Biology subjects

Pike, D.

Publications and source records attributed to Pike, D..

4 recordsLinked to original sources

bioRxiv2025

Protein Barcoding and Next-Generation Protein Sequencing for Multiplexed Protein Selection, Analysis, and Tracking

Protein barcoding has emerged as a powerful tool for the multiplexed identification and characterization of proteins, providing a mechanism for precise tracking of protein affinity, location, and expression. In this study, we describe the development of a protein barcoding workflow for use with single-molecule Next-Generation Protein Sequencing (NGPS) on the benchtop Platinum(R) and Platinum(R) Pro instruments. We present data on the validation of eight peptide barcodes, each designed to minimize detection bias and maximize sensitivity across various experimental conditions. We have also optimized the design of expression constructs to decrease both the hands-on time and input requirements of the workflow. In this workflow, affinity-tagged proteins are expressed with unique peptide barcodes. Following experimental selection or treatments, the proteins are purified, and the peptide barcodes are cleaved and sequenced on the Platinum instrument. We demonstrate that we can detect barcodes at 400 fmol of sample input concentration within the eight-plex mixture, and at 50 fmol of sample input for individual barcodes. We also show the capacity of this barcoding approach to achieve a ten-fold dynamic range, underscoring its sensitivity in recovering variants with low abundance. Through the combination of protein barcoding and NGPS, we lay the groundwork for future studies aimed at characterizing protein interactions and improving targeted drug delivery strategies.

biochemistry↗

bioRxiv2024

Detecting Amino Acid Variants Using Next-Generation Protein Sequencing (NGPS)

Next-Generation Protein Sequencing (NGPS) is a single-molecule approach for characterizing protein variants, offering detailed insight into proteoforms and amino acid substitutions not easily discerned by mass spectrometry. The novel data type produced by NGPS, which is based on binding of N-terminal amino acids by fluorescently tagged recognizer proteins, requires the development of new data analysis methods and bioinformatic tools. Here, we present ProteoVue, a comprehensive bioinformatics pipeline for Single Amino Acid Variant (SAAV) detection and quantification using the Quantum-Si Platinum(R) NGPS platform. ProteoVue integrates multiple analytical components, including robust pulse-calling, recognition segment detection, fluorescence dye classification, and a neural network-driven kinetic signature database for pulse duration prediction. These components feed into a scoring-based alignment and clustering framework that enables accurate variant calling within binary peptide mixtures. We demonstrate that ProteoVue recovers expected variant ratios across diverse substitution types including residues that lack direct amino acid recognizers. While some extreme cases remain challenging, the pipeline consistently captures the key kinetic features required for variant discrimination, underscoring its potential as a versatile and powerful tool for proteomic studies. As NGPS technology matures and recognizer libraries expand, ProteoVue provides a foundation for increasingly refined variant analysis in basic research, biomarker discovery, and clinical applications.

bioinformatics↗

bioRxiv2023

Structural Insights into Regulation of Insulin Expression Involving i-Motif DNA Structures in the Insulin-Linked Polymorphic Region.

The insulin linked polymorphic region (ILPR) is a variable number of tandem repeats (VNTR) region of DNA in the promoter of the insulin gene that regulates transcription of insulin. This region is known to form the alternative DNA structures, i-motifs and G-quadruplexes. Individuals have different sequence variants of VNTR repeats and although previous work investigated the effects of some variants on G-quadruplex formation, there is not a clear picture of the relationship between the sequence diversity, the DNA structures formed, and the functional effects on insulin gene expression. Here we show that different sequence variants of the ILPR form different DNA secondary structures and insulin expression is dependent on formation of i-motif and G-quadruplex structures. The first crystal structure and dynamics of an intramolecular i-motif also reveal sequences within the loop regions forming additional stabilising interactions, which are critical to formation of the stable i-motif structures that modulate insulin expression. The outcomes of this work reveal the detail in formation of stable i-motif DNA structures, with potential for rational based drug design for compounds to alter insulin gene expression.

biophysics↗

bioRxiv2020

Alternating handedness motifs in proteins classify structure and cofactor binding

Cofactor binding sites in proteins often are composed of favorable interactions of specific cofactors with the sidechains and/or backbone protein fold motifs. In many cases these motifs contain left-handed conformations which enable tight turns of the backbone that present backbone amide protons in direct interactions with cofactors termed cationic nests. Here, we defined alternating handedness of secondary structure as a search constraint within the PDB to systematically identify these cofactor binding nests. We identify unique alternating handedness structural motifs which are specific to the cofactors they bind. These motifs can guide the design of engineered folds that utilize specific cofactors and also enable us to gain a deeper insight into the evolution of the structure of cofactor binding sites.

bioinformatics↗