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news & events / Posters and Oral Presentations for ASMS 2005 www.asms.org |
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Rapid Characterisation of Low Molecular Weight Drug Compounds using High Energy CID MALDI MS/MS
Rachel L Martin, Andrew Bowdler, Omar Belgacem Shimadzu Biotech, Manchester, UK Traditionally, MALDI techniques have been used to analyze relatively large samples with molecular weights ranging from 1000 Daltons to several hundred thousand Daltons. Lower molecular weight analytes have largely been avoided due to interference of matrix ions in the region of 200 - 700 Daltons. However, investigations into more suitable matrices for these compounds have resulted in more successful analyses. Recently, an evaluation of a newly designed MALDI CID MS/MS system has been performed for low molecular weight samples. Interestingly, when compared with both seamless post source decay (sPSD) and fragmentation in a quadrupole ion trap, the MS/MS spectra achieved by high energy CID MS/MS in a MALDI system are greatly improved for small molecules providing significant information regarding structure. High Resolution Ion Gate For Pre-cursor Ion Selection In MALDI ToF MS/MS
Andrew R Bowdler, Katherine L Farnell, Ian Brookhouse, Emmanuel Raptakis Shimadzu Biotech, Manchester, UK Interest in MS/MS with Maldi ToF has increased dramatically with the development of tandem ToF instruments. Collision induced dissociation makes fragment spectra more complicated than from post-source decay. So for complex mixtures, fragment ions must be assigned unambiguously to their parent ions. This has increased the performance requirements of the pre-cursor ion selection gate. We report on the performance of a new ion-gate with very high mass selection resolution. Compared with a conventional design which, at 1000Da, has a selection resolution of about 15Da, the novel ion gate is able to select a range close to 2Da. Furthermore, this is achieved without changing the pulsing electronics that had previously been the limiting factor for the selection resolution. Comparison of low energy CID, high energy CID and laser induced dissociation of MALDI generated ions
Emmanuel Raptakis1, Andrew Bowdler1, Martin Resch2, Omar Belgacem1 1Shimadzu Biotech, Manchester, UK, 2Shimdzu Biotech, Duisburg, Germany Collision induced dissociation (CID) in its various forms has been used for a number of years to facilitate structural elucidation of complex molecules. In recent years tandem mass spectrometry utilising CID has been extensively used to improve the statistical significance of database identification of unknown proteins and peptides. Similar strategies are being pursued for other classes of molecules, like nucleic acids, oligosaccharides or lipids. CID tandem mass spectra are generated in various types of instruments, with varying collisional energy conditions, which affects the type of fragments generated. Recent advances of MALDI instrumentation have produced examples of tandem mass spectra where characteristic fragments of both, high energy and low energy collision processes can be observed. Partial Determination of the Disulfide Bond Pattern in the Salmon Egg Lectin 24K Isolated from the Chinook Salmon Oncorhynchus tshawytscha
Haiqiang Yu1, Kenji Murata2, Jerry L. Hedrick2, Fan Xiang3, Andreas H. Franz1 1University of the Pacific, 2University of California Davis, 3Shimadzu Biotech Disulfide bridges are very common post-translational modifications in proteins and help to stabilize the molecular structure for proper biological function.1 Lectins are carbohydrate-recognizing proteins, which are widely distributed in nature2 and have been found in the Chinook salmon eggs for example (Salmon Egg Lectin, SEL).3 SEL has three sub-units with multiple S-S-bonds and is believed to play a central role during fertilization. In the present work, one of the SEL sub-units (24K) containing 16 cysteines was isolated by C18-High-Performance Liquid Chromatography (HPLC) and trypsinized. The analysis of the tryptic peptides with Matrix-Assisted Laser Desorption/Ionization (MALDI) Time-of-Flight (TOF) and Quadrupole Ion Trap (QIT) TOF mass spectrometry (MS) leads to a partial assignment of the disulfide bond pattern in SEL 24K. A strategy for the orthogonal analysis of proteoglycans based on positive and negative lectin-selection and MALDI MSn structural dissection
Roy Edward1, Rachel L Martin1, David Gillooly2, Stine Bergholtz2 1Shimadzu Biotech, Manchester, UK, 2Dynal Biotech ASA, Oslo, Norway N- and O-linked carbohydrate moieties and GPI anchors are found on more than 50% of mammalian proteins. As such they represent the most numerous set of post-translational modifications (PTMs) in the study of protein function and disease processes. They are responsible for the protection of proteins from proteases, achievement of correct conformational states and in the case of GPI anchors the stabilisation of proteins to the cell membrane. It follows that a better understanding of the normal and aberrant carbohydrate status of proteins should be of value in determining the relationship between this status and function or protein-protein interaction. On Membrane Glycoproteomic Approach Using Micro-Dispensing of Multiple Enzymes
