Biology subjects

Chorny, I.

Publications and source records attributed to Chorny, I..

5 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

Next-generation sequencing-based liquid biopsy can be used for detection of residual disease and cancer recurrence monitoring in dogs

ObjectiveThe purpose of this study was to evaluate the performance of a next-generation sequencing-based liquid biopsy test for cancer monitoring in dogs. SamplesPre- and post-operative blood samples were collected prospectively from dogs with confirmed cancer diagnoses originally enrolled in the CANcer Detection in Dogs (CANDiD) study. A subset of these dogs also had longitudinal blood samples collected for recurrence monitoring. MethodsAll patients had a pre-operative blood sample collected (after diagnosis but prior to surgical intervention) in which a cancer signal was detected, and had at least one post-operative sample collected. Clinical data were collected for all patients and used to assign a clinical disease status for each follow-up visit. ResultsFollowing excisional surgery, in the absence of clinical residual disease at the post-operative visit, patients with Cancer Signal Detected results at that visit were 1.95-times as likely to have clinical recurrence within 6 months compared to patients with Cancer Signal Not Detected results. In the subset of patients with longitudinal liquid biopsy samples that had clinical recurrence documented during the study period, 73% (8/11; 95% CI: 39 - 93%) of patients had Cancer Signal Detected in blood prior to or concomitant with clinical recurrence; in the 6 patients where molecular recurrence was detected prior to clinical recurrence, the median lead time was 168 days (range: 47 - 238). Clinical RelevanceNext-generation sequencing-based liquid biopsy is a non-invasive tool for cancer monitoring in dogs that can be used as an adjunct to current standard-of-care clinical assessment methods.

genomics↗

bioRxiv2022

Age at cancer diagnosis by breed, weight, sex, and cancer type in a cohort of over 3,000 dogs: determining the optimal age to initiate cancer screening in canine patients

The goal of cancer screening is to detect disease at an early stage when treatment may be more effective. Until recently, cancer screening in dogs has relied upon annual physical examinations and routine laboratory tests, which are largely inadequate for detecting preclinical disease. With the introduction of non-invasive "liquid biopsy" cancer detection methods, the discussion is shifting from "How to screen dogs for cancer" to "When to screen dogs for cancer". To address this question, data from 3,452 cancer-diagnosed subjects were analyzed to determine the age at which dogs of certain breeds and weights are typically diagnosed with cancer. In the study population, the median age at cancer diagnosis was 8.8 years, with males diagnosed at younger ages than females, and spayed/neutered dogs diagnosed at significantly later ages than intact dogs. Overall, weight was inversely correlated with age at cancer diagnosis, and purebred dogs were diagnosed at significantly younger ages than mixed-breed dogs. For breeds with 10 or more subjects, a breed-based median age at diagnosis was calculated. A weight-based linear regression model was developed to predict the median age at diagnosis for breeds represented by fewer than 10 subjects and for mixed-breed dogs. The study findings support a general recommendation to start cancer screening for all dogs at the age of 7, and as early as 4 years of age for breeds with a lower median age at cancer diagnosis, in order to increase the chances of early detection and treatment.

cancer biology↗

bioRxiv2021

Blood-based liquid biopsy for comprehensive cancer genomic profiling using next-generation sequencing: an emerging paradigm for noninvasive cancer detection and management in dogs

This proof-of-concept study demonstrates that blood-based liquid biopsy using next generation sequencing of cell-free DNA can noninvasively detect multiple classes of genomic alterations in dogs with cancer, including alterations that originate from spatially separated tumor sites. Eleven dogs with a variety of confirmed cancer diagnoses (including localized and disseminated disease) who were scheduled for surgical resection, and 5 presumably cancer-free dogs, were enrolled. Blood was collected from each subject, and multiple spatially separated tumor tissue samples were collected during surgery from 9 of the cancer subjects. All samples were analyzed using an advanced prototype of a novel liquid biopsy test designed to noninvasively interrogate multiple classes of genomic alterations for the detection, characterization, and management of cancer in dogs. In 5 of the 9 cancer patients with matched tumor and plasma samples, pre-surgical liquid biopsy testing identified genomic alterations, including single nucleotide variants and copy number variants, that matched alterations independently detected in corresponding tumor tissue samples. Importantly, the pre-surgical liquid biopsy test detected alterations observed in spatially separated tissue samples from the same subject, demonstrating the potential of blood-based testing for comprehensive genomic profiling of heterogeneous tumors. Among the 3 patients with post-surgical blood samples, genomic alterations remained detectable in one patient with incomplete tumor resection, suggesting utility for noninvasive detection of minimal residual disease following curative-intent treatment. Liquid biopsy allows for noninvasive profiling of cancer-associated genomic alterations with a simple blood draw and has potential to overcome the limitations of tissue-based testing posed by tissue-level genomic heterogeneity.

genomics↗