FR AB - Science Reference

P77-M
The utilities of N-terminal sequencing in the post-genomic era.
L.R. Zieske1, S.W. Yuen1, T.A. Settineri1, D. Hawke1, C. Bloch2; 1Applied Biosystems, 850 Lincoln Centre Drive, Foster City, CA 94404,
2Embrapa-Cenargen, Brazil
Mass spectrometry continues to develop and improve methods and procedures
to make it the primary tool to identify proteins for “proteome analysis”.
The high mass accuracy and sensitivity of instruments such as hyphenated
quadrupole TOF mass spectrometers has made low level sequencing of
peptides and proteins possible. Yet even with these advancements in MS the
need for chemical sequencing still exists!
MS sequencing is very powerful, but exact location of the fragmentation patterns
are not predictable. Thus creating difficulty in assigning the exact N-termini.
The need for exact N-terminal sequence information is especially
important in determining mRNA editing and/or determining the true open
reading frames when EST database searching. Edman chemistry is the one
sure way of determining this type of information. Complementary to this is
the use of “multiple peptide sequencing” which simultaneously provides multiple
amino acid information per residue cycle. Thus providing very high
quality sequence information with which to interrogate the error prone databases
now used for searching. In addition, chemical sequencing provides the
necessary information to determine quality assurance of cloned products;
again making sure no frame shifts occurred in the processing. Another complementary
need for chemical sequencing is in assisting the assignment of the
correct ion series patterns being generated even when relatively complete
fragmentation data is recorded. Chemical sequencing also identifies difficult
to discern or unusual amino acids easily such as I/L and/or hydroxyproline.
In this work we will show some examples of how N-terminal analysis was
used in concert with mass spectrometry to unravel protein/peptide structural
information. In addition, we will show demonstration of “multiple peptide
sequencing” in inspecting the various databases available.
P79-S
Microsatellite analysis using fluorescent PCR primers synthesized
in tandem.
J. Fernandez, M. Kirchner, J. Brito, T-J. Daley, G. Dolios, B. Imai;
Rockefeller Univ., 1230 York Ave., New York, NY 11021
SDS-PAGE is typically the final purification step for isolation of small amounts
of proteins for further chemical characterization. Proteins are digested in-gel
with trypsin and the resultant peptides analyzed by 1) MALDI-TOF mass
spectrometry for tentative protein identification using database search programs
such as ProFound (http://prowl.rockefeller.edu) and/or 2) capillary
HPLC followed by Edman sequence analysis to obtain definitive primary
sequence information. While this strategy works well for Coomassie stained
proteins it poses a problem for the more sensitive silver staining method. A
technique has been published for protein identification using MALDI-TOF
mass spectrometric analysis after reversal of the silver stain (Gharahdaghi et
al., Electrophoresis 1999 20, 601–605). We have employed this technique for
preparation of proteins for internal Edman sequence analysis. Because of the
usually low amount of silver stained proteins, peptides need to be isolated
by capillary HPLC and collection techniques need to minimize peptide loss.
Silver stained gels, MALDI-TOF mass spectra, capillary HPLC, and Edman
sequence data will be presented as well as guidelines for sample handling.
POSTER ABSTRACTS
ABRF 2001 ABSTRACTS
P78-T
Increasing the capacity of a commercial Edman sequencer:
a protein auto sampler.
W. Shillinglaw, S. Wong, T. Moreno, W.J. Henzel; Genentech
We have designed an implemented a high throughput autosampler to
increase the capacity and speed of protein sequencing. The autosampler
attaches to a standard ABI Procise sequencer, enabling a single separate sample
cartridge to now hold up to six separate samples. The autosampler is
used in combination with faster Edman cycles and a rapid 12 minute PTH
separation to significantly increase the speed of sample analysis. The reaction
cartridges on the autosampler are composed of disposable Teflon tubing,
allowing for a significantly cleaner background when compared to the
reusable glass cartridge blocks, which are standard on the ABI sequencers.
The lower background reduces the ambiguity of identifying the amino acids
in the first few cycles, which is often a problem. A low cost program logic
controller which is connected through an external relay to the protein
sequencer controls the autosampler.
P80-M
Amino acid analysis 2000: a collaborative study from the
ABRF AAA Research Group.
M.A. Alterman1, P. Hunziker2, K. West3, R. Harris4, D. Chin5; 1Univ. of
Kansas, 6038 Malott Hall, Lawrence, KS 66045, 2Univ. of Zurich,
3Cleveland Clin. Fndn., 4Genentech, 5Univ. of Missouri
The AAA Research Group of the ABRF periodically provides member laboratories
with test samples in order to assist members in maintaining and
improving the quality of AAA and assess the reliability of AAA. The latest
study consists of two parts: Internet-based survey of equipment and technique
currently used for AAA, and experimental data from this year’s test
sample. The experimental part of 2000 study focused on quantitation and
identification of proteins. The participating laboratories received solutions of
4 pure proteins for the determination of their concentration by AAA as well
as by a colorimetric method (BCA, Bradford, or similar). Data obtained will
be used to examine the use of AAA for determining of the exact amount of
protein in the sample and the use of compositional data to identify the protein.
The relative precision and accuracy of the colorimetric assays will be
compared with the AAA values. The comparison of the results of this study
with the results from previous studies will show changes and improvements
in the practice of AAA.
JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000 207