Gene-centric coverage of the human liver transcriptome: QPCR, Illumina, and Oxford Nanopore RNA-Seq

It has been shown that the best coverage of the HepG2 cell line transcriptome encoded by genes of a single chromosome, chromosome 18, is achieved by a combination of two sequencing platforms, Illumina RNA-Seq and Oxford Nanopore Technologies (ONT), using cut-off levels of FPKM > 0 and TPM > 0, respectively. In this study, we investigated the extent to which the combination of these transcriptomic analysis methods makes it possible to achieve a high coverage of the transcriptome encoded by the genes of other human chromosomes. A comparative analysis of transcriptome coverage for various types of biological material was carried out, and the HepG2 cell line transcriptome was compared with the transcriptome of liver tissue cells. In addition, the contribution of variability in the coverage of expressed genes in human transcriptomes to the creation of a draft human transcriptome was evaluated. For human liver tissues, ONT makes an extremely insignificant contribution to the overall coverage of the transcriptome. Thus, to ensure maximum coverage of the liver tissue transcriptome, it is sufficient to apply only one technology: Illumina RNA-Seq (FPKM > 0)

The Size of the Human Proteome: The Width and Depth

This work discusses bioinformatics and experimental approaches to explore the human proteome, a constellation of proteins expressed in different tissues and organs. As the human proteome is not a static entity, it seems necessary to estimate the number of different protein species (proteoforms) and measure the number of copies of the same protein in a specific tissue. Here, meta-analysis of neXtProt knowledge base is proposed for theoretical prediction of the number of different proteoforms that arise from alternative splicing (AS), single amino acid polymorphisms (SAPs), and posttranslational modifications (PTMs). Three possible cases are considered: (1) PTMs and SAPs appear exclusively in the canonical sequences of proteins, but not in splice variants; (2) PTMs and SAPs can occur in both proteins encoded by canonical sequences and in splice variants; (3) all modification types (AS, SAP, and PTM) occur as independent events. Experimental validation of proteoforms is limited by the analytical sensitivity of proteomic technology. A bell-shaped distribution histogram was generated for proteins encoded by a single chromosome, with the estimation of copy numbers in plasma, liver, and HepG2 cell line. The proposed metabioinformatics approaches can be used for estimation of the number of different proteoforms for any group of protein-coding genes

Пептиды в составе препарата Лаеннек®, способствующие устранению эндотелиопатии

Objective: identification of peptides in the composition of Laennec®, which can inhibit the development of endotheliopathy (endothelial dysfunction).Material and methods. Hybrid mass spectrometry followed by data analysis based on topological recognition theory was performed. The analysis of the peptide composition of Laennec® included four stages: purification of the drug, chromatographic separation of peptides, determination of the multidimensional mass spectrum of the peptide fraction, and de novo sequencing of the isolated peptides.Results. The preparation contains peptides-inhibitors of specific target proteins (PRKCZ, PKB, PKD1, MAPK14, IKKB, PDPK1) involved in the activation of the pro-inflammatory transcription factor NF-κB. Inhibition of CDK5 and SHC1 kinases helps to reduce endothelial cell apoptosis. The peptides of the drug also block enzymes involved in the synthesis and maturation of the tumor necrosis factor alpha (MAPKAPK2/3, ADAM17).Conclusion. In the composition of Laennec®, peptides have been found that contribute to a complex pathogenetic action against endotheliopathy. Endothelial regeneration is especially important in the rehabilitation of patients who have recovered from COVID-19.Цель: выявление пептидов в составе препарата Лаеннек®, которые могут тормозить развитие эндотелиопатии (эндотелиальной дисфункции).Материал и методы. Проведена гибридная масс-спектрометрия с последующим анализом данных на основе топологической теории распознавания. Анализ пептидного состава Лаеннека® включал четыре этапа: очистка препарата, хроматографическое разделение пептидов, определение многомерного масс-спектра пептидной фракции и de novo секвенирование выделенных пептидов.Результаты. В составе препарата идентифицированы пептиды-ингибиторы специфических таргетных белков (PRKCZ, PKB, PKD1, MAPK14, IKKB, PDPK1), вовлеченные в активацию провоспалительного транскрипционного фактора NF-κB. Ингибирование киназ CDK5 и SHC1 способствует снижению апоптоза эндотелиоцитов. Пептиды препарата также блокируют ферменты, участвующие в синтезе и вызревании фактора некроза опухолей альфа (MAPKAPK2/3, ADAM17).Заключение. В составе препарата Лаеннек® найдены пептиды, которые способствуют комплексному патогенетическому действию против эндотелиопатии. Регенерация эндотелия особенно актуальна в реабилитации пациентов, переболевших COVID-19

