Seasonal changes in metabolic profiles of galls and leaves of Rhus chinensis using gas chromatography mass spectrometry and liquid chromatography quadrupole time-of-flight mass spectrometry

Many researchers have studied the potential medicinal properties of galls from Rhus chinensis because of the importance of these galls in East Asian traditional medicine. Gall formation induced by a parasitic aphid species (Schlechtendalia chinensis) occurs via a well-documented developmental progression, and traditional medicinal efficacy is thought to be maximal during a specific portion of this cycle. To investigate seasonal changes of metabolites in the galls of R. chinensis, we collected samples from the galls and leaves of R. chinensis at sites in Mt. Jiri and Mt. Cheonma in Korea between May and December, 2011. Samples were extracted and analyzed gas chromatography mass spectrometry (GC-MS) and liquid chromatography quadrupole time-offlight mass spectrometry (LC-QTOF-MS) to monitor metabolic changes. Multivariate analyses such as principle components analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) were used to find patterns in metabolite profile changes and the responsible substances for seasonal fluctuations. LC-QTOF-MS analyses showed differences of metabolites in same organisms depending on seasons, locations, and biological interactions. Additional GC-MS analyses identified approximately 28 metabolites including sugars, amino acids, and organic acids. Shikimic acid and gallic acid appear to be the major compounds contributing to the seasonal variability in metabolic profiles of R. chinensis leaves and galls. In addition, we found that shikimic acid and gallic acid content in R. chinensis galls were the highest during wintertime.     

Comparison of algorithms for pre-processing of SELDI-TOF mass spectrometry data

MOTIVATION: Surface-enhanced laser desorption and ionization (SELDI) time of flight (TOF) is a mass spectrometry technology. The key features in a mass spectrum are its peaks. In order to locate the peaks and quantify their intensities, several pre-processing steps are required. Though different approaches to perform pre-processing have been proposed, there is no systematic study that compares their performance. RESULTS: In this article, we present the results of a systematic comparison of various popular packages for pre-processing of SELDI-TOF data. We evaluate their performance in terms of two of their primary functions: peak detection and peak quantification. Regarding peak quantification, the performance of the algorithms is measured in terms of reproducibility. For peak detection, the comparison is based on sensitivity and false discovery rate. Our results show that for spectra generated with low laser intensity, the software developed by Ciphergen Biosystems (ProteinChip Software 3.1 with the additional tool Biomarker Wizard) produces relatively good results for both peak quantification and detection. On the other hand, for the data produced with either medium or high laser intensity, none of the methods show uniformly better performances under both criteria. Our analysis suggests that an advantageous combination is the use of the packages MassSpecWavelet and PROcess, the former for peak detection and the latter for peak quantification.     

Interactive feature finding in liquid chromatography mass spectrometry data

We propose a method for finding features in liquid chromatography mass spectrometry data that is based on the isotopic pattern of peaks. Our interactive approach to feature finding is carried out across many samples simultaneously and aligns features concurrently. Our scale-independent approach prioritises potential features and is easily adaptable to look for features of a particular mass and charge, paired features in isotopically labeled samples, or differentially expressed features. We demonstrate this by identifying features from normal human adult plasma. We highlight properties of plasma data that illustrate the need to visually check the quality of features found prior to further statistical analysis.     

A strategy for examining complex mixtures of deoxyoligonucleotides using ion-pair-reverse-phase high-performance liquid chromatography,matrix-assisted laser desorption ionization time-of-flight mass spectrometry,and informatics

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and ion-pair-reverse-phase high-performance liquid chromatography (IP-RP HPLC) techniques were combined to determine the sequence identity of short single-stranded deoxyoligonucleotides. This methodology is demonstrated using a commercially available multiplex set of eight primer pairs. The primer pairs were separated and collected by IP-RP HPLC. Partial sequence information for IP-RP HPLC fractions was obtained from analyzing exonuclease digestion products by MALDI-TOF MS. IP-RP HPLC, MALDI-TOF MS, exonuclease digests, and a simple computational algorithm provide an integrated strategy for determining the sequence of short nucleic acid oligomers.     

