Microsequence analysis of peptides and proteins: VI. A continuous flow reactor for sample concentration and sequence analysis

Abstract

We have designed and tested a continuous flow reactor (CFR) for microsequence analysis of peptides and proteins. The CFR forms the site for immobilization of the peptide or protein substrate and automated Edman chemistry. The CFR was constructed from 0.125-in.-o.d., 0.0625-in.-i.d. Teflon tubing (length 2–3 cm) containing 5–10 mg of Polybrene-coated, spherical, porous silica (100–200-μm particle size). The silica is retained in the CFR with porous Teflon filters (Zitex) at the bed bottom and optionally at the bed top. The i.d. of the CFR was selected for a tight press fit when 0.0625-in.-o.d. Teflon lines are inserted at the top and bottom of the CFR. This design allows the replacement of the existing cartridge/glass fiber disk found in conventional microsequencers with a CFR with a minimal amount of changes. The advantages of the CFR over the previous design include a lower background or noise level and no need to precycle Polybrene before sample application, and the entire unit is inexpensive and therefore disposable. We believe that the decrease in noise, especially the decrease in the commonly observed diphenylthiourea peak, is due to the more direct flow path and relative absence of unswept area in the CFR. Several standard peptides and proteins were sequenced in the CFR to demonstrate the improved results. A direct comparison to the cartridge/glass fiber disk design demonstrated less background and higher initial and repetitive yields for the CFR. An additional advantage is the ability to directly concentrate samples on CFRs containing reverse-phase packing. We have successfully concentrated 1.0-ml samples (200 pmol) onto 5 mg of octyldecylsilyl-derivatized silica in yields of 95–100%. The resulting samples were microsequenced after addition of Polybrene-coated silica to the CFR with high initial and repetitive yields. This methodology promises to improve sample handling and microsequence analysis of low picomole amounts of peptides and proteins.