HIGH-PERFORMANCE MALDI-TOF-TOF FOR PROTEOMICS
Marvin L Vestal, Ceo
Virgin Instruments Corporation, Box 818, 60r Union Avenue, Sudbury, Ma 01776
Grant 5R44GM079832-03 from National Institute Of General Medical Sciences
Abstract: The goal of this project is to develop a new high-performance MALDI-TOF-TOF instrument that removes a major bottleneck limiting progress in proteomics. The new TOF-TOF produces high-quality MS-MS spectra orders of magnitude faster than current MS-MS instruments. Essentially all of the spectra produced are interpreted with corresponding speed. A prototype TOF-TOF instrument for demonstrating feasibility of a high-performance MALDI instrument that provides the speed, sensitivity, and dynamic range required for high-throughput proteomics was designed and constructed in phase I. The essential elements of this new instrument include a new simplified MALDI ion source designed to operate routinely 24/7 with 5 khz laser; a high-resolution first stage MS that provides mono-isotopic precursor selection up to at least m/z 4000 with no measurable loss in ion transmission and less than 1% transmission of adjacent masses; a new TOF-TOF analyzer incorporating high-resolution precursor selection and multiplex operation that allows multiple precursors to be selected and analyzed following each laser shot; and a high resolution second stage MS that incorporates automatic internal calibration of fragment spectra providing high mass accuracy on all fragment masses. Design and performance of these elements are described in the progress report for phase I. The goal of phase II of this project is to complete the development of a fully functional, commercially viable MS-MS system including all of these elements and to demonstrate the performance in applications to biological samples. The remaining work requires not only finalizing the design and construction of the complete system, including electronics and automatic controls, but also demonstrating the long-term reliability and ruggedness of the resulting elements and system. Final testing of the prototypes developed in phase II will be carried out in laboratories of our collaborators on applications requiring rapid and reliable analysis of biological samples with high sensitivity and broad dynamic range. In phase III a high-throughput, high-resolution, multiplexed MALDI TOF-TOF mass spectrometer with the attributes described above will be commercialized, rendering all previous MALDI MS-MS instruments obsolete. Addition of separations interfaces and automation developed in related projects provide integrated systems that may replace electrospray LC-MS-MS systems for many applications. It is widely recognized that a combination of tandem mass spectrometry with efficient separation is required to approach the sensitivity and dynamic range required for global analysis of biological fluids such as plasma, serum, urine, and CSF. Analysis of proximal fluids and tissue samples is somewhat less demanding in that the dynamic range required is substantially smaller, but the amount of total sample available may be more limited so that equal ultimate sensitivity is required. Multi-dimensional separation combined with mass spectrometry can provide the required dynamic range, but a large number of fractions are required and no current MS-MS technique comes within an order of magnitude of the required speed and sensitivity
Keywords: Algorithms; Arts; Automation; Biological; Blood Plasma; Calibration; Collaborations; Complex; Computer Programs; Computer software; Data; Detection; Development; Electromagnetic, Laser; Electronics; Elements; Engineering; Engineerings; Evaluation; Fingerprint; Goals; Hand; Ions; Label; Laboratories; Lasers; Liquid substance; MALD-MS; MALDI; MALDI-MS; Mass Spectrum; Mass Spectrum Analysis; Measurable; Measurement; Methods and Techniques; Methods, Other; Mono-S; MonoS; Operation; Operative Procedures; Operative Surgical Procedures; Peptides; Performance; Phase; Photometry/Spectrum Analysis, Mass; Plasma; Price; Price Lists; Price Lists [Publication Type]; Process; Production; Programs (PT); Programs [Publication Type]; Progress Reports; Proteomics; Radiation, Laser; Relative; Relative (related person); Reliability of Results; Reports, Progress; Research Resources; Resolution; Resources; Reticuloendothelial System, Serum, Plasma; SCHED; Sampling; Schedule; Scheme; Serum, Plasma; Software; Spectrometry, Mass; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Mass; Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Speed; Speed (motion); Staging; Surgical; Surgical Interventions; Surgical Procedure; System; System, LOINC Axis 4; Techniques; Testing; Time; Tissue Sample; Transmission; Urinary System, Urine; Urine; Validation; Work; amole; amole Agave lecheguilla extract; application in practice; base; computer program/software; cost; design; design and construction; designing; discount; exhaust; fluid; instrument; ion source; liquid; mass spectrometer; matrix assisted laser desorption ionization; measurement of metabolism; metabolomics; practical application; price lists; pricing; programs; prototype; public health relevance; surgery; tandem mass spectrometry; transmission process
Project start date: 2007-03-01
Project end date: 2010-04-30
Budget start date: 1-MAY-2009
Budget end date: 30-APR-2010
PFA/PA: PA-07-280
5R44GM079832-03 (2009): $145950
Sponsored Links Excellgen http://Excellgen.com
Grants awarded to Marvin L Vestal
IDENTIFICATION AND STRUCTURAL ELUCIDATION OF PROTEINS IN MALDI-TOF-TOF MS-MS
Marvin L Vestal
Virgin Instruments Corporation, Box 818, 60r Union Avenue, Sudbury, Ma 01776
Grant 1R44GM090389-01A1 from National Institute Of General Medical Sciences
Abstract: This project is focused on developing improved instrumentation and protocols for identification and structural elucidation of large peptides and intact proteins. The proposed instrument is optimized for application to doubly charged positive ions and provides unique capabilities for MS-MS of intact proteins ionized by MALDI. Fragmentation mechanisms available include unimolecular fragmentation following excitation in the MALDI ion source (PSD), and electron transfer dissociation (ETD) from reactions of small negative ions with doubly charged intact proteins. The approach is based on recent advances in MALDI-TOF and TOF-TOF technology but requires addition of a second pulsed ion source and a new TOF analyzer for fragment ions that features up to 45 kV acceleration to achieve high performance on high mass, high energy ions. The ion optics are designed so that beams from the two sources can be merged within the collision region with the pulses accurately synchronized in time, and with the relative velocity of the beams accurately determined. For collisions between positive and negative ions the relative velocity can be set very close to zero, providing sufficient time for efficient ion chemistry to occur. A novel element in this instrument is an additional pulsed accelerator in the field-free space adjacent to the ion source that effectively reduces the velocity spread of selected ions and allows high resolution precursor selection simultaneously with high resolution measurements of fragment spectra. The first product resulting from this work is a new TOF-TOF that produces high-quality MS-MS spectra on intact proteins. This instrument generates very high quality MS-MS spectra at unprecedented speed and with very high sensitivity. In combination with a new LC interface with MALDI being developed in a separate project, an integrated LC-MS-MS system incorporating the new TOF-TOF will provide throughput more than an order of magnitude higher than any existing LC-MS-MS system, whether electrospray or MALDI, and will confidently identify many more proteins at low concentrations in complex biological samples. Because this instrument is designed for analysis of intact proteins, it will be particularly valuable for monitoring post-translational modifications that are frequently missed using bottom-up approaches for protein analysis. Functional proteomics requires characterization of proteins at the phenotypic level rather than merely gene level identifications. It is necessary to characterize the entire peptide sequence of a protein and post-translational modifications must be fully detected. The MALDI-TOF-TOF with ETD developed in this project provides accurate molecular weight and complete, unambiguous sequence, including unusual amino acids and post-translational modifications, on proteins and large peptides present at trace levels in complex mixtures. Speed, sensitivity, and dynamic range are substantially superior to the performance presently possible with other technologies