Satoshi Kimura1, Akihiko Kameyama1, Shuuichi Nakaya2, Hiromi Ito1, Hisashi Narimatsu1 1AIST, Tsukuba, Japan, 2Shimadzu Corporation, Kyoto, Japan Proteome analysis using 2-DE approaches are widely used for the separation of proteins because of their relative simplicity and high resolution. Recently, it was proposed that on-membrane direct MALDI-TOF MS analysis using piezoelectric micro-dispense technology. The technology enables multiple enzyme reaction, such as endoglycosidase to release the oligosaccharide and trypsin digestion on a single protein spot. Our group also proposed a strategy for rapid identification of the oligosaccharide structure based on MSn analysis using MALDI-QIT-TOF MS. The combination of these technologies will be convenient potential method for glycoproteomics. Here, we demonstrate that in situ analysis of both N-linked oligosaccharides and peptides on a single protein band using multiple enzyme micro-dispense technology. Determination of Phosphorylation Sites using MSn on a MALDI QIT TOF MS
Brian K Stall1+3, Etienne Waelkens2, Rachel L Martin1+3 1Shimadzu Biotech, Woburn, MA, 2K.U. Leuven, Leuven, Belgium, 3Shimadzu Biotech, Manchester, UK Phosphorylated proteins have significant biological relevance contributing to many intermediate energetic pathways. For example, redox reactions and phosphate kinases play an important role in providing energy for active transport across membranes. Analysis of phosphorylated peptides by mass spectrometry has proven difficult due to the labile bond between the phosphate group and the peptide, resulting in loss of this group in MS mode. This allows the identification of a phosphorylated peptide but not the localization of the modified site. We have recently analyzed a series of non-phosphorylated peptides and their phosphorylated counterparts by MALDI QIT TOF MS in an attempt to sequence a peptide and successfully determine the position of the modified residue. Proteomic analysis of estrogen effect on Breast Cancer development using a 2-D liquid & hybrid MALDI Ion Trap/TOF mass-mapping technique
Jia Zhao1, Fan Xiang3, Fred R. Miller2, David M. Lubman1 1University of Michigan, Ann Arbor, MI, 2Karmanos Cancer Institute,Wayne State University, Detroit, MI, 3Shimadzu Biotech, Pleasanton, CA Estrogen is a hormone which is very important for growth of breast cancer cells. MCF10AT model is an xenograft system of human breast epithelial cells and it constitutes a model of early breast cancer progression. In order to study the estrogen-induced proteome, a novel 2-D LC separation & a hybrid MALDI Ion Trap/TOF mass mapping approach is applied to analyze proteins from the premalignant human breast cell line before (MCF10AT1) and after treatment with estradiol (MCF10AT1E2). In this study, the protein identification is based on PMF, MS/MS analysis, accurate MW and pI of intact proteins. This method offers better resolution, sequence coverage, and reproducibility over the traditional 2-D gel profiling method. Derivatization of the C-Termini of Peptides with Formic Acetic Anhydride and a Phenol for C-Terminal Sequencing with MALDI-MS
Takashi Nakazawa1, Minoru Yamaguchi2, Mutsumi Oka1, Mayu Ishida1, Hiroki Kuyama2, Takashi Obama2, Eiji Ando2, Taka-aki Okamura3, Norikazu Ueyama3, Shigemi Norioka3 1Nara Women's University, 2Shimadzu Corp., 3Osaka Univ. Mass spectrometry provides a variety of rapid and sensitive methods for protein sequencing. However, to obtain the exact amino acid sequences of mature proteins, which are actually functional forms in living cells, it is necessary to establish the convenient and versatile methods for both N- and C-terminal sequencing in addition to identification of post-translational modification products. To address this issue, we have developed a method for the site-specific C-terminal activation of peptides via an oxazolone, enabling the derivatization for facilitating the C-terminal sequencing by MALDI MS. We will present the results of improvements to suppress side reactions due to the labile nature of an oxazolone by replacing the oxazolone with an active ester formed by an added phenol. High sensitive MS/MS analysis of MALDI QIT-TOF MS combined with Novel MALDI Surfaces for On-Chip Concentration
Yuzo Yamazaki1, Keisuke Shima1, Masaki Yamada1, Christopher M. Belisle2, Douglas P. Greiner2, John A. Walker II2 1Shimadzu Corp., 2LCI, Fremont, CA, USA A general in-gel digest protocol provides about 10 µl of a digest solution. However, it is quite difficult to apply all the volume onto a stainless-steel MALDI plate. The large volume of solution spreads to a large area on the plate, which leads to a decrease in sensitivity. Here we report our investigation of high sensitivity MS/MS analysis by MALDI QIT-TOFMS, which is enhanced by the concentrating properties of a surface-tension-segmented (STS) well. This well is comprised of three concentric zones with differing wettabilties. Utilizing the On-chip concentration, a method to analyze complicated mixtures will be shown through MS/MS spectra from in-gel digests that include several proteins and spiked phosphopeptide at less than 5 fmol. A new analytical method for amino acid sequencing and disulfide mapping by 1,5-diaminonaphtalene (DAN) as a reductive matrix
