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news & events / Posters for ASMS 2007 www.asms.org |
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MondayIonization efficiency study of matrix-assisted laser desorption ionization by picoliter inkjet printing sample preparation
For most of users, the sensitivity is the most important specification of mass spectrometer. In the cases of any instruments, the ion yield and ionization efficiency are directly related to the sensitivity. We tried to estimate the ionization efficiency of Matrix-assisted Laser Desorption/Ionization (MALDI), which is now one of the most important ionization methods for the mass spectrometric analysis of various kinds of materials. One of the major reasons why it is difficult to estimate the ionization efficiency of MALDI is that poor quantitativity of MALDI analysis by the heterogeneous of matrix crystal and sample distribution in sample dried spot. For solving this problem, we prepared the homogeneous and smaller diameter sample spot by picoliter inkjet printing sample preparation. An investigation into a Digital Ion Trap as an ion source for a Time of Flight mass spectrometer
Ion Trap – Time-of-flight (IT-ToF) has proved a powerful combination for many applications in mass spectrometry. In addition to marrying the advantages of both IT and ToF, the localization and cooling of the ions within the IT can further enhance ToF performance. IT-ToF instruments to date have employed high Q resonant LC circuits to generate the trapping waveform. The significant technical challenge of correctly terminating the RF trapping waveform has been subsequently overcome. However, the application of the digital ion trap technology to the IT-ToF instrument format still offers significantly more precise control of the trapping waveform termination sequence. In this work the implications for IT-ToF mass spectrometry has been investigated. Increasing fragment mass range by infrared multiphoton dissociation in a digital ion trap mass spectrometer
An Infrared multiphoton dissociation (IRMPD) is a promising candidate to identify the primary structure of proteins in tandem mass spectrometry. IRMPD does not suffer from the low-m/z cutoff limit for fragmentation, contrary to collision induced dissociation (CID), because the dissociation efficiency is not influenced on the trapping field. IRMPD can also form secondary and higher order fragments and, therefore, provides more sequence information of peptides than CID. A digital ion trap (DIT) can store higher than 10-kDa ions by changing the frequency of the waveform applied to the ring electrode with the constant amplitude. The purpose of this study is developing IRMPD technique with the DIT toward the top-down analysis of proteins. N-linked Gycosylation Profiling of Pancreatic Cancer Serum Using Capillary LC-MS (MS) and MALDI QIT TOF MS
Pancreatic cancer has the worst prognosis of any cancer, with a 5-year survival rate of <3%. The glycoproteome is one of the major subproteomes of human serum. Glycosylation plays a crucial role in various biological functions. Alterations of glycan structures are frequently observed in cancer cells of various tumors types. These alterations include increased glycan branching and increased Lewis antigen expression. The change of the cellular glycosylation profile has been shown to be causally related to aggressive cancer cell behavior such as tumor cell invasion and metastasis. Therefore, comparative studies of the carbohydrate chains of glycoproteins produced by malignant cells, as compared to their corresponding normal cells, may provide useful information for the diagnosis, prognosis and immunotherapy of tumors. Relative Quantification of Osteocyte Proteins using GLaD Chemistry combined with MALDI-ToF Analysis and Online-LC ES-IT-ToF Analysis
Osteoporosis, a disease of the bone, can be treated using bisphosphonates which prevent osteocytes, the most abundant cells in bone tissue, from undergoing apotosis. However, little is known about the molecular mechanisms of this reaction. Using a large-scale proteomics approach, combining 2D-PAGE with MS analysis, the proteome was mapped for mouse osteocytes with and without bisphosphonate treatment. Proteins identified as up- or down-regulated were subjected to differential isotopic labelling for relative quantification. One such method of relative quantification, termed GLaD(1), involves guanidination of lysine residues using isotopic variants of O-methylisourea. Here, we combine GlaD chemistry with two methods of MS analysis, MALDI-ToF, and online reversed-phase liquid chromatography prior to micro electrospray analysis, to relatively quantify osteocyte proteins. TuesdaySeparation and Identification of Chlorinated Phenols Using Liquid Chromatography with Ion Mobility Spectrometry