Keywords: ACTH; ACTH (1-39); APF-1; ATP-Dependent Proteolysis Factor 1; Acceleration; Adrenocorticotropic Hormone; Adrenocorticotropin; Affinity; Amino Acid Modification, Posttranslational; Biological; CYP; Caliber; Charge; Chemicals; Chemistry; Complex; Complex Mixtures; Computer Programs; Computer software; Corticotropin; Corticotropin (1-39); Cross Reactions; Cytochromes; DSS1; Data; Deleted In Split-Hand/Split-Foot 1 Region; Deleted in Split-Hand/Split-Foot 1 Region Gene; Detection; Development; Diameter; Dissociation; ECD; Electromagnetic, Laser; Electron Transport; Electronics; Electrons; Elements; Engineering; Engineerings; Evaluation; Gamma Globulin, 7S; Gaussian Distribution; Genes; Goals; H+ element; HMG-20; Hand; High Mobility Protein 20; Humulin R; Hydrogen Ions; IgG; Immunoglobulin G; In element; Indium; Instrumentation, Other; Insulin; Insulin (ox), 8A-L-threonine-10A-L-isoleucine-30B-L-threonine-; Insulin, Regular; Ions; Laboratories; Lasers; Liquid substance; MALD-MS; MALDI; MALDI-MS; Measurement; Methods and Techniques; Methods, Other; Molecular Weight; Monitor; Myoglobin; Negative Beta Particle; Negatrons; Normal Distribution; Normal Statistical Distribution; Novolin R; Operation; Operative Procedures; Operative Surgical Procedures; Optics; Peptides; Performance; Phase; Physiologic pulse; Post-Translational Amino Acid Modification; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Amino Acid Modification; Posttranslational Modifications; Price Lists; Price Lists [Publication Type]; Process; Production; Programs (PT); Programs [Publication Type]; Protein Analysis; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; Proteomics; Protocol; Protocols documentation; Protons; Pulse; Radiation, Laser; Reaction; Reagent; Relative; Relative (related person); Reliability of Results; Resolution; SCHED; SEM1; SHFD1; SHFDG1; SHFM1; SHFM1 gene; SHSF1; Sampling; Schedule; Science of Chemistry; Software; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Speed (motion); Surgical; Surgical Interventions; Surgical Procedure; System; System, LOINC Axis 4; Techniques; Technology; Testing; Thioredoxin; Time; Ubiquitin; V (voltage); Validation; Width; Work; aminoacid sequence of peptide; aminoacid sequence of protein; base; computer program/software; cost; design; design and construction; designing; discount; electron transfer; fluid; gene product; improved; instrument; instrumentation; ion source; ionization; liquid; matrix assisted laser desorption ionization; molecular mass; novel; peptide sequence; price lists; programs; protein aminoacid sequence; prototype; public health relevance; surgery; voltage
Relevance: Relevance Functional proteomics requires characterization of proteins at the phenotypic level rather than merely gene level identifications. It is necessary to characterize the entire peptide sequence of a protein and post-translational modifications must be fully detected. The MALDI-TOF-TOF with ETD developed in this project provides accurate molecular weight and complete, unambiguous sequence, including unusual amino acids and post-translational modifications, on proteins and large peptides present at trace levels in complex mixtures. Speed, sensitivity, and dynamic range are substantially superior to the performance presently possible with other technologies
Project start date: 2010-09-01
Project end date: 2011-02-28
Budget start date: 1-SEP-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-09-080
1R44GM090389-01A1 (2010): $100000
NEW SEPARATIONS FOR MALDI-TOF MASS SPECTROMETRY
Marvin L Vestal, Ceo
Virgin Instruments Corporation, Box 818, 60r Union Avenue, Sudbury, Ma 01776
Grant 5R44GM079833-03 from National Institute Of General Medical Sciences
Abstract: The ultimate goal of this research is to provide integrated systems that allow complex biological samples to be analyzed with high sensitivity and broad dynamic range. These systems are applicable to proteins, DNA and RNA oligomers, and metabolites from bodily fluids and tissues. This project is specifically focused on developing novel sample plates employing collimated hole structures that allow a variety of separation techniques to be efficiently coupled to MALDI-TOF and TOF-TOF mass spectrometry. Phase I established the basic feasibility of the new MALDI plates for interfacing with several widely used separation techniques including HPLC and slab gel electrophoresis, and with direct imaging of frozen tissue sections. Phase II will produce a new family of MALDI sample plates optimized for these applications and devices for interfacing with a variety of techniques for efficiently separating peptides, proteins, and metabolites. These devices integrated with new MALDI-TOF MS and MS-MS instruments being developed in parallel projects will provide orders of magnitude improvements in capabilities for global analysis of complex biological samples. Specific aims for Phase II are focused on practical MALDI sample plates employing collimated hole structures optimized for selected applications, and on practical devices for interfacing MALDI MS and MS- MS with established techniques for efficiently separating peptides, proteins, and metabolites. The definition of a practical CHS plate for these applications is that it must be sufficiently inexpensive to be disposable after single use, or there must be a simple, reliable procedure for cleaning the plates with no detectable sample carry-over. These new MALDI sample plates, parallel digestion plates, and the apparatus and reagents required for their use will provide important new products for commercialization in Phase III. In addition, complete integrated systems combining output of this project with results from other projects being pursued concurrently at Virgin Instruments Corp. will provide products with much larger potential markets. These include a fully automated LC-MALDI MS-MS system for global biological analyses; an automated "Molecular Scanner" for protein analyses using gels and MALDI MS and MS-MS; integrated global analysis laboratory; and an automated high-speed tissue imager. The human genome project has established the molecular approach to understanding biology, but present knowledge of how an organism functions at the molecular level is really very poor. Functional analyses must be carried out, not only at the level of gene expression (transcriptomics), but also at the level of protein translation and modification (proteomics), and the metabolite network (metabolomics). Further improvements in chemistry, separations science, mass spectrometry, and bio-informatics will be required to complete this revolution. The interface between separations and mass spectrometry is believed to be a major bottleneck. The proposed research will provide important new tools toward overcoming this obstacle