Yuko Fukuyama, Shinichi Iwamoto, Koichi Tanaka Shimadzu Corporation 1,5-diaminonaphtalene (DAN) has been reported as a matrix for gangliosides in MALDI-MS. However, DAN has not been used widely in proteomics because the molecular ions are not detected as the intact molecule with DAN. This is probably caused by cleavage of the disulfide bonds. On the other hand, the disulfide bonds complicate the identification of product ions of peptides and/or proteins in MSn. In this study, we propose a new simple method using DAN for amino acid sequencing and disulfide mapping of peptides and proteins with MS and MSn, without any additional pretreatments. Identification/characterization of the predominant elicitor for type I allergy to elderberry blossoms by low energy CID-MS2 experiments (MALDI-QIT/RTOF and nESI-QIT)
Martina Marchetti1, Jasmin Hirschmann1, Martin Zehl1, Emmanuel Raptakis2, Elisabeth Foerster-Waldl3, Guenter Allmaier1 1Vienna University of Technology, Vienna, Austria, 2Kratos Analytical, Manchester, United Kingdom, 3Medical University of Vienna, Vienna, Austria Black Elderberry (Sambucus nigra) trees grow in regions of moderate climate and have a long history in folk medicine to treat influenza, common cold or sinusitis. Little is known about the frequency of allergic reactions to elderberry since its anthesis overlaps with the seasonal allergy to grass and weed pollen. Furthermore patients suffering from heyfever are often not sensitised to a single allergenic plant, but appear to be polysensitised. Although type I allergic reactions with pollen of elderberry have been suspected during the last years, the diagnosis of allergy against elderberry might be underestimated. Therefore verification of the existence of elderberry allergy and identification of the allergens has to be done and are in the focus of this work. Comparative fragmentation study of polypeptide antibiotics by low energy-CID (ESI- and MALDI-ion trap) multistage MS and high energy-CID (MALDI-rTOF) MS/MS
Ernst Pittenauer1, Martin Zehl1, Omar Belgacem2, Robert Mistrik3, Guenter Allmaier1 1Vienna University of Technology, Vienna, Austria, 2Kratos Analytical, Manchester, United Kingdom, 3HighChem, Bratislava, Slovakia Chemically, polypeptide antibiotics, obtained from various microorganisms, constitute a very heterogeneous group of regularly and unusually linked polypeptides, such as N- and C-terminally blocked linear peptides, cyclic peptides, cyclic depsipeptides, side-chain cyclisized peptides, side-chain cyclisized lipopeptides as well as bicyclic peptides. Depending on the chemical nature of particular compounds, either [M+H]+- or [M+Na]+-precursor ions, in some cases also both, were selected for comparative fragmentation studies. The goal of this study is to find out the influence of different desorption/ionization techniques on the fragmentation behavior (parallels, differences) of these analytes by low energy-CID utilizing ion trap mass spectrometers. Finally, these data are compared with high-energy CID-data acquired on a vacuum MALDI-TOF/RTOF-instrument. An Automated Pre-Filtering Method to Select Mono-Isotopic Ion Peaks from MALDI Mass Spectral Data
JingWen Yao1, Mike May1, Shinichi Iwamoto2, Shigeki Kajihara2 1Shimadzu Research Lab. (Europe) Ltd. Manchester, 2Shimadzu Corporation, Kyoto, Japan In protein identification from tandem mass spectrometry, an accurate ion peak list from MS/MS spectra will provide a higher confidence in derivation of correct peptide sequence from de-novo sequencing software or database searching. De-novo sequencing software [1,2] has usually an increased requirement of peak list accuracy. A new automated pre-filtering program, specialised for MALDI_QIT and MALDI_PSD spectral data to select mono-isotopic ion peaks has been developed recently. The maximum ion peak information presented in a spectrum will be included in the final peak lists through processing the spectral raw data. The derived peak lists can be directly input into the de-novo sequencing software and database searching. Good results on the testing data have been obtained. Pattern Matching of MALDI - PSD - MS Spectra using Neural Network and Stochastic Simulation Techniques
Jennifer Broughton1, Sujeewa Alwis2, Michael P May1, Jim Austin2 1Shimadzu Research Laboratory Ltd., Manchester, UK, 2CybulaLtd, York, UK MALDI - PSD - MS/MS data is frequently utilised to try to identify the peptide sequence. However usually the raw data has to go through several processing steps. These generally consist of de-convolution of the data to determine ion peaks followed by submission to a database search engine. The search engines compare mass and/or intensity values between the experimental data and theoretical data stored in the database. However the nature of MS/MS data may vary for the same peptide with many factors including impurities in the protein substance, differences in fragmentation, differences in ionisation, and measurement inaccuracies. Therefore we have developed a pattern matching system to robustly identify similarities or partial similarities between intensity and mass distributions for non-perfect data. Development of an intact protein analytical platform for serum proteomics
