Comparison of platelet proteomic profiles between children and adults reveals origins of functional differences.

BACKGROUND: Platelets are blood cells responsible for the prevention of blood loss upon vessel wall disruption. It has been demonstrated that platelet functioning differs significantly between adult and pediatric donors. This study aimed to identify potential differences between the protein composition of platelets of pediatric, adolescent, and adult donors. METHODS: Platelet functional testing was conducted with live cell flow cytometry. Using a straightforward approach to platelet washing based on the sequential platelets centrifugation-resuspension, we were able to obtain stable and robust proteomics results, which corresponded to previously published data. RESULTS: We have identified that pediatric donors' platelets have increased amounts of proteins, responsible for mitochondrial activity, proteasome activity, and vesicle transport. Flow cytometry analysis of platelet intracellular signaling and functional responses revealed that platelets of the pediatric donors have diminished granule secretion and increased quiescent platelet calcium concentration and decreased calcium mobilization in response to ADP. We could explain the observed changes in calcium responses by the increased mitochondria protein content, and the changes in granule secretion could be explained by the differences in vesicle transport protein content. CONCLUSIONS: Therefore, we can conclude that the age-dependence of platelet functional responses originates from the difference in platelet protein content. IMPACT: Platelets of infants are known to functionally differ from the platelet of adult donors, although the longevity and persistivity of these differences are debatable. Pediatric donor platelets have enhanced amounts of mitochondrial, proteasomal, and vesicle transport proteins. Platelets of the pediatric donors had increased cytosolic calcium in the resting state, what is explained by the increased numbers of mitochondrial proteins. Infants had decreased platelet granule release, which resolved upon adolescence. Thus, platelets of the infants should be assessed differently from adult platelets. Differences in platelet proteomic contents persisted in adolescent groups, yet, no significant differences in platelet function were observed.

Proteomic Approach to Investigating Expression, Localization, and Functions of the SOWAHD Gene Protein Product during Granulocytic Differentiation.

Cataloging human proteins and evaluation of their expression, cellular localization, functions, and potential medical significance are important tasks for the global proteomic community. At present, localization and functions of protein products for almost half of protein-coding genes remain unknown or poorly understood. Investigation of organelle proteomes is a promising approach to uncovering localization and functions of human proteins. Nuclear proteome is of particular interest because many nuclear proteins, e.g., transcription factors, regulate functions that determine cell fate. Meta-analysis of the nuclear proteome, or nucleome, of HL-60 cells treated with all-trans-retinoic acid (ATRA) has shown that the functions and localization of a protein product of the SOWAHD gene are poorly understood. Also, there is no comprehensive information on the SOWAHD gene expression at the protein level. In HL-60 cells, the number of mRNA transcripts of the SOWAHD gene was determined as 6.4 ± 0.7 transcripts per million molecules. Using targeted mass spectrometry, the content of the SOWAHD protein was measured as 0.27 to 1.25 fmol/µg total protein. The half-life for the protein product of the SOWAHD gene determined using stable isotope pulse-chase labeling was ~19 h. Proteomic profiling of the nuclear fraction of HL-60 cells showed that the content of the SOWAHD protein increased during the ATRA-induced granulocytic differentiation, reached the peak value at 9 h after ATRA addition, and then decreased. Nuclear location and involvement of the SOWAHD protein in the ATRA-induced granulocytic differentiation have been demonstrated for the first time.

Mass Spectrometric Identification of BSA Covalently Captured onto a Chip for Atomic Force Microscopy.

Mass spectrometry (MS) is one of the main techniques for protein identification. Herein, MS has been employed for the identification of bovine serum albumin (BSA), which was covalently immobilized on the surface of a mica chip intended for investigation by atomic force microscopy (AFM). For the immobilization, two different types of crosslinkers have been used: 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) and dithiobis(succinimidyl propionate) (DSP). According to the data obtained by using an AFM-based molecular detector, the SuccBB crosslinker was more efficient in BSA immobilization than the DSP. The type of crosslinker used for protein capturing has been found to affect the results of MS identification. The results obtained herein can be applied in the development of novel systems intended for the highly sensitive analysis of proteins with molecular detectors.

