click to add text overview of mass spectrometry sermin tetik, phd marmara university july 2015, new...
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Overview of Mass Overview of Mass Spectrometry Spectrometry
Sermin Tetik, PhDSermin Tetik, PhDMarmara UniversityMarmara University
July 2015, New OrleansJuly 2015, New Orleans
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An overwievAn overwievWhat is the mass spectrometer?What is the mass spectrometer?
What can mass spectrometers identify?What can mass spectrometers identify?
What are the different types of mass spectrometers?What are the different types of mass spectrometers?
Peptide fragmentationPeptide fragmentation
Databese searchingDatabese searching
Practical applications of mass spectrometryPractical applications of mass spectrometry
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What is a mass spectrometerWhat is a mass spectrometerA. It measure massA. It measure mass
B. It can give information about B. It can give information about chemical structurechemical structure
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1. Sample preparation1. Sample preparation
2. Ion source 2. Ion source
Generates ions Generates ions
3.Mass analyzer3.Mass analyzer
Separates ionsSeparates ions
4. Detector4. Detector
Mass spectrumMass spectrum
5. Analysıs5. Analysıs
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John B. Fenn Koichi John B. Fenn Koichi TanakaTanaka
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The mass-to-charge ratio is often referred The mass-to-charge ratio is often referred to as m/z and is typically unitlessto as m/z and is typically unitless
m: the mass number (atomic mass/U)m: the mass number (atomic mass/U)
z: the charge number (Q/e)z: the charge number (Q/e)
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-Drug discovery-Drug discovery
Determine structures of drugs and Determine structures of drugs and metabolitesmetabolites
Screen for metabolites in biological Screen for metabolites in biological systemssystems
-Clinical testing-Clinical testing
Perform forensic analyses such as Perform forensic analyses such as confirmation of drug abuseconfirmation of drug abuse
Detect disease biomarkers (e.g. Detect disease biomarkers (e.g. newborns screened for metabolic diseases)newborns screened for metabolic diseases)
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-Geology-Geology
Carbon Dating Carbon Dating
-Environment-Environment
-Residual gases-Residual gases
-Trace contaminants and toxins-Trace contaminants and toxins
-Test water quality or food -Test water quality or food contaminationcontamination
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B. ProteomicsB. Proteomics
Identification of biological material (proteins, Identification of biological material (proteins, nucleic acids, lipids)nucleic acids, lipids)
Determine protein structure , function, Determine protein structure , function, folding and interactionsfolding and interactions
Detect specific post-translational Detect specific post-translational modifications throughout complex biological modifications throughout complex biological mixturesmixtures
Quantitate (relative or absolute) proteins in Quantitate (relative or absolute) proteins in a given samplea given sample
Monitor enzyme reactions, chemical Monitor enzyme reactions, chemical modifications and protein digestionmodifications and protein digestion
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A. Top-down proteomics: A. Top-down proteomics: Identification of intact proteinsIdentification of intact proteins
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B. Bottom-up proteomics: Identification B. Bottom-up proteomics: Identification of intact proteinsof intact proteins
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B. Bottom-up proteomics: Identification B. Bottom-up proteomics: Identification of intact proteinsof intact proteins
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Top-Down Top-Down VSVS Bottom-up proteomics: Bottom-up proteomics:
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Top-Down (+)Top-Down (+)
Access the complete protein sequenceAccess the complete protein sequence
Abilty to locate post translational Abilty to locate post translational modifications (PTMs)modifications (PTMs)
Time consuming protein digest is eliminatedTime consuming protein digest is eliminated
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Top-Down (-)Top-Down (-)
Complex spectra obtained limits approach Complex spectra obtained limits approach
to single protein or simple mixturesto single protein or simple mixtures
Does not work well with proteins > 50 kDaDoes not work well with proteins > 50 kDa
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Bottom-up (+)Bottom-up (+)
Most widely used approach for proteın IDMost widely used approach for proteın ID
Reverse phase HPLC provides high-Reverse phase HPLC provides high-resolution separation of peptide digestsresolution separation of peptide digests
Can analyze very complex mixtureCan analyze very complex mixture
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Bottom-up (-)Bottom-up (-)
Only a fraction of the total peptide Only a fraction of the total peptide
population of a given protein is identified population of a given protein is identified (loss PTMs identified)(loss PTMs identified)
Loss of information about low abundant Loss of information about low abundant peptides is mass spectra dominated by peptides is mass spectra dominated by high abundance specieshigh abundance species
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What are the different types of mass What are the different types of mass spectrophotometers?spectrophotometers?
A. MALDI(Matrix assisted laser A. MALDI(Matrix assisted laser desorption/ionization)-TOF (Time of flight)desorption/ionization)-TOF (Time of flight)
B. LC/MS-MSB. LC/MS-MS
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A. MALDI(Matrix assisted laser A. MALDI(Matrix assisted laser desorption/ionization)-TOF (Time of flight)desorption/ionization)-TOF (Time of flight)
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LC/MS-MSLC/MS-MS
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Peptide Fragmentation by MS/MSPeptide Fragmentation by MS/MS
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Peptide Fragmentation by MS/MSPeptide Fragmentation by MS/MS
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Database Searching by MS/MSDatabase Searching by MS/MS
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Database Searching by MS/MSDatabase Searching by MS/MS
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Database Searching by MS/MSDatabase Searching by MS/MS
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Database Searching by MS/MSDatabase Searching by MS/MS
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Practical applications for mass Practical applications for mass spectrometryspectrometry
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Identify purified complexes to Identify purified complexes to generate protein-protein interactiongenerate protein-protein interaction
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Identify purified complexes to Identify purified complexes to generate protein-protein interactiongenerate protein-protein interaction
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Identify purified complexes to generate Identify purified complexes to generate protein-protein interactionprotein-protein interaction
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Thank YouThank You