Keywords: Abscission; Address; Bio-Informatics; Bioinformatics; Biological; Biology; Blood capillaries; Body Tissues; Caliber; Capillaries; Capillary; Capillary, Unspecified; Chemicals; Chemistry; Chromatography, High Performance Liquid; Chromatography, High Pressure Liquid; Chromatography, High Speed Liquid; Complex; Coupled; DNA; Deoxyribonucleic Acid; Deposit; Deposition; Detection; Development; Devices; Diameter; Digestion; Electrodeposition; Electroplating; Employee, Untrained; Enzymes, Immobilized; Excision; Extirpation; Family; Freezing; Gel; Gene Expression; Gene Products, RNA; Glass; Goals; HPLC; High Pressure Liquid Chromatography; Human Genome Project; Image; Immobilized Enzymes; Instrumentation, Other; Knowledge; Laboratories; Liquid substance; MALD-MS; MALDI; MALDI-MS; MALDI-TOF Mass Spectrometry; Manuals; Maps; Marketing; Mass Spectrum; Mass Spectrum Analysis; Matrix-Assisted Laser Desorption Ionization Time-of-Flight MS; Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry; Metal Plating; Methods and Techniques; Methods, Other; Modification; Molecular; Noise; Organism; Output; Peptides; Performance; Personnel, Untrained; Phase; Photometry/Spectrum Analysis, Mass; Procedures; Property; Property, LOINC Axis 2; Proteins; Proteomics; Protocol; Protocols documentation; R01 Mechanism; R01 Program; RNA; RNA, Non-Polyadenylated; RPG; Reagent; Recovery; Relative; Relative (related person); Removal; Research; Research Grants; Research Project Grants; Research Projects; Research Projects, R-Series; Resolution; Ribonucleic Acid; Sampling; Science; Science of Chemistry; Slice; Spectrometry, Mass; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Mass; Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Speed; Speed (motion); Spottings; Structure; Surface; Surgical Removal; System; System, LOINC Axis 4; Techniques; Technology; Testing; Tissues; Translations; Tripcellim; Trypsin; Untrained Personnel; Work; application in practice; capillary; commercialization; cost; design; design and construction; designing; detector; fluid; gel electrophoresis; gene product; imaging; improved; instrument; instrumentation; liquid; living system; mass spectrometer; matrix assisted laser desorption ionization; measurement of metabolism; metabolomics; molecular imaging; novel; practical application; professor; protein metabolite; prototype; public health relevance; resection; success; tool; transcriptomics; user-friendly; worker, untrained
Relevance: Relevance: The human genome project has established the molecular approach to understanding biology, but present knowledge of how an organism functions at the molecular level is really very poor. Functional analyses must be carried out, not only at the level of gene expression (transcriptomics), but also at the level of protein translation and modification (proteomics), and the metabolite network (metabolomics). Further improvements in chemistry, separations science, mass spectrometry, and bio-informatics will be required to complete this revolution. The interface between separations and mass spectrometry is believed to be a major bottleneck. The proposed research will provide important new tools toward overcoming this obstacle
Project start date: 2007-03-01
Project end date: 2011-04-30
Budget start date: 1-MAY-2010
Budget end date: 30-APR-2011
PFA/PA: PA-08-050
5R44GM079833-03 (2010): $375000
2R44GM079833-02A1 (2009): $375000
Low-cost High-performance MALDI-TOF MS And MS-MS
Marvin L Vestal, Associate Professor Of Chemistry
Virgin Instruments Corporation
box 818, 60r Union Avenue
sudbury, Ma 01776
Grant 1R43RR024290-01 from National Center For Research Resources IRG: ZRG1
Abstract: The goal of this project is to develop a new low-cost MALDI-TOF mass spectrometer that outperforms all existing MS-MS techniques currently employed with MALDI. In the proposed instrument multiple segments of fragment spectra are required to generate a complete fragment spectrum, each segment corresponding to a particular range of the ratio of fragment mass to precursor mass; but unlike earlier post-source decay (PSD) instruments, accurate fragment ion masses may be determined simultaneously for fragments present due to many of the precursor ions in the spectrum. Thus although 10-15 segments may be required to generate a complete fragment spectrum, 100 or more precursors can be fragmented without sacrificing sensitivity or mass accuracy. A pulse rate of 5 kHz is employed, allowing data to be acquired much faster than in existing TOF instruments typically limited to rates of 200 hz or less. The emphasis is on optimum performance in MS-MS mode, but resolving power and mass accuracy in reflector MS mode are comparable to the best currently available in commercial instruments. Either positive or negative ions can be measured in both MS and MS-MS modes. The unique features of the proposed instrument include simultaneous acquisition of fragment spectra from a large number of precursors, rapid and sensitive precursor and neutral loss scans with high resolving power and mass accuracy, and acquisition of multiplexed MS3 spectra. The design effort is focused on simplicity, reliability, and minimum cost consistent with high performance. A modular approach is employed where many of the major components are common to a family of systems. Common components include the sample plate handling system with motion control, the laser optics and controls, vacuum system, digitizer and computer, and several electronics modules. A novel method for calibrating fragment spectra is described based on inversion of the equation describing the flight time in a two-stage ion reflector to yield an "exact" equation relating fragment mass to flight time. An important initial effort is focused on validating this approach and determining the fragment mass accuracy that can be achieved. A major portion of the research effort is directed toward developing the software required for processing time-of-fight data to automatically generate fragment spectra from simultaneous measurements on multiple precursors. The software will be extended to generating precursor scans, neutral loss scans, and multiple reaction monitoring with LC separation, and to interpreting MS3 time-of-flight data. The system will be validated for applications to protein identification by MS-MS on tryptic peptides and for quantitative and qualitative measurements on complex lipid samples. The Human Proteome Organization has launched major initiatives focused on plasma, liver, and brain proteomics. The premise of these initiatives is that "A comprehensive, systematic characterization of circulating proteins in health and disease will greatly facilitate development of biomarkers for prevention, diagnosis, and therapy of cancer and other diseases". Omenn, Proteomics 2004, 4, 1235-1240. Progress on these initiatives was presented at the most recent HUPO meeting (Long Beach 2006). While many positive results were presented, an inescapable conclusion based on the lack of agreement among results from different laboratories on identical samples must be that present technology falls woefully short of that required to achieve the stated goals. This project, together with improved separations interfaces being developed in a separate project, will provide essential tools for comprehensive proteomic measurements
Keywords: ion, measurement, performance base, biomarker, brain, computer, diagnosis, family, fasting, health, human, laser, lipid, liver, neoplasm /cancer therapy, optics, peptide, phospholipid, plasma, prevention, protein, proteomics, teacher, university
Project start date: 2007-07-10
Project end date: 2008-06-30
1R43RR024290-01 (2007): $105000
HIGH-PERFORMANCE MALDI-TOF-TOF FOR PROTEOMICS
Marvin L Vestal, Ceo
Virgin Instruments Corporation, Box 818, 60r Union Avenue, Sudbury, Ma 01776
Grant 3R44GM079832-03S1 from National Institute Of General Medical Sciences