Tetsuo Tanigawa1, Hirotaka Fujimoto1, Jun Takano1, Yasuhiko Takeda2, Mitsunori Hirano2, Kouji Meno3, Reiko Takano3, Takuya Katagiri3, Kazuhiko Uchida4 1Shimadzu Corp., 2NTT Comware Corp., 3MCBI Inc., 4Graduate School of Comprehensive Human Sciences, Univ. of Tsukuba The human body fluid such as serum, plasma and cerebrospinal fluid (CSF) contains many undiscovered proteins and small molecules, which can be used as drug target and biomarkers for diagnosis. However, these proteins would be present at various concentrations. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), which has been used as a standard approach in proteomics, has disadvantages in terms of the limited dynamic range and molecular mass range. Here, we present serum intact protein profiling using novel system consists of a multicompartment electrolyzer (MCE) and multi dimensional HPLC followed by direct MALDI-TOF MS and 1D-SDS-PAGE. MALDI-TOF MS covered low molecular weight proteins including peptides and 1D-SDS-PAGE detected high molecular weight proteins in human serum. Investigation of protease and chemical fragmentation of protein; The impact on sequence coverage and identification of post translational modifications (PTM)
Alan J Barnes, Neil Loftus, Rachel L Martin Shimadzu Biotech, Manchester, UK Determining protein identity was once considered an end point in experimental investigation of a target protein. The understanding of post translational modifications has now become just as important as knowing the identity of the protein. Modifications such as glycosylation and phosphorylation can determine whether there is biological function. Characterisation of these modifications can however be challenging as the modification may be unstable or be present on a peptide fragment that cannot easily be ionised by MALDI TOF MS due to preferential ionisation of other peptides within the sample. In these experiments, soluble proteins from Saccharomyces cerevisiae were separated by 2D PAGE. Proteins from this gel were excised, digested and analysed by MALDI TOF MS. High-throughput and sensitive method for N-terminal sequencing of proteins by MALDI mass spectrometry
Minoru Yamaguchi1, Takashi Obama1, Hiroki Kuyama1, Eiji Ando1, Taka-aki Okamura2, Norikazu Ueyama2, Takashi Nakazawa3, Shigemi Norioka4 1Shimadzu Corp., 2Graduate School of Science, Osaka Univ., 3Department of Chemistry, Nara Women's Univ., 4Graduate School of Frontier Biosciences, Osaka Univ. N-terminal sequencing of proteins is very important to confirm N-terminal processing, such as removal of signal peptides and initiator methionine residues. The Edman method has commonly been used to analyze amino terminal sequence of proteins . But the method has limitations in sensitivities and throughput. Peptide mass finger purint and postsource decay analysis coupled with detabase searches has alternatively employed for identification and sequencing of proteins. However these methods can not determine the N-terminal sequence of mature proteins. Therefore we have developed a novel method by facilitating specific isolation of the N-terminal peptides from tryptic digests of protein as sulfonic acid derivatives followed by their de novo peptides sequencing by MALDI PSD analysis. Investigation by LC-MALDI of the proteomic basis of a novel Immuno-adsorption therapy for Rheumatoid arthritis patients
Helen Montgomery1, Koichi Tanaka1, Cornelia Koy2, Bruno Ringel2, Susanne Drynda3, Joern Kekow3, Koichi Tanaka4, Michael O. Glocker2 1Mass Spectrometry Research Laboratory, Shimadzu, UK, 2Proteome Center Rostock, University of Rostock, Rostock, Germany, 3Rheumatology Clinics, University of Magdeburg, Germany, 4Koichi Tanaka Mass Spectrometry Research Laboratory, Shimadzu, Japan The autoimmune disease Rheumatoid arthritis (RA) results in chronic joint inflammation with subsequent destruction of the articular tissue. Recent advances in molecular medicine have initiated development of "biologicals" which sustainably reduce the disease activity in patients. However, as with all treatment regimes some patients do not respond. The percentage of non-responders can exceed 30%. Clearly, a need exists for alternative therapies for those patients who do not respond to current medication. An alternative RA treatment is immuno-adsorption therapy where plasma proteins are adsorbed on an affinity column. During therapy, plasma is transported through the column and returned to circulation. It is postulated that this removes (pathological) antibodies and immune-complexes leading to symptom remission and reduced disease activity. MALDI-IT/RTOF, MALDI-TOF/RTOF, AP-MALDI-IT and nESI-IT CID mass spectrometry of multi-site glycosylated saponins from Bacopa monnieri: a comparison of information content