Chlorinated phenols have widely been used in the production of pesticides and are environmental pollutants of great health concern. Chlorophenols have been widely studied either using gas-liquid or high performance liquid chromatography (HPLC) with conventional methods of detection. An alternative approach for the separation and characterization of chlorophenols is to electrospray the effluent from a liquid chromatograph and subsequent detection of ions in air at ambient pressure using an ion mobility spectrometer (IMS). The combination of IMS with HPLC offers an extra dimension for analysis and the ability to relate drift time to molecular structure. In the present work a complex set of chlorophenols has been analyzed by HPLC-ESI-IMS. MALDI-TOF/RTOF mass spectrometry of peptides at collision energies of 20kV and 2kV – differences in ion intensities and/or fragment types
Bradykinin potentiating peptides (BPPs) from five different snake venoms (B. jararaca, B. jararacussu, B. alternatus, B. moojeni, C. d. terrificus), with special emphasis on peptides containing pyro-glutamic acid (pGlu), were studied by MALDI-RTOF-MS and MALDI-TOF/RTOF-MS. Collision-induced-dissociation (CID) induced fragmentation pattern were compared and evaluated for peptide identification. Fragmentation was performed at full kinetic energy provided by the ion source (Elab 20kV, of a MALDI-TOF/curved field reflectron (CFR)-MS) with differential pumping and reduced kinetic energy with a deceleration and acceleration step prior to and after CID (2 kV, MALDI-TOF/classical reflectron (RTOF)-MS). A closer look on typical high energy fragment ions was taken to track differences between these two "so called" high energy CID types in terms of fragment intensities and/or types. CID fragmentation of cyclic caprolactam adduct ions depending on ionisation mode and collision energy
It is known that protonation can trigger fragmentation in CID experiments and variation of adduct ions can result in different fragmentation spectra. Since introduction of MALDI TOF/TOF technology high energy CID experiments that were accessible only using sector field instruments can be performed on MALDI-TOF instruments. High energy CID experiments were performed to investigate the fragmentation differences between protonated species and alkali adduct ions of cyclic caprolactams (n = 4 and 5). Additionally, low energy CID MALDI and ESI spectra will be acquired for comparison. High performance structural analyses of glycopeptides by low-energy CID using MALDI-QIT-TOF mass spectrometer
Multi-stage tandem mass spectrometry (MSn) for glycopeptides is powerful tool for peptide and glycan sequencing in addition to the identification of glycosylation site. However, it is difficult to perform the structural analysis of glycopeptides by MS/MS with low-energy collision-induced dissociation (LE-CID) of the singly charged protonated ion. Because their molecular mass tend to be high due to the attachment of the glycans and fragmentation efficiency of the high molecular mass ion is generally low. In this study, we demonstrate high performance analyses of the singly charged protonated ion of high molecular mass glycopeptides (~m/z 7000) by MALDI-QIT-TOF mass spectrometer. Furthermore complementary analyses of glycopeptides by positive/negative ion-MSn are shown, and the advantage of negative ion-MSn will be discussed. Structure elucidation of Sildanafil analogues by MSn and accurate mass measurement
Sildenafil is the active ingredient in Viagra (Pfizer), a prescription medicine used for erectile dysfunction. Compounds with chemical structures similar to that of sildenafil have been illegally added to health foods, predominantly herbal preparations, which then pose potentially serious health threats. In this paper, we have developed a new approach in detecting Sildenafil-like compounds from an alcohol beverage that may have been contaminated. High accuracy MSn data acquired on a quadrupole ion trap-time of flight mass spectrometer was used in combination with multivariate analysis statistical tools to identify related components based on the relationship between common fragmentation ions and neutral loss information. Mass spectrometric elucidation of N-glycosylation to the light chain of a crystallizing cryoglobulin
Cryoglobulin generally has precipitating property at low temperature, which is thought to be a result of some conformational difference in the immunoglobulin. However the structural feature of the crystallizing immunoglobulin (cryocrystalglobulin) is still unclear. Here we report a structural profile of a crystallizing serum cryogloblin observed in a myeloma patient with skin ulcer. N-glycosylation on the light chain of the cryoglobulin has been elucidated by 1D SDS-PAGE of peptide N-glycosidase F (PNGase F) treatment. The N-glycan structure and N-glycosylation site were determined by mass spectrometry and chromatography. Development of a universal method for selective isolation of N-terminal peptides from proteins and their de novo sequencing
