology (NIST). Two of these, SRM 2583 (Trace Elements in Indoor Dust) and SRM 2584 (Trace Elements in Indoor Dust), have been certified previously for lead and other inorganic constituents. A third, SRM 2585 (Organics in Indoor Dust), is a new indoor dust reference material prepared by NIST which will be certified for various organic compounds (polycyclic aromatic hydrocarbons, pesticides and polychlorinated biphenyls) in 2005 including certified concentrations for 16 individual PBDE congeners and reference values for an additional three PBDE congeners. Dust SRMs were analyzed for 30 PBDE congeners using high-resolution gas chromatography combined with low-resolution mass spectrometry operated in both negative chemical ionization (GC/ECNI-MS) and electron impact ionization (GC/EI-MS) modes. Sensitivity was an order of magnitude higher using GC/ECNI-MS relative to GC/EI-MS. These SRMs have been characterized and compared to the three PBDE commercial products (pentaBDE, octaBDE and decaBDE). PentaBDE and DecaBDE were present in all three SRMs and were the dominant commercial products, making up approximately 33% and 58%, respectively. Recent studies suggest that house dust may be a leading source of human exposure to PBDEs. These SRMs are the first reference materials with certified concentrations for PBDEs, which will aid in validating future measurements of PBDEs in house dust and other similar matrices.

Reference values of coplanar and non-coplanar PCBs in serum samples from two Italian population groups

The main goal of this study is to establish the reference values of individual Polychlorinated biphenyl (PCB) congeners in non-occupationally exposed subjects. Since the PCB pattern in human serum is related to the living area, two different population groups from North and Central Italy, were compared. Serum concentrations of both coplanar and non-coplanar PCB congeners were measured by using gas chromatography coupled with low-resolution mass spectrometry (HRGC–LRMS). A fast and reliable method for the determination of 60 congeners had been previously validated. Its reliability was further verified by using high-resolution mass spectrometry. Thirty-one congeners out of 60 were found at detectable concentrations in at least one sample. The mean value for total PCBs was found to be 2.48 and 3.93 μg/L for the two population groups. Eight dioxin-like PCBs were detected. In accordance with the findings from the literature, the most abundant congeners were found to be 153, 138, 180, and 170. Both univariate and multivariate analysis showed that age is a significant determinant of PCB concentrations. The correlation increased with increasing chlorination. Slight differences in the PCB pattern were observed in the two population groups.

Presence of Chlorinated Paraffins in Sediments from the North and Baltic Seas

Chloroparaffins (CPs) were determined in sediments collected from the North and Baltic Seas during monitoring campaigns in 2001-2003. Electron ionization tandem mass spectrometry (MS/MS) was used for a first screening. It allowed the simultaneous determination of short (SCCP) and medium chain chlorinated paraffins (MCCP). SCCP+MCCP concentrations between 5 and 499 ng/g dry weight were found. In general, Baltic Sea sediments were more highly contaminated by CPs than the North Sea was. However, concentrations related to the total organic carbon content were on the same order of magnitude due to the higher organic content in the Baltic Sea. Additional information about the congener and homologue pattern was obtained for selected samples from the Baltic Sea by high-resolution gas chromatography combined with negative ion chemical ionization and low-resolution mass spectrometry, Concentrations in the North Sea were in general too low for this approach. In the Baltic Sea, MCCP concentrations were 1.7-2.4 times higher than for SCCPs. Lower chlorinated C(13) and C(14) compounds were the main CP compounds. The CP congener and homologue patterns showed similarities with technical SCCP and MCCP mixtures when compared using principal component analysis.

Enricher for Fraction of High-Pressure Liquid Chromatography

A device is developed for concentrating a dilute solution without losing the components with boiling points slightly higher than the solvent. The device consists of an evaporator, receptor, and approximately 100 capillaries. A dilute solution is introduced into the evaporator and heated at a lower temperature than the boiling point of the solvent with the addition of a helium gas flow. As a result, mostly only the solvent evaporates, passes through the capillaries, and enters into the receptor. The low-boiling-point components in the solute, with boiling points slightly higher than the solvent, are trapped at the inlet of the capillaries. These components are then recovered by a small amount of solvent supplied from the receptor through the capillaries, with the main components of the solute concentrated at the bottom of the evaporator. A diesel fuel is separated into aliphatic and aromatic fractions by high-pressure liquid chromatography using a silica gel column. These fractions are then analyzed by low-resolution field ionization mass spectrometry, following concentration using the described device. The analytical results show that the final composition of the fractions is almost the same as that of the aliphatic or aromatic hydrocarbons in the original fuel.

Inadequate Attempts to Measure the Microheterogeneity of Transthyretin by Low-Resolution Mass Spectrometry - Reply

Inadequate Attempts to Measure the Microheterogeneity of Transthyretin by Low-Resolution Mass Spectrometry - Reply

Inadequate Attempts to Measure the Microheterogeneity of Transthyretin by Low-Resolution Mass Spectrometry - Reply

Inadequate Attempts to Measure the Microheterogeneity of Transthyretin by Low-Resolution Mass Spectrometry - Reply

Inadequate Attempts to Measure the Microheterogeneity of Transthyretin by Low-Resolution Mass Spectrometry

Recently, the authors of 2 studies have claimed to measure accurately the microheterogeneity of transthyretin (TTR) variants by surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) immunoassays (1)(2). Here we report that the mass spectrometric performance in those studies was not adequate to measure the respective TTR variants. A mass resolution of at least 1000 is required to separate the majority of the currently known posttranslational and mutation-based modifications of TTR (3). Wang et al.(1) performed their analysis at a resolution of 270 [estimated as follows: m /Δ m FWHM = 13828/52 = 266 in Fig. 1A of their report (1)], whereas Schweigert et al. (2) achieved a resolution of 90 [estimated as follows: m /Δ m FWHM = 13851/158 = 88 in panel A2 of Fig. 2 of their report (2)]. We therefore question whether these studies can justify their reported claims. To provide arguments, we performed MALDI-TOF MS analysis on a gold chip ProteinChip® array of a commercially available TTR preparation with an instrument typically used in SELDI-TOF MS (PBS IIc analyzer; Ciphergen). We applied …

Strategies to avoid the mis-identification of anatoxin-a using mass spectrometry in the forensic investigation of acute neurotoxic poisoning

Anatoxin-a (AN) is a potent neurotoxin, produced by a number of cyanobacterial species, and consumption of freshwater contaminated with this toxin has led to animal deaths. Forensic investigations of suspected AN poisonings are frequently hampered by difficulties in detecting this toxin in biological matrices due to its rapid decay. In addition, detection of AN using single quadrupole mass spectrometry (MS) is suspect due to the presence of the amino acid, phenylalanine (Phe), since these compounds are isobaric and elute similarly in reversed phase liquid chromatography (LC). Approaches to prevent the misidentification of AN that have been explored in these studies included: (a) fluorimetric LC following derivatisation using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F); (b) methylation using diazomethane prior to LC–MS determination; (c) multiple tandem MS using a quadrupole ion-trap (LC–MS 3); and (d) hybrid quadrupole time-of-flight (QqTOF). Interference from Phe was not observed in any of procedures, (a)–(c), and the high mass accuracy obtained in method (d), readily distinguished between AN (165.11536) and Phe (165.07898). LC–MS n was also employed to study the fragmentation pathway of Phe and multi-stage MS spectra provided characteristic fragmentation information that clearly distinguished between AN and Phe. The difficulties associated with the over reliance on low resolution MS without MS/MS data in forensic toxicology are discussed.