Massive Proteogenomic Reanalysis of Publicly Available Proteomic Datasets of Human Tissues in Search for Protein Recoding via Adenosine-to-Inosine RNA Editing.

The proteogenomic search pipeline developed in this work has been applied for reanalysis of 40 publicly available shotgun proteomic datasets from various human tissues comprising more than 8000 individual LC-MS/MS runs, of which 5442 .raw data files were processed in total. This reanalysis was focused on searching for ADAR-mediated RNA editing events, their clustering across samples of different origins, and classification. In total, 33 recoded protein sites were identified in 21 datasets. Of those, 18 sites were detected in at least two datasets, representing the core human protein editome. In agreement with prior artworks, neural and cancer tissues were found to be enriched with recoded proteins. Quantitative analysis indicated that recoding the rate of specific sites did not directly depend on the levels of ADAR enzymes or targeted proteins themselves, rather it was governed by differential and yet undescribed regulation of interaction of enzymes with mRNA. Nine recoding sites conservative between humans and rodents were validated by targeted proteomics using stable isotope standards in the murine brain cortex and cerebellum, and an additional one was validated in human cerebrospinal fluid. In addition to previous data of the same type from cancer proteomes, we provide a comprehensive catalog of recoding events caused by ADAR RNA editing in the human proteome.

Blood Plasma Proteome: A Meta-Analysis of the Results of Protein Quantification in Human Blood by Targeted Mass Spectrometry.

A meta-analysis of the results of targeted quantitative screening of human blood plasma was performed to generate a reference standard kit that can be used for health analytics. The panel included 53 of the 296 proteins that form a "stable" part of the proteome of a healthy individual; these proteins were found in at least 70% of samples and were characterized by an interindividual coefficient of variation <40%. The concentration range of the selected proteins was 10−10−10−3 M and enrichment analysis revealed their association with rare familial diseases. The concentration of ceruloplasmin was reduced by approximately three orders of magnitude in patients with neurological disorders compared to healthy volunteers, and those of gelsolin isoform 1 and complement factor H were abruptly reduced in patients with lung adenocarcinoma. Absolute quantitative data of the individual proteome of a healthy and diseased individual can be used as the basis for personalized medicine and health monitoring. Storage over time allows us to identify individual biomarkers in the molecular landscape and prevent pathological conditions.

Peptide ligands on the PEGylated nanoparticle surface and human serum composition are key factors for the interaction between immune cells and nanoparticles.

The architecture of a nanoparticles' surface formed due to a modification with a ligand and protein corona formation in biofluids is critical for interactions with cells in vivo. Here we studied interactions of immune cells with magnetic nanoparticles (MNPs) covalently modified with polyethylene glycol (PEG) and their counterparts conjugated with peptides: a pH (low) insertion peptide (pHLIP) and cycloRGD as a targeting ligand in human serum. The conjugation of MNPs-PEG with pHLIP, but not with cycloRGD, enhanced the association of these particles with mononuclear phagocytic cells in vitro and in vivo. We did not find a clear difference in protein corona composition between the pHLIP-modified and parental PEGylated nanoparticles. Analysis of the effect of autologous human serum on MNP uptake by monocytes showed that the efficiency of endocytosis varies among healthy donors and depends on intrinsic properties of serum. Nevertheless, using classic blood, coagulation, biochemical tests, and anti-PEG IgG serum level, we failed to identify the cause of the observed interdonor variation. These individual differences should be taken into consideration during testing of nanotherapeutics.

MS Identification of Blood Plasma Proteins Concentrated on a Photocrosslinker-Modified Surface.

