GENE 500 Lecture Notes - Lecture 13: Tandem Mass Spectrometry, Mass Spectrometry, Molecular Mass

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8 Nov 2018
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Lecture 13
Molecular Mass Protein Sequencing
Molecular mass can be determined by matrix-
assisted laser desorption/ionization timeof-
flight (MALDI-TOF) mass spectrometry.
Step 1: In a MALDI-TOF mass spectrometer,
pulses of light from a laser ionize a protein or
peptide mixture that is absorbed on a metal
target.
Step 2 & 3: An electric field in the mass
analyzer accelerates the ions in the sample
toward the detector.
**The time it takes an ion to reach the
detector is proportional to the square root of
the mass-to-charge (m/z) ratio. Among ions having the same charge, the smaller ions move
faster (shorter time to the detector). The molecular weight of each ion from the sample is
calculated using the time of flight of a standard.**
This is also used to detect modifications because a phosphorylated protein will have a
different molecular weight compared to the native protein; if the protein is from a species with
a sequenced genome, the computer generates all possible peptides that enzyme can generate
and then finds the mass match
Complex Mixture
Identification
You can use
chromatography to
separate the complex
mixture The mixture of
resulting peptides is
fractionated by liquid
chromatography (LC) into
multiple, less complex,
fractions, which are slowly
but continuously injected
by electrospray ionization
into a tandem mass
spectrometer.
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