Abstract: The goal of this project is to develop a new high-performance MALDI-TOF-TOF instrument that removes a major bottleneck limiting progress in proteomics. The new TOF-TOF produces high-quality MS-MS spectra orders of magnitude faster than current MS-MS instruments. Essentially all of the spectra produced are interpreted with corresponding speed. A prototype TOF-TOF instrument for demonstrating feasibility of a high-performance MALDI instrument that provides the speed, sensitivity, and dynamic range required for high-throughput proteomics was designed and constructed in phase I. The essential elements of this new instrument include a new simplified MALDI ion source designed to operate routinely 24/7 with 5 khz laser; a high-resolution first stage MS that provides mono-isotopic precursor selection up to at least m/z 4000 with no measurable loss in ion transmission and less than 1% transmission of adjacent masses; a new TOF-TOF analyzer incorporating high-resolution precursor selection and multiplex operation that allows multiple precursors to be selected and analyzed following each laser shot; and a high resolution second stage MS that incorporates automatic internal calibration of fragment spectra providing high mass accuracy on all fragment masses. Design and performance of these elements are described in the progress report for phase I. The goal of phase II of this project is to complete the development of a fully functional, commercially viable MS-MS system including all of these elements and to demonstrate the performance in applications to biological samples. The remaining work requires not only finalizing the design and construction of the complete system, including electronics and automatic controls, but also demonstrating the long-term reliability and ruggedness of the resulting elements and system. Final testing of the prototypes developed in phase II will be carried out in laboratories of our collaborators on applications requiring rapid and reliable analysis of biological samples with high sensitivity and broad dynamic range. In phase III a high-throughput, high-resolution, multiplexed MALDI TOF-TOF mass spectrometer with the attributes described above will be commercialized, rendering all previous MALDI MS-MS instruments obsolete. Addition of separations interfaces and automation developed in related projects provide integrated systems that may replace electrospray LC-MS-MS systems for many applications. It is widely recognized that a combination of tandem mass spectrometry with efficient separation is required to approach the sensitivity and dynamic range required for global analysis of biological fluids such as plasma, serum, urine, and CSF. Analysis of proximal fluids and tissue samples is somewhat less demanding in that the dynamic range required is substantially smaller, but the amount of total sample available may be more limited so that equal ultimate sensitivity is required. Multi-dimensional separation combined with mass spectrometry can provide the required dynamic range, but a large number of fractions are required and no current MS-MS technique comes within an order of magnitude of the required speed and sensitivity
Keywords: Algorithms; Arts; Automation; Biological; Blood Plasma; Calibration; Collaborations; Complex; Computer Programs; Computer software; Data; Detection; Development; Electromagnetic, Laser; Electronics; Elements; Engineering; Engineerings; Evaluation; Fingerprint; Goals; Hand; Ions; Label; Laboratories; Lasers; Liquid substance; MALD-MS; MALDI; MALDI-MS; Mass Spectrum; Mass Spectrum Analysis; Measurable; Measurement; Methods and Techniques; Methods, Other; Mono-S; MonoS; Operation; Operative Procedures; Operative Surgical Procedures; Peptides; Performance; Phase; Photometry/Spectrum Analysis, Mass; Plasma; Price; Price Lists; Price Lists [Publication Type]; Process; Production; Programs (PT); Programs [Publication Type]; Progress Reports; Proteomics; Radiation, Laser; Relative; Relative (related person); Reliability of Results; Reports, Progress; Research Resources; Resolution; Resources; Reticuloendothelial System, Serum, Plasma; SCHED; Sampling; Schedule; Scheme; Serum, Plasma; Software; Spectrometry, Mass; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Mass; Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Speed; Speed (motion); Staging; Surgical; Surgical Interventions; Surgical Procedure; System; System, LOINC Axis 4; Techniques; Testing; Time; Tissue Sample; Transmission; Urinary System, Urine; Urine; Validation; Work; amole; amole Agave lecheguilla extract; application in practice; base; computer program/software; cost; design; design and construction; designing; discount; exhaust; fluid; instrument; ion source; liquid; mass spectrometer; matrix assisted laser desorption ionization; measurement of metabolism; metabolomics; practical application; price lists; pricing; programs; prototype; public health relevance; surgery; tandem mass spectrometry; transmission process
Project start date: 2007-03-01
Project end date: 2010-04-30
Budget start date: 1-MAY-2009
Budget end date: 30-APR-2010
PFA/PA: PA-07-280
3R44GM079832-03S1 (2009): $229050
New Separations For MALDI-TOF Mass Spectrometry
Marvin L Vestal, Associate Professor Of Chemistry
Virgin Instruments Corporation Box 818, 60r Union Avenue Sudbury, Ma 01776
Grant 1R43GM079833-01 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: Summary The ultimate goal of this research is to provide integrated systems that allow complex biological samples to be analyzed with high sensitivity and broad dynamic range. These systems are applicable to proteins, DNA and RNA oligomers, and metabolites from bodily fluids and tissues. The proposed project is specifically focused on developing novel sample plates employing collimated hole structures that allow a variety of separation techniques to be efficiently coupled to MALDI-TOF and TOF-TOF mass spectrometry. The first specific aim for the proposed research is an improved interface between HPLC and MALDI MS-MS that is compatible with 1 mm ID columns, operating at flow rates up to 50 ?L/min without sacrificing sensitivity for trace components. This will allow larger sample loadings and is expected to increase the dynamic range by at least a factor of fifty and allow faster, high-resolution separations. The second specific aim is to develop a direct interface between slab gel electrophoresis and MALDI-TOF-TOF that allows extraction, digestion, and concentration of samples on the MALDI plate with minimal sample loss and no loss in spatial resolution. A third specific aim is to develop a similar interface directly with frozen tissue samples both for imaging the intact proteins present, and for digesting and identifying the proteins by MALDI-TOF MS-MS. This project addresses a major bottleneck limiting the utility of mass spectrometry as an essential tool needed for truly global analyses of biological systems. It is now generally recognized that mass spectrometry interfaced with efficient separation technologies is essential for large-scale analyses of complex biological samples in functional proteomics and metabolomics; but currently available systems are inadequate. Improved MALDI-TOF and TOF-TOF mass spectrometry can provide the MS performance needed, but improved interfaces with separations are equally important. This Phase I project will establish the feasibility of a new approach to MALDI that allows parallel processing of large numbers of samples in a novel micro- fluidic system to achieve high throughput and broad dynamic range. A future Phase II effort will focus on developing practical systems that can be used by biologists in a variety of research applications. These will be developed and evaluated working with collaborators directly involved in such research. Recent advances in MALDI-TOF and TOF-TOF technology make this approach an attractive alternative to the established electrospray LC-MS-MS systems for quantitation and identification of proteins and peptides in complex biological samples. Despite some early success, adoption has been slow, probably because the potential advantages have not yet been clearly demonstrated. This project, together with improved MALDI-TOF-TOF instrumentation being developed in a separate project, will demonstrate the real advantages of this new approach.
