'On-the-fly' optical encoding of combinatorial peptide libraries for profiling of protease specificity

Marcon, Lionel, Battersby, Bronwyn J., Ruhmann, Andreas, Ford, Kym, Daley, Matthew, Lawrie, Gwendolyn A. and Trau, Matt (2010) 'On-the-fly' optical encoding of combinatorial peptide libraries for profiling of protease specificity. Molecular Biosystems, 6 1: 225-233. doi:10.1039/b909087h


Author Marcon, Lionel
Battersby, Bronwyn J.
Ruhmann, Andreas
Ford, Kym
Daley, Matthew
Lawrie, Gwendolyn A.
Trau, Matt
Title 'On-the-fly' optical encoding of combinatorial peptide libraries for profiling of protease specificity
Journal name Molecular Biosystems   Check publisher's open access policy
ISSN 1742-206X
1742-2051
Publication date 2010
Year available 2009
Sub-type Article (original research)
DOI 10.1039/b909087h
Open Access Status Not Open Access
Volume 6
Issue 1
Start page 225
End page 233
Total pages 9
Place of publication Cambridge, United Kingdom
Publisher Royal Society of Chemistry
Collection year 2011
Language eng
Formatted abstract
Large solid-phase combinatorial libraries currently play an important role in areas such as infectious disease biomarker discovery, profiling of protease specificity and anticancer drug discovery. Because compounds on solid support beads are not positionally-encoded as they are in microarrays, innovative methods of encoding are required. There are many advantages associated with optical encoding and several strategies have been described in the literature to combine fluorescence encoding methods with solid-phase library synthesis. We have previously introduced an alternative fluorescence-based encoding method ("colloidal barcoding''), which involves encoding 10-20 mm support beads during a split-and-mix synthesis with smaller 0.6-0.8 mm silica colloids that contain specific and identifiable combinations of fluorescent dye. The power of this 'on-the-fly' encoding approach lies in the efficient use of a small number of fluorescent dyes to encode millions of compounds. Described herein, for the first time, is the use of a colloid-barcoded library in a biological assay (i.e., protease profiling) combined with the use of confocal microscopy to decode the colloidal barcode. In this proof-of-concept demonstration, a small focussed peptide library was optically-encoded during a combinatorial synthesis, incubated with a protease (trypsin), analysed by flow cytometry and decoded via confocal microscopy. During assay development, a range of parameters were investigated and optimised, including substrate (or probe) loading, barcode stability, characteristics of the peptide-tagging fluorophore, and spacer group configuration. Through successful decoding of the colloidal barcodes, it was confirmed that specific peptide sequences presenting one or two cleavage sites were recognised by trypsin while peptide sequences not cleavable by trypsin remained intact. © The Royal Society of Chemistry 2010
Keyword Resin beads
Chemistry
Fluorescent
Fluorophores
Colloids
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ
Additional Notes Available online 6th October 2009

Document type: Journal Article
Sub-type: Article (original research)
Collections: 2010 Higher Education Research Data Collection
Australian Institute for Bioengineering and Nanotechnology Publications
 
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Created: Sun, 10 Jan 2010, 00:03:27 EST