Анализ лёгкой пептидной фракции Лаеннека методами современной протеомики

Laennec preparation is based on standardized human placenta hydrolyzate and is highly effective in the regeneration of tissues. This study presents the results of analysis of the light peptide fraction of Laennec (Препарат Лаеннек основан на стандартизованном гидролизате плаценты человека и отличается высокой эффективностью при регенерации тканей. В настоящем исследовании проведён анализ лёгкой пептидной фракции препарата Лаеннек до 3 000 Да тремя методами: (1) высокоточной масс-спектрометрией на масс-спектрометре Q-Exactive (Thermo Scientific, Германия); (2) иммуноферментным анализом на содержание эпитопов белков посредством иммуноферментного анализа (ИФА, ELISA) с использованием моноклональных антител к IGF-1, TGF-1, HGF, VEGF, PDGF, EGF и др. и хемокинов (IL-8, IL-1a, IL-1b, TNFa, IL-12) и (3) секвенирование выделенных пептидов. Установлена высокая степень стандартизации препарата по пептидам. С использованием стандартного программного обеспечения были установлены аминокислотные последовательности 47 пептидов. Установлено наличие в составе Лаеннека пептидов альбумина, коллагенов Ia2, Va2, XIXa1, «цинковых пальцев», а также активных пептидных фрагментов ростовых факторов IGF-1, TGF-1, HGF, VEGF, PDGF, EGF и др. Наличие этих пептидов в составе Лаеннека позволило сформулировать ряд ранее неизвестных молекулярных механизмов действия препарата

Dataset of target mass spectromic proteome profiling for human chromosome 18

Proteome profiling is a type of quantitative analysis that reveals level of protein expression in the sample. Proteome profiling by using selected reaction monitoring is an approach for the Chromosome-centric Human Proteome Project (C-HPP). Here we describe dataset generated in the course of the pilot phase of Russian part of C-HPP, which was focused on human Chr 18 proteins. Proteome profiling was performed using stable isotope-labeled standards (SRM/SIS) for plasma, liver tissue and HepG2 cells. Dataset includes both positive and negative results of protein detection.These data were partly discussed in recent publications, “Chromosome 18 Transcriptome Profiling and Targeted Proteome Mapping in Depleted Plasma, Liver Tissue and HepG2 Cells” [1] and “Chromosome 18 transcriptoproteome of liver tissue and HepG2 Cells and targeted proteome mapping in depleted plasma: Update 2013” [2], supporting the accompanying publication “State of the Chromosome 18-centric HPP in 2016: Transcriptome and Proteome Profiling of Liver Tissue and HepG2 Cells” [3], and are deposited at the ProteomeXchange via the PASSEL repository with the dataset identifier PASSEL: PASS00697 for liver and HepG2 cell line

Geroprotective properties of neuroprotective and neurotrophic peptides

Objective: to elucidate whether cerebrolysin contains peptide fragments that promote geroprotection.Material and methods. The peptide composition of cerebrolysin underwent a comprehensive mass spectrometric analysis, followed by a systemic biological assessment.Results and discussion. Thirty-six peptides with geroprotective properties were isolated in the multipeptide composition of cerebrolysin. These peptides included those of mimetics of adrenomedullin and enkephalins; those of inhibitors of seven targeted human proteins (the protein kinases MAPK1, VPRBP, and PKC, the gamma-secretase PS1, the kinases CDK1, SGK1 and mTOR). The established cerebrolysin peptides were shown to be competitive inhibitors of the seven targeted proteins. In particular, inhibition of PKC and mTOR stimulated the increased autophagy (the utilization of waste and abnormal proteins), which contributes to an increase in the survival of cells and model organisms.Conclusion. Cerebrolysin can have geroprotective effects, by inhibiting the seven targeted proteins, by activating endorphinergic neurotransmission, and can be indirectly involved in vascular tone normalization

