mass-spectrometry for protein sequencing
TRANSCRIPT
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Luiza Niyazmetova, SST NU
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Outline What is mass spectrometry?
How it works?
Types
Application
Protein characterization and sequencing
ESI
MALDI
AMS
Conclusion
References
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Mass Spectrometry Analytical technique that is used to measure mass-to-
charge ratio of charged particles
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How it works
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How it works Sample is loaded onto the mass spectrometer, and
undergoes vaporization
Components of the sample are ionized (e.g., by impacting them with an electron beam)
Ions are separated according to their mass-to-charge ratio in analyzer by electromagnetic fields
Ions are detected by a quantitative method
Ion signal is processed into mass spectra
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Types AMS (Accelerator Mass Spectrometer (MS)) or
Tandem MS
MALDI (matrix-assisted laser desorption/ionization source)
Electrospray ionization (ESI) MS
TOF (time-of-flight MS)
Inductively coupled plasma-mass spectrometry (ICP-MS)
Thermal ionization-mass spectrometry (TIMS)
Spark source mass spectrometry (SSMS)
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Application Identifying unknown compounds
Determining the isotopic composition of elements in molecule
Determining the structure of a compound by observing its fragmentation
Respiratory gas analysis in hospitals
Space exploration (analysis of plasma)
Proteomics research (protein characterization & sequencing)
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Protein characterization and sequencing
Protein sequencing - determining amino acid sequence & conformation that protein adopts and extent to which it is complexed with non-peptide molecules
Important for creating protein databases
Characterization and quality control of recombinant proteins (ESI, MALDI)
Protein identification (sequencing) (ESI, MALDI, AMS)
Detection and characterization of posttranslational modifications (AMS)
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Electro-spray ionization Softest ionization technique
Best technique for polar non‐volatile compounds (proteins, peptides, nucleic acids, Pharmaceuticals, natural products)
Not very sensitive to non-volatile salts
Allows producing multiple charged ions and determining high molecular weight proteins
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Electro Spray ionization Sample is dissolved in polar volatile buffer
It is dispersed by electro spray into fine aerosol
Aerosol is directed through regions of higher vacuum until droplets evaporate to near atomic size
Molecules are transferred into the mass spectrometer with high efficiency for analysis
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Matrix-assisted laser desorption/ ionization (MALDI)
1. Desorption: triggered by UV laser beam
- Matrix material heavily absorbs UV laser light, leading to the ablation of upper layer of matrix material
- Hot plume produced during the ablation contains neutral and ionized matrix molecules, protonated & deprotonated matrix molecules, matrix clusters & nanodroplets
2. Ionization (protonation or deprotonation):
- Takes place in the hot plume
- Some ablated species participate in protonation (deprotonation) of analyte molecules
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Tandem MS Consists of two analyzers:
quadrupole - quadrupole
magnetic sector - quadrupole
magnetic sector - magnetic sector
quadrupole - time-of-flight
One peptide species out of mixture is selected in the first mass spectrometer
Dissociated by collision with inert gas (argon or nitrogen)
Resulting fragments are separated in the second part of the tandem mass spectrometer, producing the tandem mass spectrum
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Protein sequencing by Tandem Mass Spectrometry
Protein sequencing - determining amino acid sequence & conformation the protein adopts and extent to which it is complexed with non-peptide molecules
1. Proteases break protein into peptides 2. Tandem Mass Spectrometer breaks the peptides down into
fragment ions & measures the mass of each piece 3. NH-CH, CH-CO, and CO-NH bonds can fragment along
amino acid backbone 4. Each bond breakage gives rise to two species, one neutral
and the other one charged 5. Only Charged species is monitored by the mass
spectrometer
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Conclusion Protein sequencing is used for creating protein
databases
Common Mass spectrometry techniques used for protein sequencing:
- ESI
- MALDI
- Tandem (AMS) mass specrtometry
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References Ashcroft, A.E. (2008). An introduction to mass spectrometry.
Retrieved from http://www.astbury.leeds.ac.uk/facil/MStut/mstutorial.htm
Basics of mass spectrometry. (2012). Retrieved from http://masspec.scripps.edu/mshistory/whatisms_details.php#Basics
Protein sequencing and identification with mass spectrometry. (2004). Retrieved from http://cseweb.ucsd.edu/classes/wi05/cse206b/notes/L4.pdf
The mass spectrometer. (2000). Retrieved from http://www.chemguide.co.uk/analysis/masspec/howitworks.html