Reference: Bioinformatics, a pratical guide to the analysis of genes and proteins. Edited by Andreas D.Baxe
Different combinations of amino acids can add up to the same peptide mass, any single peptide mass can be produced by many proteins. While Peptide spectum, entire set of constituent peptide masses observed, is typically unique to a specific protein.
1.Peptide mass fingerprinting (PMF)
Programs: Mascot, PeptIdent, Profound, Genome Fingerprint Scanning.
2. Tandem MS
It contains a second stage of ms analysis in which selected peptides are broken down into even smaller pieces by collision-induced dissociation (CID: high-speed particles collide and cause peptide bonds to break). It results a set of charged peptides with one or more amino acids removed from either C- or N- terminus of the selected peptide. two method to analyze the Tamdem MS spectrum.
a. de novo approach
Mass spectrum data -----------Amino acid sequence------------BLAST search
in this method, each peak in the ms/ms spectrum is assumed to be all possible ion type.
Problems: PTMs, weak signal
b. uninterpreted ms/ms spectral matching
Use databases to produce hypothetical spectral, then compare with the observed experimental spectra and give a score indicating how likely it is that the spectrum was produced by each possible peptide in the database. Program: Sequest
Problems: computational intensive
Thursday, November 3, 2011
Wednesday, October 19, 2011
Introduction to Mass Spectrometry
Reference: http://www.ohsu.edu/xd/research/research-cores/proteomics/educational-links/upload/Mass_Spectrometry_Based_Proteomics.pdf
Mass Spectrometry is an analytical technique that measures the mass to charge ratio of charged molecules. It is used to determine the mass of molecules.
A mass spectrometer contains three components: ion source, mass analyzer, detector.
An ion source, which produces intact gas phase ions
a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields
a detector, which measure the intensity signal of each ion and provide data for calculating abundance of ions.
There are two kinds of ion source that are most commonly used, MALDI and ESI.
In MALDI a laser is used to ionize and vaporize a small amount of sample, which is then drawn into the mass spectrometer for analysis.This setup doesn’t have much sensitivity,but it is very useful for screening samples.
In ESI, a stream of liquid containing the sample is ionized by applying an electrical charge to it.
Types of mass analyzer
-Quadrupole
One common component used to sort ions is the quadrupole.This consists of 2 pairs of charged rods. There is an electrical potential between each pair of rods which draws the ions towards one rod. The polarity of this electrical field is oscillated rapidly, which causes the ions travel through the quadrupole in a spiral trajectory. Each oscillation frequency allows ions with a particular m/z to pass through, while the other ions crash into the poles and lose their charge, or are ejected from the quadrupole. By varying the oscillation frequency ions with different m/z ratios will get through. The number of ions passing through at any given is
measured by the mass spectrometer’s detector and a graph of intensity vs. m/z is created from this data. This is referred to as an MS Spectra.
-Ion trap
Another component used to filter ions is the ion trap. In an ion trap ions are collected and held either in a 3-dimensional space or a 2-dimentional plane. Once a certain number of ions have been collected, or after a set time, the ions are ejected from the trap. This ejection voltage is ramped in a way that allows different m/z ions to be ejected at slightly different times. This time difference creates an MS Spectra. Because a greater number of ions is collected, this method typically has a higher sensitivity than using a quadrupole mass filer.
One more common method of sorting ions is the Time-of-Flight (TOF) analyzer.
Mass Spectrometry is an analytical technique that measures the mass to charge ratio of charged molecules. It is used to determine the mass of molecules.
A mass spectrometer contains three components: ion source, mass analyzer, detector.
An ion source, which produces intact gas phase ions
a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields
a detector, which measure the intensity signal of each ion and provide data for calculating abundance of ions.
There are two kinds of ion source that are most commonly used, MALDI and ESI.
In MALDI a laser is used to ionize and vaporize a small amount of sample, which is then drawn into the mass spectrometer for analysis.This setup doesn’t have much sensitivity,but it is very useful for screening samples.
In ESI, a stream of liquid containing the sample is ionized by applying an electrical charge to it.
Types of mass analyzer
-Quadrupole
One common component used to sort ions is the quadrupole.This consists of 2 pairs of charged rods. There is an electrical potential between each pair of rods which draws the ions towards one rod. The polarity of this electrical field is oscillated rapidly, which causes the ions travel through the quadrupole in a spiral trajectory. Each oscillation frequency allows ions with a particular m/z to pass through, while the other ions crash into the poles and lose their charge, or are ejected from the quadrupole. By varying the oscillation frequency ions with different m/z ratios will get through. The number of ions passing through at any given is
measured by the mass spectrometer’s detector and a graph of intensity vs. m/z is created from this data. This is referred to as an MS Spectra.
-Ion trap
Another component used to filter ions is the ion trap. In an ion trap ions are collected and held either in a 3-dimensional space or a 2-dimentional plane. Once a certain number of ions have been collected, or after a set time, the ions are ejected from the trap. This ejection voltage is ramped in a way that allows different m/z ions to be ejected at slightly different times. This time difference creates an MS Spectra. Because a greater number of ions is collected, this method typically has a higher sensitivity than using a quadrupole mass filer.
One more common method of sorting ions is the Time-of-Flight (TOF) analyzer.
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