Use of Biotinylated Ubiquitin for Analysis of Rat Brain Mitochondrial Proteome and Interactome

Applicability of in vitro biotinylated ubiquitin for evaluation of endogenous ubiquitin conjugation and analysis of ubiquitin-associated protein-protein interactions has been investigated. Incubation of rat brain mitochondria with biotinylated ubiquitin followed by affinity chromatography on avidin-agarose, intensive washing, tryptic digestion of proteins bound to the affinity sorbent and their mass spectrometry analysis resulted in reliable identification of 50 proteins belonging to mitochondrial and extramitochondrial compartments. Since all these proteins were bound to avidin-agarose only after preincubation of the mitochondrial fraction with biotinylated ubiquitin, they could therefore be referred to as specifically bound proteins. A search for specific ubiquitination signature masses revealed several extramitochondrial and intramitochondrial ubiquitinated proteins representing about 20% of total number of proteins bound to avidin-agarose. The interactome analysis suggests that the identified non-ubiquitinated proteins obviously form tight complexes either with ubiquitinated proteins or with their partners and/or mitochondrial membrane components. Results of the present study demonstrate that the use of biotinylated ubiquitin may be considered as the method of choice for in vitro evaluation of endogenous ubiquitin-conjugating machinery in particular subcellular organelles and changes in ubiquitin/organelle associated interactomes. This may be useful for evaluation of changes in interactomes induced by protein ubiquitination under norm and various brain pathologies

Proteomic Signature of Extracellular Vesicles for Lung Cancer Recognition

The proteins of extracellular vesicles (EVs) that originate from tumors reflect the producer cells’ proteomes and can be detected in biological fluids. Thus, EVs provide proteomic signatures that are of great interest for screening and predictive cancer diagnostics. By applying targeted mass spectrometry with stable isotope-labeled peptide standards, we assessed the levels of 28 EV-associated proteins, including the conventional exosome markers CD9, CD63, CD81, CD82, and HSPA8, in vesicles derived from the lung cancer cell lines NCI-H23 and A549. Furthermore, we evaluated the detectability of these proteins and their abundance in plasma samples from 34 lung cancer patients and 23 healthy volunteers. The abundance of TLN1, TUBA4A, HSPA8, ITGB3, TSG101, and PACSIN2 in the plasma of lung cancer patients was measured using targeted mass spectrometry and compared to that in plasma from healthy volunteers. The most diagnostically potent markers were TLN1 (AUC, 0.95), TUBA4A (AUC, 0.91), and HSPA8 (AUC, 0.88). The obtained EV proteomic signature allowed us to distinguish between the lung adenocarcinoma and squamous cell carcinoma histological types. The proteomic cargo of the extracellular vesicles represents a promising source of potential biomarkers

Impact of p53 Knockout on Protein Data Set of HaCaT Cells in Confluent and Subconfluent Conditions

The immortalized keratinocytes, HaCaT, are a popular model for skin research (toxicity, irritation, allergic reactions, or interaction of cells). They maintain a stable keratinocyte phenotype and respond to keratinocyte differentiation stimuli. However, programs of stratification and expression of differentiation markers in HaCaT keratinocytes are aberrant. In HaCaT cells, there are two mutant p53 alleles (i.e., R282Q and H179Y) that contain gain-of-function (GOF) mutations resulting from spontaneous immortalization (mutp53). At the same time, mutp53 acts as a transcription factor and also affects the interaction of p63 protein with its transcription targets. Proteins of the p53 family are crucial for regulation of proliferation and differentiation processes in human keratinocytes, although the involvement of mutp53 in these processes is not fully clear. We present data sets obtained as a result of high-performance proteomic analysis of immortalized HaCaT keratinocytes with p53 knockout in two different states, subconfluent and confluent, which are characterized by different intensites of cell differentiation processes. To obtain the proteomic profiles of the cells, we applied LC-MS/MS measurements processed with MaxQuant software (version 1.6.3.4)