Biology subjects

Chernobrovkin, A. L.

Publications and source records attributed to Chernobrovkin, A. L..

5 recordsLinked to original sources

bioRxiv2025

Towards a species search engine: KISSE offers a rigorous statistical framework for bone collagen tandem mass spectrometry data

DNA and bone collagen are two key sources of resilient molecular markers used to identify species from their remains. Collagen is more stable than DNA, and thus it is preferred for ancient and degraded samples. Current mass spectrometry-based collagen sequencing approaches are empirical and lack a rigorous statistical framework. Based on the well-developed approaches to protein identification in shotgun proteomics, we introduce a first approximation of the species search engine (SSE). Our SSE named KISSE is based on a species-specific library of collagenous peptides that uses both peptide sequences and their relative abundances. The developed statistical model can identify the species and the probability of correct identification, as well as determine the likelihood of the analyzed species not being in the library. We discuss the advantages and limitations of the proposed approach and the possibility of extending it to other tissues.

zoology↗

bioRxiv2021

Abnormal (hydroxy)prolines deuterium content redefines hydrogen chemical mass

Analysing the {delta}2H in individual amino acids of proteins extracted from vertebrates, we unexpectedly found in some samples, notably bone collagen from seals, more than twice as much deuterium in proline and hydroxyproline residues than in seawater. This corresponds to at least four times higher {delta}2H than in any previously reported biogenic sample. We ruled out diet as a plausible mechanism for such anomalous enrichment. This finding puts into question the old adage that you are what you eat. SUMMARYThe chemical mass of hydrogen is defined as an interval from the lowest to the highest content of deuterium 2H, hydrogens heavy stable isotope. Measurements of the deviations {delta}2H in the deuterium content from the standard (ocean water, {delta}2H = 0{per thousand}) are used to characterise biological samples, such as animal bone collagen. The results are often interpreted in terms of the trophic level and diet of the animal as well as prevailing climate during its lifetime. The majority of the published bone collagen {delta}2H data fall into a narrow {delta}2H range limited to {+/-}100{per thousand}. Using novel analysis method, we unexpectedly found greatly higher {delta}2H values, up to 1500{per thousand}, in seal bone collagen. Such anomalous deuterium enrichment is detected only in two amino acid residues, proline and its derivative hydroxyproline, while other residues show much smaller {delta}2H values. Anomalously high {delta}2H values, albeit of lower magnitudes, are also found for these residues in other biological sources. This finding substantially expands the upper bound of the hydrogen chemical mass for biogenic sources. Since neither diet nor environment explain these mysteriously high enrichment levels amounting to more than twice deuterium content in sea water, our understanding of stable isotopes in nature, as well as the old adage "you are what you eat", are put in question.

biochemistry↗

bioRxiv2020

A tale of two tails - efficient profiling of protein degraders by specific functional and target engagement readouts

Targeted protein degradation represents an area of great interest, potentially offering improvements with respect to dosing, side effects, drug resistance and reaching undruggable proteins compared to traditional small molecule therapeutics. A major challenge in the design and characterization of degraders acting as molecular glues is that binding of the molecule to the protein of interest (PoI) is not needed for efficient and selective protein degradation, instead one needs to understand the interaction with the responsible ligase. Similarly, for proteasome targeting chimeras (PROTACs) understanding the binding characteristics of the PoI alone is not sufficient. Therefore, simultaneously assessing the binding to both PoI and the E3 ligase as well as the resulting degradation profile is of great value. The Cellular Thermal Shift Assay (CETSA) is an unbiased cell-based method, designed to investigate the interaction of compounds with their cellular protein targets by measuring compound-induced changes in protein thermal stability. In combination with mass spectrometry (MS) CETSA can simultaneously evaluate compound induced changes in the stability of thousands of proteins. We have used CETSA MS to profile a number of protein degraders, including molecular glues (e.g. IMiDs) and PROTACs to understand mode of action and to deconvolute off-target effects in intact cells. Within the same experiment we were able to monitor both target engagement by observing changes in protein thermal stability as well as efficacy by simultaneous assessment of protein abundances. This allowed us to correlate target engagement (i.e. binding to the PoI and ligases) and functional readout (i.e. degrader induced protein degradation).

molecular biology↗

bioRxiv2020

In-depth characterization of Staurosporine induced proteome thermal stability changes

Cellular thermal shift assay (CETSA) coupled with high-resolution mass-spectrometry (MS) has proven to be indispensable tool to track thermal stability changes in cellular proteins caused by various external or internal perturbations. One of the major applications of CETSA MS is still binding profile characterization of small molecule drugs. Before applying the method for characterization of novel compound it is crucially important to understand the limitations, sensitivity and ways to improve the throughput of the different method implementations. Here we present deep comprehensive profiling of Staurosporine induced proteome thermal stability alteration utilizing different experiment layouts. By applying unbiased straightforward sample preparation and data analysis approaches we were able to compare and benchmark different experiment layouts for detecting compound-induced protein stability changes in CETSA MS experiments.

molecular biology↗

bioRxiv2017

Proteogenomics of adenosine-to-inosine RNA editing in fruit fly

Adenosine-to-inosine RNA editing is one of the most common types of RNA editing, a posttranscriptional modification made by special enzymes. We present a proteomic study on this phenomenon for Drosophila melanogaster. Three proteome data sets were used in the study: two taken from public repository and the third one obtained here. A customized protein sequence database was generated using results of genome-wide adenosine-to-inosine RNA studies and applied for identifying the edited proteins. The total number of 68 edited peptides belonging to 59 proteins was identified in all data sets. Eight of them being shared between the whole insect, head and brain proteomes. Seven edited sites belonging to synaptic vesicle and membrane trafficking proteins were selected for validation by orthogonal analysis by Multiple Reaction Monitoring. Five editing events in cpx, Syx1A, Cadps, CG4587 and EndoA were validated in fruit fly brain tissue at the proteome level using isotopically labeled standards. Ratios of unedited-to-edited proteoforms varied from 35:1 (Syx1A) to 1:2 (EndoA). Lys-137 to Glu editing of endophilin A may have functional consequences for its interaction to membrane. The work demonstrates the feasibility to identify the RNA editing event at the proteome level using shotgun proteomics and customized edited protein database.

biochemistry↗