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Tsybin, Yury O.

Publications and source records attributed to Tsybin, Yury O..

8 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Top-down proteomics

Proteoforms arising from posttranslational modifications, genetic polymorphisms, and RNA splice variants, play a pivotal role as the key drivers in biology. Thus, a comprehensive understanding of proteoforms is essential for unraveling the intricacies of biological systems and bridging the gap between genotype and phenotype. By analyzing whole proteins without digestion, top-down proteomics (TDP) provides a holistic view of the proteome and presents a next-generation approach for deciphering protein function, uncovering disease mechanisms, and advancing precision medicine. This Primer embarks on a journey into the world of TDP by encapsulating its historical context, underlying principles, recent advances, and an outlook on the future of TDP. The experimental section navigates instrumentation, sample preparation, intact protein separation, tandem mass spectrometry techniques, and data collection. Results decipher raw data, visualize intact protein spectra, unravel data analysis, and explain proteoform identification, characterization, and quantitation, as well as statistical analysis. Various applications of TDP spanning the human proteoform project, biomedical, biopharmaceutical, and clinical applications are described. These are complemented by discussions on measurement reproducibility, limitations, and a forward-looking perspective outlining uncharted waters where the field can advance, and potential exciting future applications of TDP.

Roberts, David S.↗
DOE OSTI2021

The Human Proteoform Project: Defining the human proteome

Proteins are the primary effectors of function in biology, and thus, complete knowledge of their structure and properties is fundamental to deciphering function in basic and translational research. The chemical diversity of proteins is expressed in their many proteoforms, which result from combinations of genetic polymorphisms, RNA splice variants, and posttranslational modifications. This knowledge is foundational for the biological complexes and networks that control biology yet remains largely unknown. We propose here an ambitious initiative to define the human proteome, that is, to generate a definitive reference set of the proteoforms produced from the genome. Several examples of the power and importance of proteoform-level knowledge in disease-based research are presented along with a call for improved technologies in a two-pronged strategy to the Human Proteoform Project.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Interlaboratory Study for Characterizing Monoclonal Antibodies by Top-Down and Middle-Down Mass Spectrometry

The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product’s primary structure reliably and reproducibly. The major objective of the present study was to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up approaches, such as confirming protein integrity, sequencing variable domains, ruling out artefacts, and revealing modifications and their locations. A panel of three mAbs was selected and centrally provided to twenty laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). A variety of different MS instrument platforms and ion dissociation techniques were employed. Overall, the present study confirms that MD/TD MS strategies are valuable and readily available tools in laboratories worldwide. They provide complementary information to the bottom-up approach and can add unique information, which may be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. One of the limitations revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS, but the number of TD/MD MS experts is still very low.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

A five-level classification system for proteoform identifications

The term proteoform, introduced in these pages in 2013, has rapidly gained acceptance in the proteomics community. The challenge and importance of comprehensively identifying proteoforms in complex samples has been recognized, and reports have begun to appear of new platforms towards that end. However, one interesting central ambiguity has emerged, namely determining precisely what is meant by a “proteoform identification”. At present, the only practical approaches for establishing the exact primary structure of a proteoform employ mass spectrometry (MS), and there is a wide range of MS results that provide “proteoform identifications”. This seemingly small matter has significant impact, as the ambiguity in knowing what is meant by an “identification” makes it difficult to compare results from different laboratories and approaches. This situation hinders the ability of the community to evaluate technological progress and to efficiently expand biological knowledge. To address this issue, we propose a five-level system for classifying proteoform identifications. Here, the classification scheme stems directly from a consideration of the four types of possible ambiguity possible for a proteoform identification, ranging from the most subtle (i.e., precise localization of a post-translational modification, or PTM) to the most dramatic (i.e., ambiguity in the gene of origin). The five classes then correspond to the level of ambiguity present in the identification, ranging from no ambiguity at all (Level 1), to ambiguity of all four types (Level 5). Details of the scheme are provided in Table 1 and Supplementary Table 1, with specific use cases and examples provided in Supplementary Figure 1.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Best practices and benchmarks for intact protein analysis for top-down mass spectrometry

One gene can give rise to many functionally distinct proteoforms, each of which has a characteristic molecular mass. Many laboratories, however, forgo the challenging mass analysis of intact proteins. Instead, these researchers resort to well-established methods for proteolytic digests. In this work, the Consortium for Top-Down Proteomics (CTDP) addresses the unmet need for robust protocols for obtaining intact protein mass spectra. We quantify and rationalize the signal suppression associated with common interfering substances. A decision tree, based upon sample composition and experimental goals, guides researchers to a given protocol. Cross-platform analytical benchmarks are provided to allow users to gauge their proficiency and to promote method standardization. This work should permit users at any experience level to obtain high-quality intact mass spectra of native and denatured proteins from mixtures of varying complexity, including intact antibodies and membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

ProForma: A Standard Proteoform Notation

The Consortium for Top-Down Proteomics (CTDP) proposes a standardized notation, ProForma, for writing the sequence of fully characterized proteoforms. ProForma provides a means to communicate any proteoform by writing the amino acid sequence using standard one-letter notation and specifying modifications or unidentified mass shifts within brackets following certain amino acids. The notation is unambiguous, human readable, and can easily be parsed and written by bioinformatic tools. This system uses seven rules and supports a wide range of possible use cases, ensuring compatibility and reproducibility of proteoform annotations. Standardizing proteoform sequences will simplify storage, comparison, and reanalysis of proteomic studies, and the Consortium welcomes input and contributions from the research community on the continued design and maintenance of this standard.

Environmental Molecular Sciences Laboratory↗
DOE OSTI2018

Multiparticle Simulations of Quadrupolar Ion Detection in an Ion Cyclotron Resonance Cell with Four Narrow Aperture Detection Electrodes

The current paradigm in FT-ICR cell design is to approximate the ideal three-dimensional quadratic trapping potential as closely as possible to maintain ion cloud spatial coherence and achieve long transients, either with hyperbolically shaped electrodes, shimming electrodes, or by dynamic harmonization. In sharp contrast, the FT-ICR analyzer cell with four narrow aperture detection electrodes (NADEL) introduces significant anharmonic terms to the trapping potential. This analyzer cell is capable of quadrupolar detection by which one can measure a signal that is close to the unperturbed cyclotron frequency. This is far less sensitive to trapping potential and space charge shifts than the reduced cyclotron frequency measured in conventional ICR cells. The quadrupolar mode of ion detection in NADEL cells has been examined previously by SIMION simulations of ion clouds with up to 500 ions per simulation. Here, the behavior of the NADEL analyzer cell is examined through particle-in-cell (PIC) simulations, which allows us to examine the behavior of large populations (tens of thousands) of ions with space charge considerations, and to calculate the induced charge on the NADEL detection electrodes, and thus the transient signal. PIC simulations confirm a unique spatial distribution of the ions, with a coherent motion that results in long transient signals. Dependence of the ion cloud and image current signal on cell design, ion energy, and magnetron radius are examined. Coalescence effects are compared with those found in a dynamically harmonized cell. The insensitivity of the measured cyclotron frequency to space-charge is demonstrated both with simulations and experimentally. .

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