Both peptide mass fingerprinting and MS/MS ion search analysis have been used as powerful approaches to protein identification. But, they are not enough to resolve real primary structures of proteins in mature form, because many proteins undergo post-translational modifications (PTMs) to play their functions within living cells. As one of such PTMs, it has been elucidated that processing at N-termini of proteins deeply affects on half-life of each protein. Therefore, N-terminal analysis of proteins is important not only to identify target proteins but also to understand their roles in a cell. Here, we have developed a universal method for N-terminal sequencing of proteins. This method is effective for both blocked and unblocked proteins at their N-termini. C-Terminal derivatization of proteins via hydrazides for sequencing with MALDI-MS
The C-terminal sequencing of proteins suffers from the lack of reliable method for modification of the C-terminal carboxyl group, because the N-terminal amino groups of mature proteins are often blocked by post-translational modifications. Our previous method for the specific C-terminal modification of proteins based on oxazolone chemistry has been improved by successively converting the oxazolone to active ester, and finally to hydrazide, which can be used as a sole site in a protein for attaching to a compound for the preferential detection and facilitating sequencing with MALDI-MS. We will present the results of C-terminal charge- and fluorescent tagging of peptides using this method. Its application to C-terminal protein sequencing is also presented. Mass spectrometric determination of the N- and C- termini of zona pellucida glycoproteins from X. laevis eggs
Zhiguo Li1, Ruben T. Almaraz2, Jerry L. Hedrick2, Fan Xiang3, Andreas Franz1 1Department of Chemistry, University of the Pacific, Stockton, California, 2Department of Animal Sciences, UC Davis, Davis, California, 3Shimadzu Biotech Corp., Pleasanton, California The extracellular glycoprotein matrix of mature unfertilized Xenopus laevis eggs, which is called the vitelline envelope (VE), plays an important role in the specific sperm-egg binding during fertilization and in the protection of the early embryo. The VE consists of 5 zona pellucida (ZP) glycoproteins: ZPA, ZPB, ZPC, ZPD and ZPX. Among these glycoproteins, the N- and C-termini of ZPB and ZPC have been confirmed in our lab with MALDI-TOF and MALDI-QIT-TOF mass spectrometry. The objective here was to demonstrate a convenient method for the determination of N- and C-termini through trypsin digestion in the presence of 18O-water and mass spectrometry. N- and C-terminal sequence information and its relevance to biochemical processing of the ZP-proteins will be presented. WednesdayA statistical based approach in metabolite identification by high mass accuracy MSn analysis
Metabolites characterization is often performed at a relatively late stage of drug development. As part of a general recognition by the pharmaceutical industry and the regulatory authorities that more metabolites information needs to be gathered earlier than currently practiced there have been a number of approaches in enhancing metabolite identification with non-radio labeled parent drug. These include off-line analysis and the development of software tools to help accelerate metabolite identification. This paper describes the application of high mass accuracy MSn data analysis together with a partial least squares approach in data mining for novel metabolites. Structure and Performance of PCB Ion Trap Mass Spectrometer(PCBITMS)
A new type of linear ion trap mass spectrometer is described and tested. The ion trap mass spectrometer is built by 4 piece printed circuit boards, and its cross section has a rectangular shape. The surface conductor of each printed circuit board is fabricated to five sections, and each section is applied with different RF voltage for producing variable electric field within the ion trap for transferring, trapping and analyzing ions. We present some mass analysis results using this simple and low cost Printed Circuit Board Ion Trap Mass Spectrometer(PCB IT-MS). The preliminary experiments indicate that the mass spectrum has a reasonable signal-to-noise ratio, and a mass resolution of half height of 500 can be achieved at proper voltage distribution. A Density Functional Investigation on the Secondary Processes in Matrix-assisted Laser Desorption Ionization
