0:04 tandem mass spectrometry is a powerful
0:06 method for analyzing and identifying
0:08 peptides and proteins
0:10 some proteins can be identified by
0:12 comparing their mass to a database of
0:15 known proteins
0:17 in other cases all or part of the amino
0:20 acid sequence must be determined before
0:22 a protein can be identified
0:25 when a protein is large the ability to
0:27 uniquely identify it with a single mass
0:29 measurement becomes more difficult
0:32 to make this easier one can break down
0:35 the peptide into smaller components
0:38 all methods of mass spectrometry can
0:40 determine a protein's mass
0:43 however tandem mass spectrometry can
0:45 also rapidly and accurately determine
0:48 both the mass and amino acid sequence of
0:50 a protein
0:54 all mass spectrometers consist of three
0:56 essential components the ion source
0:59 the mass analyzer and the detector
1:03 as its name suggests
1:05 tandem mass spectrometry utilizes two
1:08 spectrometers arranged in tandem
1:11 the two spectrometers are separated by a
1:14 collision cell which contains an ionized
1:17 inert gas such as helium or argon
1:21 as with any mass spectrometer the first
1:24 step is to convert the protein sample
1:26 into gaseous ions
1:28 for example flashing a prepared sample
1:31 with a laser pulse excites and vaporizes
1:34 some of the sample into a hot gas
1:37 when the vaporized protein molecules
1:39 collide with one another
1:41 charges are transferred from one
1:43 molecule to another
1:44 and the gaseous protein molecules become
1:47 ionized
1:49 with tandem mass spectrometry these
1:51 initial gaseous protein ions are called
1:54 precursor ions
1:56 applying an electrostatic field
1:58 accelerates the precursor ions through
2:01 the first mass spectrometer
2:04 after the precursor ions have been
2:06 analyzed by the first mass spectrometer
2:09 they enter a collision cell which
2:11 contains an inert gas such as helium or
2:14 argon
2:16 the inert gas atoms collide with and
2:18 fragment the precursor ions into smaller
2:21 peptide chains
2:23 these new smaller fragments are called
2:25 product ions
2:27 the product ions then pass through the
2:29 second mass spectrometer
2:33 the fragmentation of precursor ions into
2:36 product ions occurs in chemically
2:38 predictable ways
2:40 fragmentation is due to the disruption
2:43 of individual peptide bonds
2:46 the cleavage of a peptide bond breaks
2:48 the original protein ion into two
2:50 smaller peptide ions
2:53 these two smaller product ions contain
2:56 the amino acid sequences before and
2:59 after the cleavage site
3:01 in this example the disruption of a
3:03 peptide bond cleaves a single amino acid
3:06 residue from the amino terminus of the
3:08 peptide
3:10 the precursor peptide has an initial
3:12 mass to charge ratio of about 621
3:16 following the cleavage of the terminal
3:18 residue the mass to charge ratio is
3:21 reduced to about 492
3:24 this change in the mass to charge ratio
3:27 of 129
3:28 corresponds with the calculated mass to
3:31 charge value of a glutamate residue
3:34 in this way the tandem mass spectrometer
3:37 can identify the terminal amino acid
3:39 residue
3:41 the collisions between the precursor
3:43 ions and the inert gas atoms create a
3:46 family of product ions
3:48 each product ion represents a fragment
3:51 of the original peptide with one or more
3:53 amino acids removed from one end
3:57 by comparing the mass to charge ratio of
3:59 these various product ions the tandem
4:02 mass spectrometer can determine the
4:04 entire amino acid sequence of the
4:06 original peptide
4:07 for simplicity only the carboxyl
4:10 terminal peptide fragments are shown
4:14 the mass spectrum of the product ions
4:16 formed by the fragmentation of this
4:18 peptide shows five different peaks
4:21 each peak corresponds to a different
4:23 sized fragment
4:25 the differences in the mass to charge
4:27 ratio between the peaks can be used to
4:29 identify specific amino acid residues
4:33 in this way the mass spectrum of the
4:35 product ions reveals the amino acid
4:38 sequence of the original peptide
4:45 you
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