Keywords: mass spectrometry, protein, DNA, RNA, capillary, capillary electrophoresis, digestion, fasting, fluid, forensic medicine, freezing, gel, gel electrophoresis, metabolomics, parallel processing, peptide, performance, proteomics, small molecule, solution, success, tissue
Project start date: 2007-03-01
Project end date: 2007-08-31
1R43GM079833-01 (2007): $100000
ISOTOPE RATIOS AT PPT LEVEL BY TOF-MS
Marvin L Vestal, Ceo
Virgin Instruments Corporation, Box 818, 60r Union Avenue, Sudbury, Ma 01776
Grant 1R43RR025257-01A1 from National Center For Research Resources
Abstract: Quantitative Measurement of Isotope Ratios at the part-per-trillion Level by TOF MS Summary This project is aimed at developing and demonstrating the performance of a new multi-stage laser desorption time-of-flight mass spectrometer that provides accurate measurement of the relative abundance of isotopes at very low levels. This instrument will ultimately provide performance at least equal to that achieved by established methods employing accelerator mass spectrometers at a very small fraction of the cost. The proposed benchtop instrument is small, highly automated and suitable for use by relatively untrained operators in a hospital or medical research laboratory. The major focus in this work is on those applications that require measurements of specific isotopes at levels below the part-per-billion level. These applications generally involve radioactive isotopes with very long half-lives (>1000 years). Specific examples include 14C for radiocarbon dating and biological tracer studies, and 41Ca used as a tracer for monitoring bone long term metabolic status in human patients. These applications often require precise determination of the relative abundance of isotopes at levels below 10-10 and extending down to 10-15. At present these measurements require use of a very large and expensive "accelerator mass spectrometer" (AMS) located in a central facility. Accelerator mass spectrometry has demonstrated the utility of long- lived radioisotopes as biological tracers, but applications have been limited by the relatively high cost and inaccessibility of complex instruments housed in central facilities. Nevertheless, support from the NIH, the pharmaceutical industry, and several AMS-based businesses led to a 2006 FDA guidance statement including AMS-based 14C microdosing (i.e., first-in-human "Phase 0" studies) of drug pharmacokinetics and pharmacodynamics, and AMS studies of 41Ca for cancer diagnostics have also seen early support from the NIH. The technique is limited to solid samples deposited on a suitable target, and it appears that the sample preparation procedures that have been developed for AMS can be employed with little or no modification in the proposed instrument. Building on the basic scientific work conducted using accelerator mass spectrometry (AMS), this project will enable broader application of these state-of-the-art research and diagnostic methods by providing inexpensive instruments with competitive performance that are suitable for routine use in clinical and medical research laboratories. For example, measurement of the ratio of 41Ca/Ca in urine and serum to evaluate the calcium metabolic status of patients may enable early detection and improved clinical management of osteoporosis, multiple myeloma, and cancer metastatic to the bone. It has been estimated that as much as 30% of the population may benefit from this test and this could lead to better quality of life and lower medical care costs. Multiple small business opportunities may be enabled by development of an affordable and easy to use analytical platform, versus the multi-million dollar facilities needed for AMS-based 41Ca
Keywords: Arts; Biological; Blood Coagulation Factor IV; Blood Serum; Bone Metastasis; Bone cancer metastatic; Bony metastasis; Businesses; C element; Ca++ element; Calcium; Cancer Diagnostics; Carbon; Characteristics; Clinical; Clinical Management; Coagulation Factor IV; Complex; Costs, Medical Care; Dependence; Deposit; Deposition; Detection; Development; Diagnostic Method; Diagnostic Procedure; Diagnostic Technique; Diagnostics, Cancer; Drug Industry; Drug Kinetics; Drugs; Early Diagnosis; Electromagnetic, Laser; Factor IV; HOSP; Half-Life; Half-Lifes; Hospitals; Housing; Human; Human, General; Industry, Pharmaceutic; Ions; Isotopes; Laboratories; Lasers; Lead; Life; Long Bone; Man (Taxonomy); Man, Modern; Measurement; Measures; Medical Care Costs; Medical Research; Medication; Metabolic; Metals; Metastasis to bone; Metastatic Cancer to the Bone; Metastatic Neoplasm to the Bone; Metastatic Tumor to the Bone; Metastatic malignant neoplasm to bone; Methods; Methods and Techniques; Methods, Other; Modification; Monitor; Morphology; Multiple Myeloma; Myeloma, Plasma-Cell; NIH; National Institutes of Health; National Institutes of Health (U.S.); Osseous metastasis; Osteoporosis; Patients; Pb element; Performance; Pharmaceutic Preparations; Pharmaceutical Industry; Pharmaceutical Preparations; Pharmacodynamics; Pharmacokinetics; Phase; Population; Preparation; Procedures; Property; Property, LOINC Axis 2; QOL; Quality of life; Radiation, Laser; Radioactive Isotopes; Radioisotopes; Radionuclides; Relative; Relative (related person); Research; Research, Medical; Sample Size; Sampling; Secondary cancer of bone; Secondary malignancy of bone; Secondary malignant neoplasm of bone; Serum; Skeletal metastasis; Solid; Spottings; Staging; Structure of long bone; System; System, LOINC Axis 4; Tag; Techniques; Testing; Time; Tracer; Transmission; United States National Institutes of Health; Urinary System, Urine; Urine; Work; accelerator mass spectrometry; base; bone loss; bone neoplasm secondary; cost; design; designing; drug/agent; early detection; heavy metal Pb; heavy metal lead; improved; instrument; ion source; ionization; long bone; mass spectrometer; myeloma; myelomatosis; public health relevance; stable isotope; transmission process
Relevance: Relevance Building on the basic scientific work conducted using accelerator mass spectrometry (AMS), this project will enable broader application of these state-of-the-art research and diagnostic methods by providing inexpensive instruments with competitive performance that are suitable for routine use in clinical and medical research laboratories. For example, measurement of the ratio of 41Ca/Ca in urine and serum to evaluate the calcium metabolic status of patients may enable early detection and improved clinical management of osteoporosis, multiple myeloma, and cancer metastatic to the bone. It has been estimated that as much as 30% of the population may benefit from this test and this could lead to better quality of life and lower medical care costs. Multiple small business opportunities may be enabled by development of an affordable and easy to use analytical platform, versus the multi-million dollar facilities needed for AMS- based 41Ca
Project start date: 2009-09-15
Project end date: 2010-03-14
Budget start date: 15-SEP-2009
Budget end date: 14-MAR-2010
PFA/PA: PA-08-050
1R43RR025257-01A1 (2009): $100000
COMPREHENSIVE DATABASE SYSTEM FOR MODERN MALDI- TOF AND TOF-TOF MS AND MS-MS
Marvin L Vestal, Ceo
Virgin Instruments Corporation, Box 818, 60r Union Avenue, Sudbury, Ma 01776
Grant 1R43RR030734-01 from National Center For Research Resources
