Biology subjects

Ocampo, D.

Publications and source records attributed to Ocampo, D..

3 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↗

bioRxiv2023

Cellular zinc status alters chromatin accessibility and binding of transcription factor TP53 to genomic sites

Zinc (Zn2+) is an essential metal required by approximately 2500 proteins. Nearly half of these proteins act on DNA, including > 850 human transcription factors, polymerases, DNA damage response factors, and proteins involved in chromatin architecture. How these proteins acquire their essential Zn2+ cofactor and whether they are sensitive to changes in the labile Zn2+ pool in cells remain open questions. Here, we examine how changes in the labile Zn2+ pool affect chromatin accessibility and transcription factor binding to DNA. We observed both increases and decreases in accessibility in different chromatin regions via ATAC-seq upon treating MCF10A cells with elevated Zn2+ or the Zn2+-specific chelator tris(2-pyridylmethyl)amine (TPA). Transcription factor enrichment analysis was used to correlate changes in chromatin accessibility with transcription factor motifs, revealing 477 transcription factor motifs that were differentially enriched upon Zn2+ perturbation. 186 of these transcription factor motifs were enriched in Zn2+ and depleted in TPA, and the majority correspond to Zn2+ finger transcription factors. We selected TP53 as a candidate to examine how changes in motif enrichment correlate with changes in transcription factor occupancy by ChIP-qPCR. Using publicly available ChIP-seq and nascent transcription datasets, we narrowed the 50,000+ ATAC-seq peaks to 2164 TP53 targets and subsequently selected 6 high-probability TP53 binding sites for testing. ChIP-qPCR revealed that for 5 of the 6 targets, TP53 binding correlates with the local accessibility determined by ATAC-seq. These results demonstrate that changes in labile zinc directly alter chromatin accessibility and transcription factor binding to DNA.

genomics↗

bioRxiv2022

Adaptive variation in avian eggshell structure and gas conductance across elevational gradients?

Many tropical species have restricted elevational distributions, which are potentially bounded by constraints imposed by physical conditions on physiological processes. Although some studies have examined variation in the physiology of adult birds with respect to elevation, little attention has been paid to the structure and function of eggshells, which mediate gas exchange between the embryo and the environment. At high elevations, dry air is expected to increase water loss from the egg; selection to avoid desiccation might therefore be expected to favor reduced gas conductance by means of increased eggshell thickness or reduced pore size. We used gas diffusion experiments and scanning electron microscopy to examine water vapor conductance rates and eggshell structures in 197 bird species distributed along an elevational gradient in the Andes. As predicted, water vapor conductance across the eggshell declined in a narrow range with elevation among all species and among species within families, but not among individuals within species. Variation among species in eggshell conductance was lower at high-elevation sites, potentially indicating greater constraints at such sites. Structural changes in eggshells with respect to elevation varied among taxonomic families of birds, suggesting potentially different adaptive responses to common selective pressures in terms of eggshell thickness and pore density, and size. We suggest that considering functional and structural traits of eggshells, which influence embryo development, may help one to better understand the elevational distributions of species and to forecast their responses to global climate change.

evolutionary biology↗