Click to add Text Overview of Mass Spectrometry Sermin Tetik, PhD Marmara University July 2015, New...

Overview of Mass Overview of Mass Spectrometry Spectrometry

Sermin Tetik, PhDSermin Tetik, PhDMarmara UniversityMarmara University

July 2015, New OrleansJuly 2015, New Orleans

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

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

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

John B. Fenn Koichi John B. Fenn Koichi TanakaTanaka

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)

-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)

-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

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

A. Top-down proteomics: A. Top-down proteomics: Identification of intact proteinsIdentification of intact proteins

B. Bottom-up proteomics: Identification B. Bottom-up proteomics: Identification of intact proteinsof intact proteins

B. Bottom-up proteomics: Identification B. Bottom-up proteomics: Identification of intact proteinsof intact proteins

Top-Down Top-Down VSVS Bottom-up proteomics: Bottom-up proteomics:

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

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

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

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

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

A. MALDI(Matrix assisted laser A. MALDI(Matrix assisted laser desorption/ionization)-TOF (Time of flight)desorption/ionization)-TOF (Time of flight)

LC/MS-MSLC/MS-MS

Peptide Fragmentation by MS/MSPeptide Fragmentation by MS/MS

Peptide Fragmentation by MS/MSPeptide Fragmentation by MS/MS

Database Searching by MS/MSDatabase Searching by MS/MS

Database Searching by MS/MSDatabase Searching by MS/MS

Database Searching by MS/MSDatabase Searching by MS/MS

Database Searching by MS/MSDatabase Searching by MS/MS

Practical applications for mass Practical applications for mass spectrometryspectrometry

Identify purified complexes to Identify purified complexes to generate protein-protein interactiongenerate protein-protein interaction

Identify purified complexes to Identify purified complexes to generate protein-protein interactiongenerate protein-protein interaction

Identify purified complexes to generate Identify purified complexes to generate protein-protein interactionprotein-protein interaction

Thank YouThank You