Proton transfer is probably the most important type of secondary reactions observed in MALDI. Many analytes especially peptides and proteins are detected predominantly in protonated form. Recently, esterification was used to investigate how introduction of aliphatic chains varying in length can affect the MALDI response. It was found that while derivatization tended to increase detection in negative mode modification of acidic character showed a deleterious effect on formation of deprotonated species[1]. Here, we use a density functional theory method to calculate the energies of the gas-phase processes occurring after expansion in vacuo. The absolute protonation and deprotonation energies of native and modified peptides are determined considering that these neutral molecules react with the most common protonated matrices in the gas-phase. Metabolomic Profiling in Wheat Grains Using Automated Sample Preparation
Although it is well known that the spatial arrangement of metabolites in complex tissues is required before regulation of metabolism can be understood, this is not possible using existing technologies. Matrix assisted laser desorption ionisation mass spectrometric imaging (MALDI-MSI) is an emerging technology that avoids problems of sample disruption associated with extraction and has the potential to provide a metabolite profile at the single cell level. This would allow not only the metabolite profile to be determined but also more importantly the known heterogeneity of tissues to be examined. We have previously reported the examination of metabolite distribution in wheat using MALDI-MSI. Development of High-Vacuum MALDI-DIT-MS and Improvement of Throughput with Novel Ion Introduction Technique
Digital Ion Trap (DIT) is driven by rectangular wave high voltage. For mass scan, frequency of the wave voltage is scanned with fixed amplitude. Previously, usability of DIT with atmospheric ion source (ESI and AP-MALDI) was reported. AP-MALDI-DIT produced practical MS and MSn data for high mass proteins and peptides. It suggested that DIT-MS could be powerful tool in protein research. However, ions from atmospheric source spread spatially and fly slowly. To utilize rapid driving voltage start of DIT, faster ion source like High-Vacuum MALDI source would be better. In this study, we developed a High-Vacuum MALDI-DIT-MS and evaluated the performance. Moreover, we investigated “additional ion introduction” with experiments and simulation studies. Optimization of a MALDI interface for the Digital Ion Trap using simulations
The Digital Ion Trap (DIT) employs a rectangular waveform with fixed high voltage pulse amplitude. A mass spectrum is obtained by means of a highly accurate frequency scan rather than voltage amplitude as in all commercial ion trap instruments. Consequently, the upper mass limit during a scan is frequency limited. A prototype DIT instrument was designed and constructed with an ESI source, which efficiently provides multiply charged ions in the range up to 2kTh. Further development of the DIT instrument incorporates a MALDI source, where ions up to hundreds of thousands of Da units can be generated and the benefits of efficient high mass range trapping can be appreciated. Thermalization of MALDI ions in a High-Vacuum QIT-TOF Mass Spectrometer
Emmanuel Raptakis1, Dimitris Papanastasiou1, Omar Belgacem1, Michael Sudakov2 1Shimadzu Biotech, Kratos, Manchester, United Kingdom, 2Shimadzu Research Laboratories, Manchester, United Kingdom In modern MS, MALDI is one of the few methods of choice for obtaining intact gas phase biomolecular ions. MALDI ions are internally excited and can readily undergo fragmentation within a few tens of microseconds. In ion trap MS incorporating a high vacuum MALDI source, ions are susceptible to such uncontrolled fragmentation during injection. Inelastic ion-neutral collisions in the trap can further collisionally activate the ions and/or promote internal energy relaxation via vibrational-to-translational energy transfer. Thermalization can be favored over activation by an elevated pressure environment, which is however associated with many technical challenges such as maintaining a low pressure in the source as well as the high-vacuum requirements for ejection into a TOF mass analyzer. Mass Analysis of Ion Trap Array (ITA)
Xiao-Xu Li1, Gongyu Jiang1, Fuxin Xu1, Peng Yang1, Chan Luo1, An Hu1, Li Ding2, Yuan-yuan Wang1, Chuan-Fan Ding1 1Fudan University, Shanghai, China, 2Shimadzu Research Laboratory (Europe), Manchester, United Kingdom Ion Trap Array (ITA) is constructed by two parallel printed circuit board plates.Each plate is fabricated to several parallel electric segments which are placed symmetrically to those on the opposite plate.Every electric segment is supplied with a RF voltage,and the voltage polarity of adjacent segment is opposite.Without partitions between the djacent trap regions,it still produces mainly quadrupole field between the pposite electric segments and forms multiple trapping regions.It differs from the Multiplexed Rectilinear Ion Trap[1] where ions are collected in separated cells.The preliminary experiments indicate that each ion trap in ITA can have normal property of an ion trap.Hundreds mass resolution can be easily achieved at this moment. Identification and mass spectrometric characterization of isomeric isoflavone aglycones by ion trap time-of-flight mass spectrometry
