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MICROSEQUENCE ANALYSIS OF
NATURAL PEPTIDES
The Peptide Biochemistry and
Molecular Probes Core does not operate a microsequence analysis
instrument, but Dr. Reeve has collaborated on the microsequence
analysis of more than 150 peptides. Peptides purified from reverse
phase HPLC are evaluated by HPCE to insure they are suitable
for microsequence analysis. Purified fractions that contain primarily
a single peptide and are sufficiently concentrated (50 pmol/5ml)
are loaded directly onto a microsequencer. Peptides that are
too dilute are concentrated on a microbore HPLC before they are
loaded on the microsequencer. The Core director assists CURE
investigators in purifying samples until they are suitable for
microsequence analysis, assures that the samples are properly
delivered, and helps investigators interpret results.
Several CURE investigators'
projects require microsequence analysis of proteins after purification
by 2D gel electrophoresis. Proteins can be sequenced from gels
after washing away the dye by rinsing with digestion buffer.
The protein is then digested with trypsin and the products are
either analyzed by on-line LC/MS/MS4 or purified by HPLC and
fractions characterized by microsequence analysis. The minimum
amounts for this type of protein analysis are 300 picomole, which
can easily be recovered from a single 2D gel. Our collaborators
at the City of Hope have determined the entire sequence of some
proteins using this type of technique with multiple specific
endopeptidases. However, for most analysis it will be quicker
and more cost effective to make molecular probes to sequences
determined by analysis of tryptic peptides and determine the
entire sequence through molecular biological techniques. The
director and co-director of the Core will assist investigators
in deciding which approach to use in determining the sequence
of new proteins.
The director of the Peptide
Biochemistry and Molecular Probes Core has collaborated for more
than 15 years with John E. Shively on microsequence analysis
at the City of Hope Research Institute, Duarte, California, and
has also worked extensively with the UCLA Microsequence Facility,
directed by Audree Fowler. The core director advises investigators
on which facility is most suitable for individual samples. Peptide
microsequence analysis is provided on a charge-back basis paid
directly to the facility performing the service.
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