Abstract: Our research is focused on developing integrated systems that provide analytical tools needed for truly global analyses of biological systems. The approach employs multi-dimensional separations coupled to high- performance MALDI TOF mass spectrometers via new three-dimensional sample plates using collimated hole structures (CHS) and monolithic supports as an alternative to established LC-MS-MS systems employing electrospray ionization. Recent advances in high performance ion trap mass spectrometers employing electrospray ionization and nanoflow liquid chromatography have significantly improved capabilities for analyzing biological samples, but this technology has serious inherent limitations on dynamic range and speed. Early applications of MALDI were limited by relatively low resolving power and mass accuracy of available TOF instruments, and the lack of a commonly available interface with liquid chromatography. Our recent work has overcome these limitations and high performance MALDI-TOF and TOF-TOF instruments interfaced with high capacity separations are now available. These instruments provide speed, resolution, dynamic range, and sensitivity orders of magnitude superior to the performance of commercially available instruments. These instruments and interfaces provide the performance required for global analysis of biological samples, but the database and bioinformatics tools proposed for development in this project are essential for converting this massive volume of data to useful information. Although global proteomics is clearly much more challenging than sequencing the human genome, the technology developed in this and related projects will provide the tools required for significant progress in the proteomics analog to the human genome project. The major remaining challenge involves converting the massive volume of data than can be produced by these instruments into information that can be readily used to address biological problems. This is the focus of the proposed project. Major initiatives by The Human Proteome Organization are focused on plasma, liver, and brain proteomics. The premise of these initiatives is that "A comprehensive, systematic characterization of circulating proteins in health and disease will greatly facilitate development of biomarkers for prevention, diagnosis, and therapy of cancer and other diseases." This project will demonstrate that the technology described in this proposal provides a feasible route to achieving the goals of these initiatives
Keywords: 2PP2A; Address; Amino Acids; Archives; Bio-Informatics; Bioinformatics; Biological; Blood Plasma; Blood Serum; Body Tissues; Brain; Campylobacter; Cancer Treatment; Chemical Fractionation; Chemicals; Complex; Complex Mixtures; Computer Programs; Computer software; Consensus; Coupled; Data; Data Banks; Data Bases; Databank, Electronic; Databanks; Database, Electronic; Databases; Detection; Development; Diagnosis; Digestion; Disease; Disorder; ESI; Electrospray Ionization; Encephalon; Encephalons; Enzymes; FRACN; Family; Fingerprint; Fractionation; Fractionation Radiotherapy; Genes; Genome, Human; Goals; HLA-DR Associated Protein II; Health; Human; Human Genome; Human Genome Project; Human, General; I2PP2A; IGAAD; Inhibitor of GZMA-Activated DNase; Ions; Isotopes; Label; Lipids; Liquid Chromatography; Liver; MALD-MS; MALDI; MALDI-MS; Malignant Neoplasm Therapy; Malignant Neoplasm Treatment; Man (Taxonomy); Man, Modern; Maps; Measurement; Modeling; Modification; Molecular Weight; Nervous System, Brain; Organ; Organism; PHAPII; Peptides; Performance; Phase; Phosphatase 2A Inhibitor I2PP2A; Plasma; Population; Post-Translational Modifications; Post-Translational Protein Processing; Posttranslational Modifications; Prevention; Process; Protein Modification; Protein Modification, Post-Translational; Protein Processing, Post-Translational; Protein Processing, Posttranslational; Protein/Amino Acid Biochemistry, Post-Translational Modification; Proteins; Proteome; Proteomics; Relative; Relative (related person); Reproducibility; Research; Resolution; Reticuloendothelial System, Serum, Plasma; Route; SET Translocation Inhibitor-2 of Protein Phosphatase-2A; Sampling; Sensitivity and Specificity; Serum; Serum, Plasma; Set protein; Software; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Speed (motion); Structure; System; System, LOINC Axis 4; TAF-IBETA; Technology; Template Activating Factor I Beta; Testing; Tissues; Tripcellim; Trypsin; Validation; Work; aminoacid; aminoacid sequence of peptide; aminoacid sequence of protein; analog; analytical method; analytical tool; anticancer therapy; application in practice; biological systems; biomarker; body system, hepatic; cancer therapy; clinical data repository; clinical data warehouse; computer program/software; data repository; database structure; design; designing; disease/disorder; experiment; experimental research; experimental study; gene product; improved; instrument; living system; mass spectrometer; matrix assisted laser desorption ionization; measurement of metabolism; metabolomics; multiple reaction monitoring; novel; organ system, hepatic; pathogen; peptide sequence; practical application; professor; protein aminoacid sequence; public health relevance; relational database; research study; response; small molecule; tool; virtual
Relevance: Relevance: Major initiatives by The Human Proteome Organization are focused on plasma, liver, and brain proteomics. The premise of these initiatives is that "A comprehensive, systematic characterization of circulating proteins in health and disease will greatly facilitate development of biomarkers for prevention, diagnosis, and therapy of cancer and other diseases." This project will demonstrate that the technology described in this proposal provides a feasible route to achieving the goals of these initiatives
Project start date: 2010-05-15
Project end date: 2010-11-14
Budget start date: 15-MAY-2010
Budget end date: 14-NOV-2010
PFA/PA: PA-09-080
1R43RR030734-01 (2010): $106041
MERGED BEAM MALDI TOF-TOF USING ION-ION REACTIONS TO DETERMINE STRUCTURE OF
Marvin L Vestal, Ceo
Virgin Instruments Corporation, Box 818, 60r Union Avenue, Sudbury, Ma 01776
Grant 1R21RR026206-01 from National Center For Research Resources
Abstract: Merged Beam MALDI TOF-TOF Using Ion-Ion Reactions to Determine Structure of Biological Molecules Summary The ultimate goals of this research are practical techniques for MALDI-TOF MS-MS on biologically significant molecules using electron transfer dissociation (ETD) of positive ions and proton transfer dissociation (PTD) of negative ions to determine molecular structure. This technology will provide the ability to study ion-ion collision processes under single collision conditions with direct measurement of the results in terms of charge transfer and fragmentation. Reaction cross sections for competing processes will be measured for several types of ion-ion collision processes. The emphasis in the fundamental studies is on reactions that lead to excited ions that dissociate following collision to produce structurally informative fragment ions. Singly charged precursors may be analyzed by employing double charge transfer to first produce excited neutrals that fragment efficiently, and then ionize the fragments by a second collision to produce fragment ions with charge opposite that of the