Martin Zehl1, Ernst Pittenauer1, Leopold Jirovetz2, Omar Belgacem3, Emmanuel Raptakis3, Vijay Kaul4, Guenter Allmaier1 1Vienna University of Technology, Vienna, Austria, 2University of Vienna, Vienna, Austria, 3Shimadzu Biotech Kratos Analytical, Manchester, UK, 4Inst. of Himalayan Biresource Technology, Palampur, India Bacopa monnieri is a plant used in India’s ayurvedic system of medicine. It is found in marshy areas of India/USA. Being classified as a drug it is applied in the treatment of e.g. epilepsy besides having antipyretic properties. The plant has recently gained attention in the west as well, and commercial products are available claiming as having cognitive enhancing effects comparable to those of Ginkgo. Its bioactivity is considered to be related to triterpenoid saponins. The characterization of these compounds by various desorption/ionization techniques in combination with low and high energy CID mass spectrometry have been performed. The information content of the MSn spectra, particular from multi-site glycosylated saponins, and the applicability of the three desorption/ionization techniques, will evaluated. Simultaneous separation and detection of purine metabolites in biological samples by LC/MS
Satoshi Yamaki, Tomio Fujita CSC Shimadzu Corp. There are several inborn errors of nucleotide metabolism in humans that are mainly the result of abnormal catabolism of purine and pyrimidine. Catabolism of the purine nucleotides leads finally to production of uric acid which is insoluble and is excreted in urine as sodium urate crystals. Excess accumulation of uric acid leads to hyperuricemia, commonly known as gout. Nucleic acids and related components in urine and plasma therefore are the metabolites that are of diagnostic importance in inborn errors of purine metabolism. Measurement of these components currently performed by HPLC in food analysis and clinical practice. A sensitive and rapid LC/MS coupled with electrospray ionization method for simultaneous separation and determination of purine metabolites in body fluids was developed. Identification of Molecular Species of Phospholipids by combination of Neutral Loss Scanning and MS3
Mayuko Ishida1, Toshiaki Houjou2, Hiroki Nakanishi2, Shinichi Yamaguchi1, Junichi Taniguchi1 Yusuke Inohana1, Junko Iida1, Kozo Miseki1, Takao Shimizu2, Ryo Taguchi2 1Shimadzu Corp., 2Univ. of Tokyo To elucidate the function of phospholipids, it is necessary to analyze not only their classes and subclasses but also molecular species. Recently, in the analysis of phospholipids, the application of mass spectrometry (MS) has become increasingly popular. We found that electrospray ionization (ESI) MS3 analysis is effective for more detailed or accurate annotation of each molecular species. We established the system for analyses of molecular species of phospholipids with neutral loss scanning of the head group-relating mass values and succeeding MS3 analyses by selecting the resulting product ions as precursor ions for MS3 analyses. This method can be effectively applicable without preliminary LC separation of phospholipid mixture. Characterization of the lipids non-covalently bound to plasma derived human serum albumin using ultraviolet high-energy CID MALDI.
Omar Belgacem1, Gerald Stubiger3, Gunter Allmaier3, Christoph Kannicht2, Andrea Buchacher2, Katharina Pock2 1Shimadzu Biotech, Manchester, UK, 2Octapharma, Vienna, Austria, 3Institute for chemical Technologies and analysis, Vienna, Austria Human serum albumin (HSA) accounts for nearly 50 % of all human plasma proteins and has multiple physiological roles. It maintains the colloid osmotic pressure and transports fatty acids (FA), bilirubin, bile acids, drugs and toxins. The monitoring of hydrophobic ligands bound to albumin is of particular interest for the clinical and pharmaceutical use of albumin but it is also an important part of the protein characterization itself. Our interest was focused on the identification of esterified and non-esterified FA ligands of HSA prepared from a human plasma pool of approximately 2400 donors. The identification of complex lipids was our aim. In order to characterize these ligands, it was necessary to use different analytical techniques combined with MALDI MS-MS. A System for Rapid and Accurate Identification of Oligosaccharide Structures Using Observational MSn Spectral Library of Human Glycans
Akihiko Kameyama1, Norihiro Kikuchi3, Shuuichi Nakaya2, Hiromi Ito1, Takashi Sato1, Yoriko Takahashi3, Hisashi Narimatsu1 1AIST, Tsukuba, JAPAN, 2Shimadzu Corporation, Kyoto, JAPAN, 3Mitsui Knowledge Industry Co., Ltd., Tokyo, JAPAN Glycomics lags far behind proteomics because of the difficulties arisen from their structural complexities such as the variations of branching, linkage and stereo-chemistry. Recently tandem mass spectrometric techniques have been revealing that oligosaccharides might have characteristic fragment patterns. However, no practical method for glycan structural analysis with a wide range applicable to human glycomics currently exists. We describe here a strategy for the rapid and accurate identification of the oligosaccharide structures using only mass spectrometry. It is based on a comparison of the signal intensity profiles of multi-stage tandem mass spectra between the analyte and a library of observational mass spectra which are acquired from the large variety of the structurally defined oligosaccharides prepared by human glycosyltransferase library. Simulation of Characteristic Fragment Patterns on Tandem Mass Spectra towards Distinction of Complex type of N-Linked Oligosaccharide Isomers
