YouTube Transcript: Tandem Mass Spectrometer Animation I CSIR NET Life Science I GATE Life Science I DBT JRF | YouTubeToText
YouTube Transcript: Tandem Mass Spectrometer Animation I CSIR NET Life Science I GATE Life Science I DBT JRF
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tandem mass spectrometry is a powerful
method for analyzing and identifying
peptides and proteins
some proteins can be identified by
comparing their mass to a database of
known proteins
in other cases all or part of the amino
acid sequence must be determined before
a protein can be identified
when a protein is large the ability to
uniquely identify it with a single mass
measurement becomes more difficult
to make this easier one can break down
the peptide into smaller components
all methods of mass spectrometry can
determine a protein's mass
however tandem mass spectrometry can
also rapidly and accurately determine
both the mass and amino acid sequence of
a protein
all mass spectrometers consist of three
essential components the ion source
the mass analyzer and the detector
as its name suggests
tandem mass spectrometry utilizes two
spectrometers arranged in tandem
the two spectrometers are separated by a
collision cell which contains an ionized
inert gas such as helium or argon
as with any mass spectrometer the first
step is to convert the protein sample
into gaseous ions
for example flashing a prepared sample
with a laser pulse excites and vaporizes
some of the sample into a hot gas
when the vaporized protein molecules
collide with one another
charges are transferred from one
molecule to another
and the gaseous protein molecules become
ionized
with tandem mass spectrometry these
initial gaseous protein ions are called
precursor ions
applying an electrostatic field
accelerates the precursor ions through
the first mass spectrometer
after the precursor ions have been
analyzed by the first mass spectrometer
they enter a collision cell which
contains an inert gas such as helium or
argon
the inert gas atoms collide with and
fragment the precursor ions into smaller
peptide chains
these new smaller fragments are called
product ions
the product ions then pass through the
second mass spectrometer
the fragmentation of precursor ions into
product ions occurs in chemically
predictable ways
fragmentation is due to the disruption
of individual peptide bonds
the cleavage of a peptide bond breaks
the original protein ion into two
smaller peptide ions
these two smaller product ions contain
the amino acid sequences before and
after the cleavage site
in this example the disruption of a
peptide bond cleaves a single amino acid
residue from the amino terminus of the
peptide
the precursor peptide has an initial
mass to charge ratio of about 621
following the cleavage of the terminal
residue the mass to charge ratio is
reduced to about 492
this change in the mass to charge ratio
of 129
corresponds with the calculated mass to
charge value of a glutamate residue
in this way the tandem mass spectrometer
can identify the terminal amino acid
residue
the collisions between the precursor
ions and the inert gas atoms create a
family of product ions
each product ion represents a fragment
of the original peptide with one or more
amino acids removed from one end
by comparing the mass to charge ratio of
these various product ions the tandem
mass spectrometer can determine the
entire amino acid sequence of the
original peptide
for simplicity only the carboxyl
terminal peptide fragments are shown
the mass spectrum of the product ions
formed by the fragmentation of this
peptide shows five different peaks
each peak corresponds to a different
sized fragment
the differences in the mass to charge
ratio between the peaks can be used to
identify specific amino acid residues
in this way the mass spectrum of the
product ions reveals the amino acid
sequence of the original peptide
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