precursor. In addition to fundamental knowledge, this research will provide practical techniques for determining molecular structure of singly and doubly charged molecular ions produced by MALDI. The approach should be applicable to a wide variety of molecules including intact proteins, peptides, oligonucleotides, oligosaccharides, glycoconjugates, lipids, and metabolites, and may provide speed, sensitivity, and specificity for identification and structural elucidation of unknowns that surpasses that available with electrospray ionization and ion traps. The instrument features addition of a second pulsed ion source to a recently developed high- performance MALDI-TOF-TOF MS-MS system. Pulsed beams from the two sources are merged within a collision cell with the pulses accurately synchronized in time, and with the relative velocity of the beams accurately determined and very near zero. The proposed apparatus will allow sensitive measurements of reactions between ions of opposite polarity and between ions and excited neutrals at low translational energies not previously accessible. The internal temperature (or excitation) of the reactants may be quite high but the translational temperatures are very low leading to very high cross sections for these processes. Collisions with reduced mass of 100 Da and relative velocity of 1 m/s correspond to translational energy of 5x10-7 eV, and translational temperature of 6x10-3 K. We are not aware of any previous work on ion-ion collision processes under these conditions. PUBLIC HEALTH RELEVANCE The "Holy Grail" of mass spectrometry for biological applications is an instrument that provides accurate molecular weight and complete, unambiguous sequence, including unusual amino acids and post- translational modifications, on proteins present at trace levels in complex mixtures. It should also provide similar information on mass and structure of other biologically significant molecules, including metabolites, lipids, oligosaccharides, glycoconjugates, oligonucleotides, and peptides. This may be it
Keywords: Affinity; Amino Acid Modification, Posttranslational; Biological; Cells; Charge; Chemicals; Complex Mixtures; Cross Reactions; Dissociation; ESI; Electromagnetic, Laser; Electron Transport; Electrons; Electrospray Ionization; Elements; Funding; Glycoconjugates; Goals; H+ element; Hydrogen Ions; Ions; Knowledge; Lasers; Lead; Lipids; MALD-MS; MALDI; MALDI-MS; Macromolecular Structure; Mass Spectrum; Mass Spectrum Analysis; Measurement; Measures; Methods and Techniques; Methods, Other; Molecular; Molecular Structure; Molecular Weight; Monitor; Negative Beta Particle; Negatrons; Oligo; Oligonucleotides; Oligosaccharides; Optics; Pb element; Peptides; Performance; Phase; Photometry/Spectrum Analysis, Mass; Physiologic pulse; Post-Translational Amino Acid Modification; Posttranslational Amino Acid Modification; Process; Production; Property; Property, LOINC Axis 2; Proteins; Protocol; Protocols documentation; Protons; Pulse; Radiation, Laser; Reaction; Reagent; Relative; Relative (related person); Research; Sensitivity and Specificity; Source; Spectrometry, Mass; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Mass; Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Speed; Speed (motion); Structure; System; System, LOINC Axis 4; Techniques; Technology; Temperature; Time; Work; design; designing; driving force; electron transfer; gene product; heavy metal Pb; heavy metal lead; insight; instrument; interest; ion source; ionization; matrix assisted laser desorption ionization; prototype; public health relevance
Relevance: Relevance The "Holy Grail" of mass spectrometry for biological applications is an instrument that provides accurate molecular weight and complete, unambiguous sequence, including unusual amino acids and post- translational modifications, on proteins present at trace levels in complex mixtures. It should also provide similar information on mass and structure of other biologically significant molecules, including metabolites, lipids, oligosaccharides, glycoconjugates, oligonucleotides, and peptides. This may be it
Project start date: 2010-01-01
Project end date: 2012-12-31
Budget start date: 1-JAN-2010
Budget end date: 31-DEC-2011
PFA/PA: RFA-RR-09-001
1R21RR026206-01 (2010): $156250
HIGH RESOLUTION MALDI-TOF MASS SPECTROMETER
Marvin L Vestal, Ceo
Virgin Instruments Corporation, Box 818, 60r Union Avenue, Sudbury, Ma 01776
Grant 5R44RR025705-02 from National Center For Research Resources
Abstract: High-performance MALDI-TOF MS for Peptides and Small Molecules Summary. This project will build on recent advances in MALDI-TOF mass spectrometry to develop new instruments and software that provide resolving power and mass accuracy competitive with electrospray ionization on FTICR and Orbitrap instruments, but with speed, throughput, sensitivity and dynamic range orders of magnitude greater than are possible with any electrospray system. In preliminary work a prototype instrument has been developed that provides greater than 30,000 resolving power at a selected mass, and ppm mass accuracy over a broad mass range on known protein digests. Based on preliminary results and theoretical calculations the ultimate resolving power is expected to be at least 100,000 in a small, inexpensive benchtop instrument with better than 1ppm RMS mass accuracy for all spots on a large format MALDI plate using automated internal calibration with no operator intervention and sensitivity to detect and accurately determine masses at the 1 attomole level. The long-range goal of this project is to provide practical commercial instruments that make the full power of high-performance MALDI-TOF mass spectrometry routinely available for difficult biological applications that are not satisfactorily served by the tools presently available. This effort will include technical advances in TOF MS and will also focus on the needs of specific applications and on developing reliable and cost-effective means for satisfying these needs. The proposed instrument is a vital component of new integrated analytical systems based on MALDI that will make large-scale proteomics and metabolomics practical. Completion of the human genome project and improved methods for analyzing proteins and small molecules in complex biological samples have been widely predicted to dramatically change drug discovery and clinical practice of medicine in the near future, but this has not yet been realized. Current technology is clearly not up to the task
