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Rapid Characterisation of Low Molecular Weight Drug Compounds using High Energy CID MALDI MS/MS
| Session: Metabolite Identification using Hyphenated MS Techniques |
Code: MP08 |
Poster: 119 |
Rachel L Martin, Andrew Bowdler, Omar Belgacem
Shimadzu Biotech, Manchester, UK
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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.
Determination of Phosphorylation Sites using MSn on a MALDI QIT TOF MS
| Session: Proteins: Phosphoproteins |
Code: MP29 |
Poster: 491 |
Brian K Stall1+3, Etienne Waelkens2, Rachel L Martin1+3
1Shimadzu Biotech, Woburn, MA, 2K.U. Leuven, Leuven, Belgium, 3Shimadzu Biotech, Manchester, UK
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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.
High sensitive MS/MS analysis of MALDI QIT-TOF MS combined with Novel MALDI Surfaces for On-Chip Concentration
| Session: Proteomics: New & Improved Methods |
Code: MP32 |
Poster: 552 |
Yuzo Yamazaki1, Keisuke Shima1, Masaki Yamada1, Christopher M. Belisle2, Douglas P. Greiner2, John A. Walker II2
1Shimadzu Corp., 2LCI, Fremont, CA, USA
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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
| Session: Proteomics: New & Improved Methods |
Code: MP32 |
Poster: 558 |
Yuko Fukuyama, Shinichi Iwamoto, Koichi Tanaka
Shimadzu Corporation
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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.
Development of an intact protein analytical platform for serum proteomics
| Session: Proteomics: Biomarkers |
Code: TP24 |
Poster: 428 |
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
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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)
| Session: Proteomics: Fundamental - 2D Electrophoresis |
Code: TP25 |
Poster: 440 |
Alan J Barnes, Neil Loftus, Rachel L Martin
Shimadzu Biotech, Manchester, UK
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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
| Session: Proteomics: Fundamental - Other New |
Code: TP26 |
Poster: 442 |
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.
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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
| Session: Proteomics: Fundamental - Other New |
Code: TP28 |
Poster: 483 |
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
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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.
Simultaneous separation and detection of purine metabolites in biological samples by LC/MS
| Session: Clinical Chemistry |
Code: WP02 |
Poster: 032 |
Satoshi Yamaki, Tomio Fujita
CSC Shimadzu Corp.
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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
| Session: Lipids: Structural Analysis |
Code: WP13 |
Poster: 227 |
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
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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.
A System for Rapid and Accurate Identification of Oligosaccharide Structures Using Observational MSn Spectral Library of Human Glycans
| Session: Glycomics |
Code: WP14 |
Poster: 244 |
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
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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.
Peak classification in MS/MS using high precursor resolution ion trap
| Session: Computer App: Deconvolution |
Code: WP19 |
Poster: 345 |
Shigeki Kajihara, Shinichi Iwamoto
Shimadzu Corp.
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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.
Novel derivatization for stabilizing sialic acids in MALDI-MS
| Session: Carbohydrates/Oligosaccharides |
Code: ThP02 |
Poster: 025 |
Sadanori Sekiya1, Yoshinao Wada2, Koichi Tanaka1
1Shimadzu Corp., 2Research Institute, Osaka MCH
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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
| Session: Glycomics |
Code: ThP02 |
Poster: 026 |
Junko Amano1, Koichi Tanaka2
1The Noguchi Institute, 2Shimadzu Corporation
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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.
Advances of Tandem MS Functions with a Digital Ion Trap
| Session: Instrumentation: Mass Analyzers (Quadrupoles & Traps) |
Code: ThP13 |
Poster: 204 |
Li Ding, Francesco Brancia, Roger Giles, Sergey Smirnov, Eugeny Nicholaev
SRL
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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
| Session: Peptides: Fragmentation and Sequencing |
Code: ThP21 |
Poster: 333 |
Matthew E. Openshaw, Joanne B. Connolly
Shimadzu Biotech, Manchester, UK
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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
| Session: Peptides: Fragmentation and Sequencing |
Code: ThP21 |
Poster: 336 |
Joanne B Connolly1, Hakan Larsson2, Matthew Openshaw1
1Shimadzu Biotech, Manchester, UK, 2Pharmacia Diagnostics, Uppsala, Sweden
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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
| Session: Biomarkers and Mass Spectrometry |
Code: ThP28 |
Poster: 441 |
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.
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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.
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