Easy parallel screening of reagent stability, quality control, and metrology in solid phase peptide synthesis (SPPS) and peptide couplings for microarrays

Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS...

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Bibliographic Details
Published in:Journal of peptide science 2015-10, Vol.21 (10), p.751-757
Main Authors: Achyuthan, Komandoor E., Wheeler, David R.
Format: Article
Language:English
Subjects:
(3-aminopropyl)triethoxysilane
absorption
APTES
COMU
fluorescence
Immunochemistry
INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY
metrology
Peptides
Peptides - chemical synthesis
Peptides - chemistry
Propylamines - chemistry
Quality Control
Silanes - chemistry
solid phase peptide synthesis
Solid-Phase Synthesis Techniques - methods
SPPS
YGGFL
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Summary:Evaluating the stability of coupling reagents, quality control (QC), and surface functionalization metrology are all critical to the production of high quality peptide microarrays. We describe a broadly applicable screening technique for evaluating the fidelity of solid phase peptide synthesis (SPPS), the stability of activation/coupling reagents, and a microarray surface metrology tool. This technique was used to assess the stability of the activation reagent 1‐{[1‐(Cyano‐2‐ethoxy‐2‐oxo‐ethylidenaminooxy)dimethylamino‐morpholinomethylene]}methaneaminiumHexafluorophosphate (COMU) (Sigma‐Aldrich, St. Louis, MO, USA) by SPPS of Leu‐Enkephalin (YGGFL) or the coupling of commercially synthesized YGGFL peptides to (3‐aminopropyl)triethyoxysilane‐modified glass surfaces. Coupling efficiency was quantitated by fluorescence signaling based on immunoreactivity of the YGGFL motif. It was concluded that COMU solutions should be prepared fresh and used within 5 h when stored at ~23 °C and not beyond 24 h if stored refrigerated, both in closed containers. Caveats to gauging COMU stability by absorption spectroscopy are discussed. Commercial YGGFL peptides needed independent QC, due to immunoreactivity variations for the same sequence synthesized by different vendors. This technique is useful in evaluating the stability of other activation/coupling reagents besides COMU and as a metrology tool for SPPS and peptide microarrays. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd. Microwell plate‐based absorption and fluorescence assays were developed to evaluate the quality and shelf life of peptide activation reagents in order to establish metrology during solid phase peptide synthesis, peptide couplings, and the building of peptide microarrays.
ISSN:1075-2617
1099-1387
DOI:10.1002/psc.2806