Keywords: Area; Biological; Calibration; Chromatography; Chromatography / Separation Science; Collaborations; Complex; Computer Programs; Computer software; Detection; Drug Formulations; ESI; Electrospray Ionization; Elements; Engineering; Engineerings; Evaluation; Family; Formulation; Formulations, Drug; Fourier transform ion cyclotron resonance; Future; Gel; Goals; Human Genome Project; Instrumentation, Other; Intervention; Intervention Strategies; MALD-MS; MALDI; MALDI-MS; MALDI-TOF Mass Spectrometry; Maintenance; Maintenances; Marketing; Matrix-Assisted Laser Desorption Ionization Time-of-Flight MS; Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry; Measurement; Medicine; Methods; Operation; Operative Procedures; Operative Surgical Procedures; Peptides; Performance; Phase; Preparation; Price Lists; Price Lists [Publication Type]; Procedures; Production; Protein Analysis; Proteins; Proteomics; Protocol; Protocols documentation; Relative; Relative (related person); Reliability of Results; Research Design; Resolution; Sampling; Science of Medicine; Software; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Mass, Matrix-Assisted Laser Desorption-Ionization; Speed; Speed (motion); Spottings; Study Type; Surgical; Surgical Interventions; Surgical Procedure; System; System, LOINC Axis 4; Technology; Time; TimeLine; Work; base; clinical practice; commercialization; computer program/software; cost; design; designing; develop software; developing computer software; drug discovery; engineering design; fibrinopeptide; gene product; improved; instrument; instrumentation; interventional strategy; mass spectrometer; matrix assisted laser desorption ionization; measurement of metabolism; member; metabolomics; performance tests; price lists; prototype; public health relevance; small molecule; software development; study design; surgery; tool
Relevance: Relevance The proposed instrument is a vital component of new integrated analytical systems based on MALDI that will make large-scale proteomics and metabolomics practical. Completion of the human genome project and improved methods for analyzing proteins and small molecules in complex biological samples have been widely predicted to dramatically change drug discovery and clinical practice of medicine in the near future, but this has not yet been realized. Current technology is clearly not up to the task
Project start date: 2009-03-01
Project end date: 2012-02-28
Budget start date: 1-MAR-2010
Budget end date: 28-FEB-2011
PFA/PA: PA-08-050
5R44RR025705-02 (2010): $360740
Sponsored Links Excellgen http://Excellgen.com
High-performance MALDI-TOF-TOF For Proteomics
Marvin L Vestal, Associate Professor Of Chemistry
Virgin Instruments Corporation, Box 818, 60r Union Avenue, Sudbury, Ma 01776
Grant 1R43GM079832-01 from National Institute Of General Medical Sciences IRG: ZRG1
Abstract: DESCRIPTION () The goal of the goal of this project is to develop a new high-performance MALDI-TOF-TOF instrument that removes a major bottleneck limiting progress in proteomics. It is widely recognized that a combination of tandem mass spectrometry with efficient separation is required to approach the sensitivity and dynamic range required for global analysis of biological fluids such as plasma, serum, urine, and CSF. Analysis of proximal fluids and tissue samples is somewhat less demanding in that the dynamic range required is substantially smaller, but the amount of total sample available may be more limited so that equal ultimate sensitivity is required. Multi-dimensional separation combined with mass spectrometry can provide the required dynamic range, but a large number of fractions are required, and the MS and MS-MS measurements must be very fast and reliable or throughput is insufficient for practical applications. Several separation schemes have been developed or proposed that may produce up to 100,000 fractions/day for MS and MS-MS analysis with ca. 10-20 peptides per fraction on average. MS techniques are available that can approach the speed required for the MS measurement, but no current MS-MS technique comes within an order of magnitude of the required speed. The new TOF-TOF will produce high-quality MS-MS spectra orders of magnitude faster than current MS-MS instruments. Essentially all of the spectra produced will be interpreted with corresponding speed. The high speed is achieved by using a laser rate 25 times faster than previous MALDI instruments and multiple precursor ion selection following each laser shot. This instrument is unique in that it provides high-resolution precursor selection with MALDI MS-MS. Single isotopes can be selected and fragmented up to m/z 4000 with no detectable loss in ion transmission and less than 1% contribution from adjacent masses. This instrument also allows up to 50 fold multiplexing in MS-MS. Selected masses must differ by at least 1%, and are preferably within an order of magnitude range in intensity. This allows the generation of very high quality MS-MS spectrum at unprecedented speed. All of the peptides present in a complex peptide mass fingerprint containing a hundred or more peaks can be fragmented and identified without exhausting the sample. This allows speed and sensitivity of the MS-MS measurements to keep pace with the MS results. The combination of high-resolution precursor selection with high laser rate and multiplexing will allow high-quality, interpretable MS-MS spectra to be generated on detected peptides at the 10 attomole/?L level.Following completion of the human genome it was widely felt that "proteomics was the next big thing." Unfortunately, the proteomics factories spawned by this euphoria have failed to deliver on their promises and have gone out of business. The major problem is that the dynamic range is inadequate. The most abundant proteins are routinely detected by a variety of techniques, but lower abundance proteins are usually not detected, particularly if stringent conditions for confident identification are imposed. The limited dynamic range of mass spectrometry can be overcome by extensive fractionation of the sample, but this then requires that very large numbers of fractions must be analyzed. The new TOF-TOF system provides a practical solution to this problem.
Project start date: 2007-03-01
Project end date: 2007-08-31
1R43GM079832-01 (2007): $100000
METAL ISOTOPE RATIOS BY LD/TOF MASS SPECTROMETRY
Marvin L Vestal, Associate Professor Of Chemistry
Vestec Corporation 9299 Kirby Dr Houston, Tx 77054
Grant 1R43HD029334-01 from National Institute Of Child Health And Human Development IRG: SSS
Abstract: Adapted from Applicant s )The overall goal of this project is to develop a new laser desorption time-of-flight (LD/TOF) mass spectrometric technique for the determination of metal isotope ratios that can be readily applied to studies of the metabolism of biologically significant bulk and trace elements.The Phase I goals of this project are to demonstrate the feasibility of an instrument that combines the features of the high accuracy and precision of thermal ionization with the sensitivity, speed, and freedom from memory effects of the LD/TOF instrument. This new instrument should provide broad applicability to metals similar to inductively coupled plasma (ICP) ionization or FAB/SIMS mass spectrometry without the interfering matrix effects. Successful completion of the proposed research will provide a generally applicable mass spectrometric technique for metal tracer studies with better performance than that obtained by thermal ionization, and which is faster and easier to use and requires smaller absolute sample amounts.
Keywords: laser spectrometry, mass spectrometry, metal, method development, stable isotope, calcium, copper, iron, magnesium, potassium, trace element, zinc, isotope dilution method
Project start date: 1992-06-01
Project end date: 1993-03-31
1R43HD029334-01 (1992): $49990
ELEMENT AND ISOTOPE SPECIFIC DETECTION FOR HPLC
Marvin L Vestal, Associate Professor Of Chemistry
Vestec Corporation 9299 Kirby Dr Houston, Tx 77054
Grant 5R44GM043669-03 from National Institute Of General Medical Sciences IRG: SSS
Project start date: 1989-09-28
Project end date: 1994-03-31
5R44GM043669-03 (1992): $222957