This article explores how peptide barcodes are utilized in Next-Generation Protein Sequencing™ (NGS).
Sponsored Content by Quantum-SIMar 11 2024Reviewed by Aimee Molineux Barcoding technologies using the high information content encoded in oligonucleotide sequences have aided various applications in biotechnology, especially when paired with next-generation DNA sequencing or NGS, whereby they can decode this information in a low-cost and high-throughput manner.
This platform is user-friendly and simple to use, combining DNA-based approaches with the innovation of peptide-based barcoding methods to enable real-time single-molecule sequencing. The temporal order of NAA recognition and associated kinetic properties throughout sequencing are highly characteristic of any given peptide and are called its kinetic signature . KSs are analyzed with Platinum to deliver high-assurance alignments to individual sequences.5
After cloning, the barcode plasmids were transformed into SHuffle T7 express competent E. coli and cultivated overnight on suitable antibiotic selection media. Using Sanger sequencing, the sequence of the barcode plasmids was confirmed. The supernatant solution was applied to the pre-equilibrated Ni-NTA resin then incubated for 30 minutes at an ambient temperature and mixed. The resin was then pelleted by centrifugation at 1000 x g for two minutes and the supernatant was collected.
For the purification of the azide-labeled PB product, 20 µL of Magne HaloTag beads were used per barcode. First, the HaloTag beads were washed four times for five minutes with 200 µL of HEB Buffer using a magnetic stand for the bead collection during the wash phase. The following day, the beads were washed with HEB buffer three times as described. To elute the PBs from the HaloTag, 25 µL of HEB Buffer supplemented with 1 mM DTT and 1 U of Sumo Protease was introduced to each barcode.
Affinity purification selection was performed on the barcoded model nanobody library using commercially purchased GFP immobilized on M-280 tosylactivated Dynabeads . The barcoded nanobody library was incubated with the GFP-coated Dynabeads for five minutes at room temperature in 50 mM Tris pH 7.5 and 0.5 % Tween-20.
To examine if this group of peptides had the particular qualities needed for use as a barcode set, synthetic peptides were generated and their sequencing performance was individually analyzed. The Levenshtein distance is a common measure of sequence similarity in bioinformatics defined as the minimum number of edit operations required to transform one sequence into another.8 The L was computed for every pair of sequences, and L ≥ 3 was observed for all pairs, with a mean L=8.7 .
Barcode sets were created and ranged from 6-member sets for 3-residue barcodes to 1,600-member sets for 8-residue barcodes . Other design parameters, depending on the application, could be used to align with the desired throughput, accuracy and sensitivity. An innovative solution to enrich and specifically conjugate the K-linker to the PB from a complex sample has been developed.
The next task involves developing a method which specifically enriches and elutes the barcodes from a co-expressed protein of interest because digestion is no longer involved in the workflow. This was addressed by the incorporation of a SUMO tag at the N-terminus of the barcode sequence. The fully enzymatic library preparation workflow was tested on barcodes recombinantly expressed in E. coli, with the aim to establish that these barcodes can be identified with a minimal FDR in a controlled mixture of PBs with varying ratios. An 8-PB mixture library, in which each barcode was added to the mixture at the indicated amounts shown in Table 1, was prepared and sequenced.
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