The main fragmentation pathway of isoflavones was RDA reaction(Retro Diels–Alder reaction). Fragments such as [M+H]+, 1,3A+, 1,3B+, [1,3A-28]+ were observed in the spectra. For most of the methy-lated isoflavones, the loss of a CH3 radical appeared to be prevalent. Accordingly, the [M+H-CH3]+ ion dominated the whole spectrum (Cuyckens et al., 2004; Ma et al., 1997; Wolfender et al., 2000). How-ever, during the current MSn analysis, under the same experimental conditions, compared to the isofla-vones with C-4’methoxylic substituent, it was found that methylated isoflavones with single meth-oxylic group in the C-7 site presented RDA fragments as base peaks and lacked fragments of [M+H-CH3]+ in the positive mode. Comparison of different algorithms for determining peptide abundance ratios in online LC based proteomics experiments
Relative protein quantification relies largely on the use of differential stable isotope labeling. This strategy can be achieved in vivo through metabolic labeling, or in vitro via chemical derivatization of the protein or their corresponding proteolysis products. Determination of the abundance ratio between the light and heavy version of an isotopically labeled peptide can be obtained from the corresponding extracted ion chromatogram. In order to quantify accurately, a clear understanding of the effect of the parameters used in data processing is crucial. Here, a comparison between methods employing both i)the type of data used (area vs intensity) and ii)the algorithms for determining peptide abundance ratios is made using peptide mixtures labeled with the GLaD chemistry. Improvement of oligosaccharide and glycopeptide analyses by using an ionic liquid matrix
Since Armstrong et al. introduced effective ionic liquid matrices (ILMs), several ILMs were applied in the MALDI analyses. One of the attractive advantages of ILMs is homogeneity of spot surface and thereby high reproducibility. In 54th ASMS, MP455, 1,1,3,3-tetramethylguanidium (TMG) salt of α–cyano–4–hydroxy–cinnamic acid (G2CHCalifornia) was reported as ILM for sulfated oligosaccharides to suppress the loss of sulfate in positive ion extraction, where more than 10 pmol amount of samples was used. Herein, we demonstrate high sensitive identification of sulfated/sialylated/neutral oligosaccharides and preferential ionization of glycopeptides among digests of glycoprotein by optimizing the new ILM: TMG salt of p–coumaric acid (GCalifornia) and G2CHCalifornia in positive and negative ion extractions. New approaches for the structural characterization of a complex, sulfated oligosaccharide fraction isolated from human salivary MUC5b.
Sarah Robinson1, Akraporn Prakobphol1, Rachel Martin2, H. Ewa Witkowska3, Steve C. Hall3, Susan J. Fisher1 1Department of Cell and Tissue Biology, UCSF, San Francisco, California; 2Shimadzu-Biotech, Manchester, United Kingdom; 3BRC Mass Spectrometry Facility, UCSF, San Francisco, California The high-molecular-weight human salivary mucin MG1 (MUC5b) is modified by specialized glycan structures that play a role in coordinating bacterial and leukocyte adhesion, which, in turn, we theorize is an important determinant of the oral ecology. Previously, this laboratory characterized the neutral and sialylated species that this mucin carries. The sulfated fraction has proven more resistant to structural elucidation due to its complexity, polydispersity (with estimated chain lengths of up to 41 residues) and chemical lability. Thus, we needed to develop novel approaches for the structural elucidation of these glycans, information that is crucial for drawing functional correlates. Characterisation of metabolites generated by mutant cytochromes P450 enzymes using a 3D ion trap-time of flight mass spectrometer
The cost of clinical development has escalated over recent years primarily as a result of the greater drug attrition rates. Clinicians and regulatory agencies are requiring more data from clinical trials, particularly concerning the pharmacodynamic behaviour of drugs. Cytochromes P450 (P450s) play a critical role in hepatic biotransformation of drugs and other xenobiotics. Their ability to form metabolites with altered pharmacologic or toxicologic characteristics has resulted in the need to fully understand the properties and activities of the P450s. In this paper wild type and mutant P450 isoforms are used to generate metabolites that have been characterised by reversed phase liquid chromatography coupled to high accuracy MSn analysis and novel software tools. Global Metabolite Profiling of Human Urine using LC-MS on an Ion-Trap Time of Flight hybrid Mass Spectrometer.