Serum metabolic profiling of abnormal savda by liquid chromatography/mass spectrometry

Abnormal savda is a special symptom in Uigur medicine. The understanding of its metabolic origins is of great importance for the subsequent treatment. Here, a metabonomic study of this symptom was carried out using LC-MS based human serum metabolic profiling. Orthogonal signal correction partial least-squares discriminant analysis (OSC-PLS-DA) was used for the classification and prediction of abnormal savda. Potential biomarkers from metabonomics were also identified for a metabolic understanding of abnormal savda. As a result, our OSC-PLS-DA model had a satisfactory ability for separation and prediction of abnormal savda. The potential biomarkers including bilirubin, bile acids, tryptophan, phenylalanine and lyso-phosphatidylcholines indicated that abnormal savda could be related to some abnormal metabolisms within the body, including energy metabolism, absorption of nutrition, metabolism of lecithin on cell membrane, etc. To the best of our knowledge, this is the first study of abnormal savda based on serum metabolic profiling. The LC/MS-based metabonomic platform could be a powerful tool for the classification of symptoms and for the development of this traditional medicine into an evidence-based one.     

Forensic DNA fingerprinting by liquid chromatography-electrospray ionization mass spectrometry

The determination of the molecular mass of a DNA sequence has several benefits over conventional fragment-length analysis that are advantageous to the forensic field: (i) sequence variation is captured that increases the power of discrimination compared with that obtained by conventional fragment-length analysis. First experiments showed that this increase makes up to 20%-30% for STR analysis. The new technical approach does not invalidate established developments and data, but adds to this information with additional discriminative categories. (ii) ICEMS is faster and cheaper than electrophoresis, does not require internal size standards, allelic ladders, or spectral calibration, which are necessary for fluorescence-based electrophoresis. (iii) ICEMS can unequivocally detect any single sequence variation in DNA molecules with lengths up to 250 nucleotides. This allows for maximum discrimination of forensically relevant DNA fragments, covering all sorts of STRs, SNPs, and also the analysis of the hypervariable segments of mtDNA. More effort, however, needs to be put into software development that escorts the analysis and data interpretation processes to make this technology manageable for the practical user.     

A novel wavelet-based thresholding method for the pre-processing of mass spectrometry data that accounts for heterogeneous noise

In recent years there has been an increased interest in using protein mass spectroscopy to discriminate diseased from healthy individuals with the aim of discovering molecular markers for disease. A crucial step before any statistical analysis is the pre-processing of the mass spectrometry data. Statistical results are typically strongly affected by the specific pre-processing techniques used. One important pre-processing step is the removal of chemical and instrumental noise from the mass spectra. Wavelet denoising techniques are a standard method for denoising. Existing techniques, however, do not accommodate errors that vary across the mass spectrum, but instead assume a homogeneous error structure. In this paper we propose a novel wavelet denoising approach that deals with heterogeneous errors by incorporating a variance change point detection method in the thresholding procedure. We study our method on real and simulated mass spectrometry data and show that it improves on performances of peak detection methods.     

Screening for N-glycosylated proteins by liquid chromatography mass spectrometry

In the last few years mass spectrometry has become the method of choice for characterization of post-translationally modified proteins. Whereas most protein chemical modifications are binary in the sense that only one change can be associated with a given residue, many different oligosaccharides can be attached to a glycosylation site residue. The detailed characterization of glycoproteins in complex biological samples is extremely challenging. However, information on N-glycosylation can be gained at an intermediary level. Here we demonstrate a procedure for mapping N-glycosylation sites in complex mixtures by reducing sample complexity and enriching glycoprotein content. Glycosylated proteins are selected by an initial lectin chromatography step and digested with endoproteinase Lys-C. Glycosylated peptides are then selected from the digest mixture by a second lectin chromatography step. The glycan components are removed with N-glycosidase F and the peptides digested with trypsin before analysis by on-line reversed-phase liquid chromatography mass spectrometry. Using two different lectins, concanavalin A and wheat germ agglutinin, this procedure was applied to human serum and a total of 86 N-glycosylation sites in 77 proteins were identified.     

Two-dimensional liquid chromatography system for online top-down mass spectrometry

An online metal-free weak cation exchange-hydrophilic interaction LC/RPLC system has been developed for sensitive, high-throughput top-down MS. Here, we report results for analyzing PTMs of core histones, with a focus on histone H4, using this system. With just ～24?μg on-column of core histones (H4, H2B, H2A, and H3) purified from human fibroblasts, 41 H4 isoforms were identified, with the type and location of PTMs unambiguously mapped for 20 of these variants. Compared to corresponding offline studies reported previously, the online weak cation exchange-hydrophilic interaction LC/RPLC platform offers significant improvement in sensitivity, with several orders of magnitude reduction in sample requirements and a reduction in the overall analysis time. To the best of our knowledge, this study represents the first online 2-D LC-MS/MS characterization of core histone mixture at the intact protein level.     

Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry

Metabolism has an essential role in biological systems. Identification and quantitation of the compounds in the metabolome is defined as metabolic profiling, and it is applied to define metabolic changes related to genetic differences, environmental influences and disease or drug perturbations. Chromatography-mass spectrometry (MS) platforms are frequently used to provide the sensitive and reproducible detection of hundreds to thousands of metabolites in a single biofluid or tissue sample. Here we describe the experimental workflow for long-term and large-scale metabolomic studies involving thousands of human samples with data acquired for multiple analytical batches over many months and years. Protocols for serum- and plasma-based metabolic profiling applying gas chromatography-MS (GC-MS) and ultraperformance liquid chromatography-MS (UPLC-MS) are described. These include biofluid collection, sample preparation, data acquisition, data pre-processing and quality assurance. Methods for quality control-based robust LOESS signal correction to provide signal correction and integration of data from multiple analytical batches are also described.     

Profiling of Arabidopsis secondary metabolites by capillary liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry

Large-scale metabolic profiling is expected to develop into an integral part of functional genomics and systems biology. The metabolome of a cell or an organism is chemically highly complex. Therefore, comprehensive biochemical phenotyping requires a multitude of analytical techniques. Here, we describe a profiling approach that combines separation by capillary liquid chromatography with the high resolution, high sensitivity, and high mass accuracy of quadrupole time-of-flight mass spectrometry. About 2000 different mass signals can be detected in extracts of Arabidopsis roots and leaves. Many of these originate from Arabidopsis secondary metabolites. Detection based on retention times and exact masses is robust and reproducible. The dynamic range is sufficient for the quantification of metabolites. Assessment of the reproducibility of the analysis showed that biological variability exceeds technical variability. Tools were optimized or established for the automatic data deconvolution and data processing. Subtle differences between samples can be detected as tested with the chalcone synthase deficient tt4 mutant. The accuracy of time-of-flight mass analysis allows to calculate elemental compositions and to tentatively identify metabolites. In-source fragmentation and tandem mass spectrometry can be used to gain structural information. This approach has the potential to significantly contribute to establishing the metabolome of Arabidopsis and other model systems. The principles of separation and mass analysis of this technique, together with its sensitivity and resolving power, greatly expand the range of metabolic profiling.     

Terpenoid metabolic profiling analysis of transgenic Artemisia annua L. by comprehensive two-dimensional gas chromatography time-of-flight mass spectrometry

Amorpha-4,11-diene synthase (ADS) is a very important enzyme which catalyzes the committed step of artemisinin biosynthesis. In this work, two lines of transgenic Artemisia annua L. which ADS was over-expressed (line A9) and suppressed (line Amsi), respectively, were utilized. And the transgenic line GUS with β-Glucuronidase gene was regarded as the control. Their terpenoid metabolic profiling was investigated by using GC × GC–TOFMS. The metabolic profiling method established included simple extraction, two-dimension separation and multivariate analysis. Partial least squares discriminant analysis (PLS-DA) was used to classify two transgenic lines and the control line. Eleven important compounds in classification were identified. Most of them were sesquiterpenoids including monoterpenoid, diterpenoid and four bioprecursors of artemsisnin. Compared with the control, artemisinin and bioprecursors in the line A9 increased as a result of over-expressing ADS. Borneol and phytol also increased in the line A9, but (E)-β-farnesene and germacrene D were reversely altered. The result indicated that over-expression of the ADS affected not only artemisinin biosynthesis, but also the whole metabolic network of terpenoid. Compared with the line A9, no opposite change of artemisinin and related derivatives was observed in the line Amsi, the ADS inhibition had no significant effect on artemisinin biosynthesis in the line Amsi.     

Analysis of carbohydrate heterogeneity in a glycoprotein using liquid chromatography/mass spectrometry and liquid chromatography with tandem mass spectrometry

High-performance liquid chromatography with on-line electrospray ionization mass spectrometry (ESI-LC/MS) was investigated for the analysis of carbohydrate heterogeneity using RNase B as a model glycoprotein. Oligosaccharides released from RNase B with endoglycosidase H were reduced and separated on a graphitized carbon column (GCC). GCC-HPLC/MS in the positive-ion mode was successful in the identification of one Man5GlcNAc, three Man6GlcNAc, three Man7GlcNAc, three Man8GlcNAc, one Man9GlcNAc, and an oligosaccharide having six hexose units (Hex) and two N-acetylhexosamine units (HexNAc). The branch structures of the three Man7GlcNAc isomers were determined by liquid chromatography with tandem mass spectrometry (LC/MS/MS). LC/MS/MS analysis was shown to be useful for the detection and identification of a trace amount of Hex6HexNAc2 alditol as a hybrid-type oligosaccharide. Its structure was confirmed by the combination of LC/MS with enzymatic digestion using beta-galactosidase and N-acetyl-beta-glucosaminidase. The relative quantities of high-mannose-type oligosaccharides in RNase B detected by ESI-LC/MS are in reasonable agreement with those by UV, high-pH anion-exchange chromatography with pulsed amperometric detection, fluorophore-assisted carbohydrate electrophoresis. Our results indicate that LC/MS and LC/MS/MS can be utilized to elucidate the distribution of oligosaccharides and their structures, which differ in molecular weight, sugar sequence, and branch structure.     