Shuuichi Nakaya1, Akihiko Kameyama2, Norihiro Kikuchi3, Hiromi Ito2, Hide-ki Ishida4, Mitsuru Nakamura2, Takashi Angata2, Hisashi Narimatsu2 1Shimadzu Corporation, Kyoto, JAPAN, 2AIST, Tsukuba, JAPAN, 3Mitsui Knowledge Industry Co., Ltd., Tokyo, JAPAN, 4The Noguchi Institute, Tokyo, JAPAN Glycosylation is the most wide-spread post-translational modification in eukaryotes, however the role of oligosaccharides attached to proteins has been studied little because of the lack of a sensitive and easy analytical method for oligosaccharide structures. To develop a novel glycomics tool which can allow anyone to identify oligosaccharide very easily and quickly, we have recently started the construction of an observational MSn spectral library of oligosaccharides. However, since this approach requires the preparation of large variety of the structurally defined oligosaccharides, simulation of the fragmentation patterns for any given structures is desired as another powerful approach. We report here the novel approach to the simulation of fragment patterns for distinction of N-linked oligosaccharide isomers. A New Approach to Small Molecular Analysis using Matrix-Less Laser Desorption and a Hybrid Mass Spectrometer, Potential for Metabonomic Applications
Fan Xiang1, Joseph D. Cuiffi2, Annie Yun Wang1, Daniel J. Hayes2 1Shimadzu Biotech, Pleasanton, CA, 2NanoHorizons inc., State College, PA High-throughput drug compound molecular weight screening and structure elucidation is dominated by relatively slow electrospray techniques, and many machines in parallel are necessary to process large libraries. Laser desorption techniques (such as MALDI) are more suited for high-throughput analysis; however, interference from matrix ions has limited the use of this technique to small molecules. This work demonstrates a low cost, disposable platform for performing high throughput small molecule analysis based on a matrix-less, laser desorption and ionization technique. An automated hybrid Ion-Trap/TOF mass spectrometer was used for obtaining molecular weight and structure information. The data presented here demonstrate the feasibility of this technique by analyzing a natural products library. Peak classification in MS/MS using high precursor resolution ion trap
Shigeki Kajihara, Shinichi Iwamoto Shimadzu Corp. MS/MS is a powerful tool to identify the peptide easily. In MS/MS, it is possible to conduct the simple separation of the peptide when an isotopic peak cluster generated from a single peptide is selected as a precursor ion. However, it is difficult to separate some peptides in MS/MS if some isotopic peak clusters are overlapped in MS. The recent advanced ion trap technique enables to isolate a precursor ion at the resolution up to 3500 [1]. We suggest a novel method to classify the isotopic peaks to each peptide in the mixture using an ion trap with extremely high resolution capability. Proteome Study on Rice Responses Under Osmotic Stress
Hong Chen1, Xiaojuan Li2, Shihua Shen2, Jihong Lin1 1Shimadzu International Trading (shanghai) Co. Ltd., Beijing, Ch, 2Institute of Botany, CAS, Beijing, Ch Water availability is a major limiting factor for plant growth. To dissect the cellular responses to osmotic stress, three-day-old seedlings of rice were cultured in mild stress and severe stress, respectively. The extracted proteins were analyzed. Selective and Efficient N-Terminal Amino Acid Sequencing by Bis(terpyridine)ruthenium(II) labelling
Taka-aki Okamura1, Akihiro Ito1, Maki Kaneko1, Taku Iwamura1, Minoru Yamaguchi2, Hitoshi Yamamoto1, Norikazu Ueyama1, Hiroki Kuyama2, Eiji Ando2, Takashi Nakazawa3, Shigemi Norioka1, Seiki Kuramitsu1 1Osaka Univ., 2Shimadzu Corp., 3Nara Women's Univ. Recent improvement of mass spectroscopy has enabled rapid and sensitive determination of amino acid sequences. MS/MS spectra of peptides show both N- and C-terminal fragment ions, which give complicated and unclear results. Enhancement of N- or C-fragments simplifies the spectra and results easy assignment and correct sequencing. For this purpose, many researchers have been encouraged to develop new methods of specific labeling or modifications by use of cation, anion, and isotope. We have recently developed a new reagent having transition metal complex, [Ru(terpyridine)2]2+, which is cationic and shows characteristic isotope pattern due to the natural abundance (96Ru, 98Ru-102Ru, and 104Ru). In this paper, efficient N-terminal amino acid sequencing by use of this complex is described. Novel derivatization for stabilizing sialic acids in MALDI-MS
