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Proceedings -Tuesday, October 8, 2002
TuOD1
The Role of Complete De Novo Peptide Sequencing in Protein Characterization
Al Yergey, National Institutes of Health
A powerful technique for protein identification is known as de novo
peptide sequencing. Proteins can be characterized either globally
(all proteins present) or specifically (by function, location
or partners). Any characterization of proteins requires the separation
and isolation of components with their subsequent identification.
Components can be separated by techniques such as immunoprecipitation,
electrophoresis and chromatography; identification can be made by
mass spectrometry, Edman sequencing or antibody reactions.
The standard approach to protein identification uses mass spectrometry
for identification. A mixture of proteins is separated on a gel, selected
proteins are removed from that gel, a digestion with protease is performed
to generate peptides and then partial or full peptide sequencing is
performed. This isolated peptide mixture is analyzed by mass spectrometry
to give a list of peptide masses that can be searched against standard
peptide databases.
Peptide sequencing uses two stage mass analysis—quad, trap,
hybrid or TOF-TOF. The peptides are charged and separated according
to their mass to charge ratios. From the first stage mass spectrum
a precursor mass and fragment are selected and fragmentation is performed
in the second stage of analysis. Since the fragmentation can occur
anywhere along the peptide, a spectrum of the observed mass to charge
ratios is generated. The different amino acids within a peptide each
have different masses and so the spectrum of a peptide is usually
characteristic of the peptide sequence.
The peptides that are identified can sometimes be adequate to identify
the protein. However, there can be problems with this technique. Note
that the quality of the "hit" depends on the length of the sequence
tag and the number of peptides matched. The majority of "hits" involve
a single peptide; isobaric peptides exist at both ends, leading to
an ambiguity in the sequence at these points. A large percentage (>50%)
of the MS/MS spectra are not used due to reasons such as a poor quality
spectrum, a non-protein contaminant or an unknown protein. In order
to maximize the information learned, the spectral information obtained
needs to be better utilized. Toward accomplishing this goal, algorithms
are used for searching protein and nucleic acid databases for similarities
to query sequences.
The main technique for tandem mass spectrometry is electrospray ionization
followed by MS/MS in an ion trap or a triple quadrupole instrument.
Many commercial MALDI mass spectrometers are used to perform sequencing
by a technique called post-source decay, in which the spectrum is
acquired in a series of steps and summed to give the product ion spectrum.
However, using newer designs, true MS/MS experiments can be conducted
using MALDI-MS. These instruments have two TOF analyzers linked in
tandem and are set up to give high throughput and MS/MS spectra with
many fragments and high mass resolution. Several examples were presented
to illustrate peptide fragmentation patterns and problem solving of
peptide sequencing using an "alligator algorithm."
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