omation mbers - Society for Laboratory Automation and Screening

9:00 am Thursday, February 5 Proteomics – Technology 2 Room B1
Wei-Wei Zhang
GenWay Biotech, Inc.
10130 Sorrento Valley Road, Suite C
San Diego, California 92121
wzhang@genwaybio.com
Polyclonal Gene-Specific IgY Antibodies for Proteomics and Abundant Plasma
Protein Depletion
91
Co-Author(s)
Xiangming Fang
Jerry Feitelson
Lei Huang
Kena Wang
Polyclonal IgY antibodies are produced based upon gene information via immunizing chickens with gene
expression vectors and/or purified protein antigens. Antibodies produced by this approach have high specificity
and typically bind to a single gene product. Gene-specific IgY antibodies have several distinct advantages over
conventional IgG antibodies due to their higher surface stability, lower cross-reactivity, stronger avidity, and
higher binding capacity. Besides being useful in conventional immunoassays, IgY antibodies have demonstrated
applicability for multiplex assays. In the Luminex-100 System and on Ciphergen’s ProteinChip, detection
sensitivities reached 10 pg/ml and 40 fg/ml, respectively. To make detection of low-abundant proteins more
effective and to facilitate human plasma proteomics studies, IgY antibodies are covalently conjugated to polymeric
beads via Fc region for selectively removing abundant plasma proteins, such as albumin, IgG, transferrin, and
fibrinogen. After short incubation of IgY beads with serum or plasma samples, target proteins are specifically
bound to the IgY beads at an approximately 1:1 molar ratio. The flow-through samples can be used for proteomics
analysis and for developing clinical diagnosis markers. This allows for highly sensitive analysis of rare proteins in
blood samples using 2D gels and mass spectrometry. Also, polyclonal IgY against human serum albumin is shown
to be capable of effectively removing the albumins from the serum samples of mouse, rat, pig, and goat. This
cross-species binding of albumin cannot be accomplished by using IgG against the same antigen, indicating the
unique and important feature of IgY in application for proteomics studies.
9:30 am Thursday, February 5 Proteomics – Technology 2 Room B1
Dhaval N. Gosalia
University of Pennsylvania
1150 Vagelos Labs
3340 Smith Walk
Philadelphia, Pennsylvania 19104
dhavalg@seas.upenn.edu
Protease Substrate Profiling Using Microarrays
Co-Author(s)
Scott L. Diamond,
Cleo M. Salisbury,
Jonathan A. Ellman
University of California, Berkeley
We developed a novel technique for microarray-based enzymatic assays for biological reactions at nanoliter
volumes. The technique allows for rapid determination of protease substrate specificity with minimal sample
usage. A peptidyl coumarin library of fluorogenic protease substrates, ACC-P1-P2-P3-P4-Ac was synthesized in
parallel using standard Fmoc-based solid-phase synthesis techniques. The amino acids at P1 and P4 were held
constant with P1=lysine and P4=alanine, and all combinations of proteinogenic amino acids (except cysteine)
were used at the P2 and P3 positions, for a total of 361 compounds. Purity of the library was demonstrated
by HPLC-MS, and the substrates were reconstituted in DMSO to a concentration of 10 mM. The library, along
with appropriate controls, was further diluted to 1 mM concentration with 50% (v/v) glycerol and microarrayed
on a standard microscope slide with a density of 400 spot/cm². The microarrayed library was treated with the
serine protease human thrombin and then scanned. The chip demonstrated that thrombin had an extremely high
specificity for proline at position P2. The remaining amino acids at the P2 position produced essentially no signal
above background. Definite specificities were observed for amino acids at the P3 position with Q, V, W, Y, R
resulting in the fastest cleavage and P3=D, P, A, H, E resulting in the slowest cleavage. The microarray specificities
agreed with previously published results, demonstrating that the technique provides accurate substrate specificity
information. This study indicates that protease substrate profiling can be done in this microarray format.
PODIUM ABSTRACTS