Sadanori Sekiya1, Yoshinao Wada2, Koichi Tanaka1 1Shimadzu Corp., 2Research Institute, Osaka MCH Mass spectrometry using MALDI is a powerful tool for the oligosaccharide analysis. However, MS analyses of the sialylated oligosaccharide often leads to the preferential loss of the sialic acid, or N-acetylneuraminic acid (NANA), moiety due to in- or post-source decay. We attempted to solve this problem by changing the carboxyl group of NANA into amide, namely amidation. The modification causes a mass decrease of 0.984016 units, but the use of nitrogen isotope 15N in an amine constituent minimizes the mass shift to +0.013019 units. This negligible mass shift significantly reduces the complexity of the mass spectrum and facilitates a database search. Herein we demonstrate that amidation is an effective derivatization for the analysis of the sialylated oligosaccharide. Negative-ion MALDI MSn for Identification and Quantification of Isomeric Fucosylated Oligosaccharide Mixtures
Junko Amano1, Koichi Tanaka2 1The Noguchi Institute, 2Shimadzu Corporation Fucosylation of sugar chains is very important for cell recognition and constantly regulated in various organs during development, differentiation and activation. Therefore, altered fucosylation is directly associated with various diseases such as infection, immunity and cancers. Identification of fucosylation is essential for diagnosis, pathogenesis and pathology. However, it is difficult to isolate fucosylated oligosaccharides and determine their structures without complicated separation procedures because several kinds of fucosyl linkages produce many isomers. To overcome these difficulties, we have developed a simple method for determination of structures and quantity of fucosylated oligosaccharides at sub-picomolar level. This method does not require prior separation even though isomeric fucosylated oligosaccharides are mixed. Structural determination and fragmentation pattern of complex carbohydrates compounds using MALDI-TOF MS
Noriyuki Ojima1, Omar Belgacem1, Martin Resch2, Andrew Bowdler1 1Shimadzu Biotech, Manchester, UK, 2Shimadzu Biotech, Duisburg, Germany Recent rapid progress of technological innovation in MS spectrometer enables to analyze carbohydrate structures by high quality MS/MS spectra of MALDI generated ions. MALDI Tof-Tof with re-accelerating mechanism increased the sensitivity and resolution of fragment ions and QIT TOF MS opened the way for MSn measurement with MALDI ion source. PSD and low energy CID are adopted as dissociation mechanism for most commercial MALDI tandem mass instrument. In these low collisional energy conditions, bond cleavage tends to occur at only weak bond. Therefore, the important structural information of compound cannot be occasionally given from MS/MS spectrum. We investigated branched carbohydrate structure as an example of complex compounds using high energy CID MS/MS spectra. Advances of Tandem MS Functions with a Digital Ion Trap
Li Ding, Francesco Brancia, Roger Giles, Sergey Smirnov, Eugeny Nicholaev SRL Driven by a digital waveform and using frequency scan, the digital ion trap provides wide mass scan range and yet very high mass resolution. Digital waveform is also more flexible so it can be tailored to suit different kind of ion manipulations. It can selectively generate a resolving DC by simply using asymmetric rectangular waveform (duty cycle other than 50%), giving rise to a fast precursor isolation. High resolution reverse scan can be achieved as a result of the fringing field correction/adjusting. The rectangular digital waveform provides a time window where the electric field in the ion trap is stationary, allowing low energy electrons to be introduced to the trapping region for ion dissociation. Improving the Fragmentation Efficiency of Tryptic Peptides by MALDI-PSD using Carboxypeptidase B Digestion
Matthew E. Openshaw, Joanne B. Connolly Shimadzu Biotech, Manchester, UK Gas-phase peptide fragmentation is thought to occur via the 'mobile proton' model in which, the ionising proton is proposed to initiate bond cleavage. In MALDI post-source decay (MALDI-PSD) experiments, the internal energy of the precursor ions is increased by increasing the laser power and the precursor ions fragment in the field free region of the TOF analyser. The high gas-phase basicity of lysine and arginine residues often results in poor fragmentation efficiency for tryptic peptides analysed by MALDI-PSD. Various methods have been described to facilitate fragmentation of peptides analysed by MALDI-PSD (1, 2). In this presentation, the use of carboxypeptidase digestion is investigated to increase the fragmentation efficiency of tryptic peptides analysed by MALDI-PSD. Improved Confidence in Protein Identification Using MASCOT via Peptide SPITC Derivatization