Global metabolite profiling is emerging as a powerful tool in a number of research and development fields including biomarker discovery, disease diagnosis and drug efficacy. NMR was first applied to profiling studies, principally as a result of strong quantitative data, however, MS provides higher sensitivity and has led to a greater acceptance of this technology in exploring profiling research. With the development robust ion source performance together with high mass accuracy and resolution, LC-MS has been applied to large sets of real samples (biological fluids, extracts etc) and enables subsequent statistical analysis by multivariate statistics to identify key biomarkers and patterns of disease. This paper describes the application of high mass accuracy MSn analysis applied to human urine profiling studies. Comparison of different sample preparation techniques for differentiation of mycotoxin-producing Fusarium species by MALDI-TOF/RTOF mass spectrometry
Identification and characterisation of intact microorganisms by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) is utilized by a number of research groups, but only few of them focus on fungal cells, and particular on fungal spores. Fungi are of special interest since some of them are producing mycotoxins on food-relevant plants (e.g. corn or wheat), toxins affecting human health. Therefore rapid identification of intact fungal spores by generating surface peptide/protein fingerprints of fungi would be a quick and easy tool for rapid species identification and differentiation and finally to evaluate the pathogenicity of individual species. This strategy is of interest not only in the medical field, but of great relevance in agriculture and food production technologies. Proteomic experiments with an Ion Trap – Time-of-Flight (IT-TOF) MS utilizing a nanointerface
In the age of various “omics” research programs, proteomics based experiments continue to be a large proportion of mass spectrometry based research. It is no surprise then that researchers are continually looking for improvements in chromatography methods and instrumentation. An ion trap – time-of-flight mass spectrometer combines a known workhorse in the proteomics field, the ion trap, with a medium resolving power detector, the reflectron time-of-flight. The advantages of both mass spectrometers are exploited in the hybridization of these instruments such as MSn capabilities and good mass accuracy and resolution. This work hopes to show the advantages of such a hybridization coupled to nanoESI for proteomics based experiments. ThursdayDetecting Compounds of Dissimilar Ionization Using Dual Source Ionization for Increased Throughput
To achieve a rapid and thorough analysis of complex or unknown samples, it is imperative to develop a system that results in a high probability of detection. Since compounds preferentially ionize based on both polarity and ionization method, it is critical to develop techniques that support multiple methods. Traditional dedicated ionization sources can hinder high throughput analysis, such as metabolic screening, by limiting the number of samples that can be examined within a set time frame. By combining electrospray and atmospheric pressure chemical ionization into a single run, instrument time can be halved allowing for a greater number of samples to be analyzed. Ion Guide and Quadrupole Mass Filters Employing a Digitally Controlled Waveform
Quadrupole mass filters and ion guides are used extensively in many types of mass spectrometers. Here, existing digital ion trap technology is applied to a quadrupole mass filter (QMF) / ion guide. Traditionally, the necessary RF trapping waveform is supplied by a highly tunes, fixed frequency, LC resonator circuit, and the QMF spectrum is achieved by scanning the RF and DC voltages with a fixed ratio. For a given instrument configuration the effective m/z range of operation is decided by the frequency of the resonator circuit. If the digital method is employed the frequency of operation can be freely chosen. Dynamic Pharmaco Metabolome of mouse brain with precise bio-molecule identification by MALDI QIT-TOF based high resolution MS microscopy
MS imaging, which can visualize the localization of molecules in the tissues, is a very attractive tool for a biological research field. In addition to an MS imaging, an identification of molecules which localize in tissues is very important. To achieve the high spatial resolution MS imaging and highly reliable identification of the biomolecule simultaneously, we developed an imaging mass spectrometer with high performance MSn Microscopy system utilizing a QIT-TOF. Using this apparatus, we analyzed brain tissues of mice with kainic acid stimulation time course. We observed the high spatial resolution MS images of the tissues and identified specific peaks which appeared at 30 minutes after the kainic acid stimulation. Forensic Identification of Ricin by MALDI-TOF/TOF Analysis
Frederick J. Cox1, E. Alex Jestel1, Joy M. Ginter2 1Battelle East. Science and Tech Center, Aberdeen, Maryland, 2Shimadzu Scientific Instruments, Inc., Columbia, Maryland Because of the ease of manufacture and previous case history, the threat of a ricin contamination incident continues to be a concern. Although field screening by immunological methods is useful, laboratory confirmation of ricin by mass spectrometric or other methods is required for forensic identification. Previous reports on mass spectrometric ricin identification in the literature have identified both MALDI-MS of the intact protein and peptide mass mapping (Darby et al.), as well as LC-ESI-MS/MS sequencing of the tryptic digest fragments (Fredriksson et al.), as suitable techniques. In this work, we attempt to combine these previous techniques using a MALDI-TOF/TOF platform, enabling rapid analysis of the intact protein, peptide mass mapping, and peptide sequencing, on a single instrument. Off-line capillary LC coupled to a glycan analysis system utilizing MALDI-QIT-TOF MS and an observed MSn spectral library