A sensitive and specific determination method for azaspiracids by liquid chromatography mass spectrometry

A liquid chromatography/mass spectrometry (LC/MS) method was developed for the sensitive and specific determination of azaspiracid and its two analogs, the causative toxins of azaspiracid poisoning that occurred in the Netherlands and Ireland. The LC/MS method provided a detection limit of 50 pg for azaspiracid. The sensitivity was approximately 8 x 10(4) times greater than the mouse bioassay. The method was used to confirm the presence of azaspiracids in toxic mussels collected at Arranmore Island, Ireland in 1997.     

Comparison of originator and biosimilar therapeutic monoclonal antibodies using comprehensive two-dimensional liquid chromatography coupled with time-of-flight mass spectrometry

As research, development, and manufacturing of biosimilar protein therapeutics proliferates, there is great interest in the continued development of a portfolio of complementary analytical methods that can be used to efficiently and effectively characterize biosimilar candidate materials relative to the respective reference (i.e., originator) molecule. Liquid phase separation techniques such as liquid chromatography and capillary electrophoresis are powerful tools that can provide both qualitative and quantitative information about similarities and differences between reference and biosimilar materials, especially when coupled with mass spectrometry. However, the inherent complexity of these protein materials challenges even the most modern one-dimensional (1D) separation methods. Two-dimensional (2D) separations present a number of potential advantages over 1D methods, including increased peak capacity, 2D peak patterns that can facilitate unknown identification, and improvement in the compatibility of some separation methods with mass spectrometry. In this study, we demonstrate the use of comprehensive 2D-LC separations involving cation-exchange (CEX) and reversed-phase (RP) separations in the first and second dimensions to compare 3 reference/biosimilar pairs of monoclonal antibodies (cetuximab, trastuzumab and infliximab) that cover a range of similarity/disimilarity in a middle-up approach. The second dimension RP separations are coupled to time-of-flight mass spectrometry, which enables direct identification of features in the chromatograms obtained from mAbs digested with the IdeS enzyme, or digestion with IdeS followed by reduction with dithiothreitol. As many as 23 chemically unique mAb fragments were detected in a single sample. Our results demonstrate that these rich datasets enable facile assesment of the degree of similarity between reference and biosimilar materials.     

Structural characterization of fucose-containing oligosaccharides by high-performance liquid chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Eight pyridylamino (PA) derivatives of fucose-containing oligosaccharides, which occur as free oligosaccharides in human milk and also are derived from glycosphingolipids, have been analyzed by high-performance liquid chromatography (HPLC) on normal-phase and reversed-phase columns, and by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Six out of eight PA-oligosaccharides were clearly separated by both normal- and reversed-phase HPLC at a column temperature of 40 degrees C, but two PA-oligosaccharides, lacto-N-fucopentaose II [Gal beta1-3(Fuc alpha1-4)GlcNAc beta1-3Gal beta1-4GIcPA] and lacto-N-fucopentaose III [Gal beta1-4(Fuc alpha1-3)GlcNAc beta1-3Gal beta1-4GIcPA], were not separated. The two unresolved PA-oligosaccharides were finally separated by reversed-phase HPLC at a column temperature of 11 degrees C. MALDI-TOF mass spectra of PA-oligosaccharides demonstrated pseudo-molecular ions as the predominant signals, therefore information about the molecular mass of each PA-oligosaccharide was easily obtained. Post-source decay (PSD) MALDI-TOF mass spectra of PA-oligosaccharides gave information about the carbohydrate sequences and carbohydrate species of each PA-oligosaccharide by detecting the ions responsible for the cleavage of the glycosidic bonds. The detection limits of the PA-oligosaccharides by HPLC, MALDI-TOF mass spectrometry, and PSD MALDI-TOF mass spectrometry were 20 fmol, 20 fmol, and 2 pmol, respectively. These results suggest that a system including HPLC and MALDI-TOF mass spectrometry or HPLC and PSD MALDI-TOF mass spectrometry is quite useful for the structural characterization of sub-pmol or pmol levels of fucose-containing oligosaccharides, and that these methods could be used for the analysis of various types of oligosaccharides derived from glycoproteins and glycosphingolipids.