FR AB - Science Reference

ABRF 2001 ABSTRACTS
P97-S
An improved procedure using immobilized metal-ion affinity
chromatography for isolation and characterization of
phosphopeptides from phosphoproteins.
K-L. Hsi, D.R. Dupont, S.W. Yuen, C.A. Settinery; Applied Biosystems,
Foster City, CA, 850 Lincoln Center Drive, Foster City, CA 94404
It has become clear that phosphorylation is the most common and important
reversible regulatory modification of proteins. Defining the sites of phosphorylation
is necessarily important for the mechanism studies of cell regulation.
However, methods to specifically isolate and characterize phosphopeptides
remain challenged, especially for those proteins separated from
1D/2D gel at low concentration. An attempt is thus made to improve the procedure
based upon immobilized metal-ion affinity chromatography (IMAC)
for working with sub-microgram amount of phosphoproteins.
A protease digest of gel-separated phosphoprotein was treated with immobilized
Fe��� on a chelating resin. The affinity bound phosphopeptides were
then eluted from the resin with ammonium acetate buffer. We use a mini-size
(250 �l) of cartridge to perform all the processes of isolation of phosphopeptides
including washing of the resin, binding and elution of the phosphopeptides,
instead of an open column as previously reported.
This improved procedure provides some advantages over the previously
reported open column method: 1. Fast, i.e. 30 minutes or less. 2. High washing
efficiency, thus high recovery of pure phosphopeptides. 3. Sensitive
working level, i.e. sub-microgram of gel separated phosphoproteins.
Several phosphoproteins with different types of phosphorylation (Ser, Thr
and Tyr) have been studied using the improved procedure. The effectiveness
of the procedure was demonstrated by chemical sequencing and MS/MS
analysis of MicroBlotter-collected phosphopeptides.
P99-T
Synthesis of lactam-bridged dipeptides mediated by
aminoacylpyroglutamates.
I. Rodionov, A. Chulin, V.T. Ivanov; Br. of Shemyakin-Ovchinnikov Inst.
of Bioorganic Chem., 8 Academy Avenue, Pushchino, Moscow Region
142290, Russian Federation
Aminoacyl incorporation reaction (AI) discoverd about 40 years ago by Shemyakin
et al. has been evaluated as a general synthetic route to variously
constrained lactam-bridged dipeptides. The AI is effectively a ring enlargement,
which involves intramolecular acylation of amino group (or different
nucleophilic functions like HS- and HO-) by the activated cyclic diacylamino
moiety via intermediate formation of bicyclic azacyclols. The most straightforward
models for studying AI are aminoacylated pyroglutamates derived
from diaminoacids
R-Xaa-Glp-OR� (I), Xaa � Lys, Orn, Dab and Dpr,
a class of unusual peptides, which remains unexplored as yet. We have studied
3 different synthetic approaches to I:
direct acylation of sodium derivative of Glp-OR�;
pyrrolidone ring closure in the plain dipeptides R-Xaa-Glu(OX)-OR� promoted
by bases and/or by activation of side chain carboxyl of Glu;
selective oxidation of the related proline dipeptides Boc-Xaa-Pro-OR� by
ruthenium tetroxide/sodium periodate.
Scope and limitation of the above approaches will be discussed and exemplified
by the synthesis of 26 protected dipeptides I. As expected, deprotection
of amino or hydroxy functions followed by exposure to potassium carbonate
buffer, pH 9.5 (water-acetonitrile) resulted in smooth AI for the
majority of the synthesized dipeptides I. No oligomerization products were
detected. In this way a number of bridged dipeptides (9–12 membered
cycles) were obtained in 35–70% yield and characterized by NMR and MS
data. NMR data suggested that in 4 cases stable azacyclols have been isolated.
POSTER ABSTRACTS
212 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P98-M
Postsynthesis labeling of agiotensin II peptide with d-rhodamine
for fluorescence monitoring of receptor binding.
S.P. Yadav, W-Z. Shen, Y. Luo, J. Zhang, S. Karnick; Lerner Res. Inst.,
Cleveland, Mail Code NC10, 9500 Euclid Avenue, Cleveland, OH 44195
Solid-phase peptide synthesis is now routinely used for drug discovery and
immunological research and for studying the structure-function relationships
of proteins. Protein-protein interactions play an important role in a wide variety
of biochemical and physiological processes and generally involve large
interfaces with many intermolecular contacts. In addition, protein-protein
interaction may also occur through small surface binding epitopes such as in
the case of human growth hormone-receptor binding and erythropietinreceptor
complex formation. Therefore, the rational design of relatively small
molecular size peptide activators/inhibitors for receptor surfaces has long
been considered a formidable challenge. These findings convincingly open
up the possibility that small peptides that mimic such small binding epitopes
may help to block a large protein-protein interface. Usefulness of this hypothesis
in drug design rationale remains to be further tested in different biological
systems. Furthermore rationalization of general approach of inhibition of
protein-protein interaction will of course have a tremendous impact on
development of new therapeutic strategies for several human diseases. That
is where fluorescent dye labeling of synthetic peptides becomes important.
Studies on labeling of a peptide with fluorescent dyes in a manner without
altering functional characteristics of the peptide are of considerable interest
for studying the peptide-receptor interactions. Strategy for synthesis of an
octa-mer angiotensin II by Fmoc chemistry and subsequent labeling of the
cleaved peptide with Rhodamine dye are discussed in this report. The results
of double labeling are presented here to show that the removal of excess free
Rhodamine is critically important in confocal microscopy.
P100-S
The characterization of prostate specific antigen activating peptide.
M. Pakkala1, P. Wu2, J. Leinonen2, U-H. Stenman2, J. Vepsäläinen1, A. Närvänen1; 1Univ. of Kuopio, P.O. Box 1627, Kuopio 70211, Finland,
2Univ. Central Hosp., Helsinki
Prostate specific antigen (PSA) is widely used as a marker of prostate cancer.
PSA is a 30 kD serine protease and belongs to Kallikrein family. Peptides
with specific binding properties to PSA were studied by using phage display
peptide libraries. Four different PSA binding clones were isolated and characterized.
One of the clones, containing 13 amino acids sequence with four
cysteines, showed the highest affinity for PSA. This peptide (C4), as a fusion
protein with glutathione-S-transferase (GST), increased the protease activity
of PSA against a synthetic substrate (Wu et al. (2000), Eur. J. Biochem. 267,
1–10)
We have synthesized the active peptide C4 (CVAYCIEHHCWTC) and its modifications
by using conventional solid phase peptide synthesis method (SPPS).
Two cysteines (2. And 3.) were protected by using Acm protecting group,
which remains uncleaved during the removal of the peptide from the resin.
The final cyclization was made during the removal of the Acm-group from
cysteines by Iodine treatment. The synthetic analog showed same activity to
PSA than the original GST fusion protein where as the activity of the peptide
with two Acm groups was less than 50% from the fully cyclized form. The
acetylation of the ?-amino group did not affect to the activity. In addition we
synthesized cyclic version with amide bond VAYCIEHHCWT instead of the
cysteines 1. and 4. by using Allyl protected E(6) (Fmoc-L-Glu-OAll) as a starting
amino acid. The final peptide was cyclic with one disulfide bond. This
modification was totally inactive.
The results suggest that the correct secondary structure of the peptide C-4
plays an important role in increased activation of PSA. Structure analysis by
NMR is under study. Based on the preliminary NMR results the prepared peptides
are rather flexible and the structures are strongly dependent on the temperature.