Glycans have great potential to be diagnostic and prognostic biomarkers. To find glyco-biomarkers, it is necessary to analyze glycans both qualitatively and quantitatively. However, we often encounter difficulties in both identifying glycans and separation of isomers due to their complexities in structure. Therefore, a convenient system which can simultaneous perform separation and identification of glycans is desired. Conventional LC-MS can not assure quantitative analysis unless stable isotope is used. In this context, we constructed a glycan analysis system which consists of a LC equipped with fluorescence detector and a MALDI-QIT-TOF MS equipped with structural analysis software utilizing MSn spectral library. Using this system, we performed the analysis of N-glycans in human IgG as an example. In vitro study of glycoxidative modified low density lipoproteins using a lipoproteomic approach
Low Density Lipoproteins (LDL) are the primary carriers of cholesterol esters (CEs) and triacylglycerols (TAGs) and their level in plasma can be correlated with cardiovascular disease (CVD). CVD is a complex pathophysiological process initiated by LDL modification in vivo. Diabetic patients who suffer from extensive and uncontrolled protein glycation are considered as persons of high CVD risk. More importantly, it now appears that modified LDL proteins or the constitutive modified lipids can trigger an uncontrolled immune response leading to CVD. We are applying a proteomics approach in parallel with a fast lipid profiling method in order to monitor changes of LDL simulated in vitro and to obtain a broader picture of the whole molecule complex. MSn analysis of natural nutraceutical supplements
Over the past 5 years, a rising trend in the use of over-the-counter alternative/complementary medicine has lead to a sharp growth in the global nutraceutical market. A recent herbal supplements market report estimated a 1.7% average annual growth rate from $5.5 billion in 2005 to $6.1 billion projected in 2011. This is highlighted by the widespread increase in the regular personal use of natural health supplements such as red-fruit anthocyanins, green-tea and ginseng. Typically, electrospray techniques have been used in the analysis of these products, here we investigate the use of MALDI-QIT-TOF-MS, in the identification and structural elucidation of biologically active ingredients in red fruit/vegetables, green-tea and ginseng; comparing and contrasting these results with those obtained using LCMS-IT-TOF-MS. Small Molecular Analysis using a TOF/TOF Mass Spectrometer, a Cationizing Matrix and µFocus MALDI Plate
In this presentation, a new approach to small molecular analysis using a TOF/TOF mass spectrometer, a cationzing matrix and µFocus MALDI plate is demonstrated. A TOF/TOF mass spectrometry was chosen for its capabilities of providing higher mass accuracy, throughput and MS/MS spectra. To limit interference of commonly used matrices in the low mass range during the analysis, a porphyrin molecule was used as a matrix in the MALDI TOF/TOF experiments. Homogeneous matrix and sample crystals were obtained by preparing a thin matrix layer on a µFocus MALDI plate and subsequent sample deposition on the matrix crystals. The data presented here demonstrate the feasibility of this approach by analyzing drug compounds. Characterization of glycoprotein isoforms separated by cIEF using MALDI-QIT with an on-plate digestion method
Variation of glycosylation modification on proteins is an interesting phenomenon and has been found to be related to a number of cell functions. With the resolution down to 0.02 pH units, cIEF is an ideal separation method to resolve glycoprotein isoforms which usually differentiate by their pIs. But the slow flow rate of CIEF and the electrolyte contained in the fractions does not allow efficient coupling of CIEF and mass spectrometry for characterizing glycoprotein isoforms with different glycosylation sites and glycan structures. In this work an on-plate digestion and enrichment strategy is developed to improve the collection of glycoprotein isoforms fractions from the capillary and enhance the signals of glycopeptides in mass spectra. Proteomic Analysis iof Markers Associated with Tumor Stage in Ovarian Serous Tissues Using MALDI-QIT-TOF-MS
Ovarian cancer remains the fifth leading cause of cancer deaths among women in the United States. The high mortality rate associated with ovarian cancer is due in part to the delay of diagnosis which results from the late clinical manifestation of ovarian tumors. There is an important need for diagnostic and prognostic markers for this disease. The heterogeneous nature of ovarian cancer is an obstacle to diagnosis of the disease. Identification of ovarian cancer biomarkers that can be used to classify tumors with respect to different stages should eventually lead to the development of more specific and effective strategies for detection, improved monitoring of tumor progression, and even targeting therapy. |
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