Joanne B Connolly1, Hakan Larsson2, Matthew Openshaw1 1Shimadzu Biotech, Manchester, UK, 2Pharmacia Diagnostics, Uppsala, Sweden Peptide fragmentation following PSD or MS/MS is not always easy to interpret - especially de-novo. Interpretation of the spectra can be complicated by the appearance of ions corresponding to multiple ion series which are hard to predict and difficult to assign. Low fragmentation efficiency and preferential fragmentation pathways can reduce the amount of sequence information. Derivatization with 4-sulfophenyl-isothiocyanate (SPITC) is a quick, easy and cost effective modification that can be used to modify peptide fragmentation. Derivatives promote efficient charge site initiated fragmentation of peptide backbone, selectively enhance detection of the y ion series, improve fragmentation efficiency, simplify interpretation of fragment spectrum, and allow de-novo sequencing. In this study we show improved confidence of protein identification from SPITC peptides using MASCOT. The automated multi-dimensional µLC-MALDI-MS peptide profiling system identified potential biomarkers for cancer
Hirotaka Fujimoto1, Tetsuo Tanigawa1, Toru Nozawa1, Takaaki Satou1, Yasuhiko Takeda2, Mitsunori Hirano2, Hideya Kuwabara3, Takahiro Inatsugi3, Kenichi Aoki3, Ken Aoshima3, Reiko Takano4, Takuya Katagiri4, Kazuhiko Uchida4,5 1Shimadzu Corp., 2NTT Comware Corp, 3Mitsui Knowledge Industry Corp., 4MCBI Inc., 5Graduate School of Comprehensive Human Sciences, Univ. of Tsukuba. Biomarkers are biological molecules that are indicators of physiologic state and also of change during a disease process. Mass spectrometry-based protein/peptide expression profiles are recently used as diagnostic biomarkers in several diseases including cancer. However, it is difficult to discover and identify the protein/peptide indicating disease status present in blood at low concentration. In order to identify sensitive and accurate diagnostic biological molecules in blood, we established a comprehensive and sensitive analytical platform using automated multi-dimensional mLC-MALDI-TOF MS. Structure determination of potential biomarker proteins derived from plasma of patients with HELLP syndrome by MALDI QIT Tof MS/MS sequencing
Cornelia Koy1, Juliane Heitner2, Martin Resch3, Michael Kreutzer1, Pablo Serrano-Fernandez1, Toralf Reimer2, Michael O. Glocker1 1Proteome Center Rostock, University of Rostock, Rostock, Germany, 2Department of Obstetrics and Gynecology, University of Rostock, Germany, 3Shimadzu Biotech, Duisburg, Germany Hemolysis, elevated liver enzymes, and low platelets syndrome (HELLP) is a serious, life-threatening variant of preeclampsia in pregnant women that can lead in up to 65% of all incidents to severe complications with both, maternal (3.5%) and infant death (25%). Neither reliable preclinical recognition nor effective prevention measures for HELLP patients are available. As the course of HELLP is incalculable, non-invasive and rapidly determinable, reliable diagnostic markers are urgently needed. We applied mass spectrometry-based screening approaches for a rapid and reliable characterization of potential marker proteins in combination with statistical methods to plasma samples from HELLP patients and from healthy controls, followed by in-depth analysis of post-translational modifications of proteins of interest using MALDI QIT ToF MSn technology. Optimisation of GLaD chemistry for comparative proteomics
Francesco Brancia1 , D Delneri2 1SRL, Shimadzu Biotech, Manchester, UK2Life Science Department University of Manchester UK Analytical techniques that combine stable isotopic protein, peptides labelling and mass spectrometric analysis are becoming increasingly popular for quantifying protein abundance. A guanidino-labelling derivatisation method, termed GLaD, was recently developed for comparative proteomics, combining differential guanidination and off-line liquid chromatography prior to MALDI analysis [1]. Concomitant isolation of both chemically modified variants of a labelled peptide is followed by mass analysis. In this paper we used this approach for characterising protein mixtures isolated from 2D gels obtained from E. coli and S. cerevisiae. In addition, a new generation of molecules which incorporate larger number of heavy atoms will be presented and applied to protein mixtures. Protein Ladder Sequencing Using MALDI-TOF MS
Keli Ou1, Chin Chin Yau2, Xin Pei Goh2, Binhui Wu3, Rupert Wilmouth3 Hiroyuki Jikuya1 1Agenica Research Pte Ltd/Shimadzu (Asia Pacific), 2Agenica Research Pte Ltd, 3Nanyang Technological Univ., Singapore In proteomics laboratories matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS) has been routinely used for intact protein molecular weight determination as well as protein identification via peptide mass fingerprinting. However amino acid sequence information is difficult to obtain using MALDI-MS. Recently, Zhong et al (Nature Biotechnology, 22, 1291-1296, 2004) reported a simple and effective method for protein ladder sequencing using microwave acid hydrolysis and MALDI-MS. Using this method we have successfully identified a 45 kDa protein and elucidated its possible post-translational modification. Also we have demonstrated that this technique can be applied to the gel-separated protein identification. |
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