This work demonstrates the use of a modified mica to concentrate proteins, which is required for proteomic profiling of blood plasma by mass spectrometry (MS). The surface of mica substrates, which are routinely used in atomic force microscopy (AFM), was modified with a photocrosslinker to allow "irreversible" binding of proteins via covalent bond formation. This modified substrate was called the AFM chip. This study aimed to determine the role of the surface and crosslinker in the efficient concentration of various types of proteins in plasma over a wide concentration range. The substrate surface was modified with a 4-benzoylbenzoic acid N-succinimidyl ester (SuccBB) photocrosslinker, activated by UV irradiation. AFM chips were incubated with plasma samples from a healthy volunteer at various dilution ratios (102X, 104X, and 106X). Control experiments were performed without UV irradiation to evaluate the contribution of physical protein adsorption to the concentration efficiency. AFM imaging confirmed the presence of protein layers on the chip surface after incubation with the samples. MS analysis of different samples indicated that the proteomic profile of the AFM-visualized layers contained common and unique proteins. In the working series of experiments, 228 proteins were identified on the chip surface for all samples, and 21 proteins were not identified in the control series. In the control series, a total of 220 proteins were identified on the chip surface, seven of which were not found in the working series. In plasma samples at various dilution ratios, a total of 146 proteins were identified without the concentration step, while 17 proteins were not detected in the series using AFM chips. The introduction of a concentration step using AFM chips allowed us to identify more proteins than in plasma samples without this step. We found that AFM chips with a modified surface facilitate the efficient concentration of proteins owing to the adsorption factor and the formation of covalent bonds between the proteins and the chip surface. The results of our study can be applied in the development of highly sensitive analytical systems for determining the complete composition of the plasma proteome.

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).

TRIM28 Is a Novel Regulator of CD133 Expression Associated with Cancer Stem Cell Phenotype.

CD133 is an extensively studied marker of the most malignant tumor cell population, designated as cancer stem cells (CSCs). However, the function of this glycoprotein and its involvement in cell regulatory cascades are still poorly understood. Here we show a positive correlation between the level of CD133 plasma membrane expression and the proliferative activity of cells of the Caco-2, HT-29, and HUH7 cancer cell lines. Despite a substantial difference in the proliferative activities of cell populations with different levels of CD133 expression, transcriptomic and proteomic profiling revealed only minor distinctions between them. Nonetheless, a further in silico assessment of the differentially expressed transcripts and proteins revealed 16 proteins that could be involved in the regulation of CD133 expression; these were assigned ranks reflecting the apparent extent of their involvement. Among them, the TRIM28 transcription factor had the highest rank. The prominent role of TRIM28 in CD133 expression modulation was confirmed experimentally in the Caco2 cell line clones: the knockout, though not the knockdown, of the TRIM28 gene downregulated CD133. These results for the first time highlight an important role of the TRIM28 transcription factor in the regulation of CD133-associated cancer cell heterogeneity.

Impact of p53 knockdown on protein dataset of HaCaT cells.

The HaCaT line of immortalized non-tumor cells is a popular model of keratinocytes used for dermatological studies, in the practice of toxicological tests, and in the study of skin allergic reactions. These cells maintain a stable keratinocyte phenotype, do not require specific growth factors during cultivation, and respond to keratinocyte differentiation stimuli. HaCaT cells bear two mutant p53 alleles - R282Q and H179Y. At least two mechanisms of GOF (gain-of-function) of mutant p53 are known: it affects functions of p63/p73 by inhibiting their binding to DNA; or it binds to new DNA sites by interacting with other transcription factors (NF-Y, E2F1, NF-KB, VDR, p63). Proteins of the P53 family play an important role in the regulation of proliferation and differentiation processes of human keratinocytes. Proteomic study of HaCaT cells with TP53 gene knockdown provides new data for understanding the limitations of HaCaT cells when using them as an experimental model of normal human keratinocytes. In this article we present datasets obtained through the high-throughput shotgun proteomics analysis of human immortalized HaCaT keratinocytes and p53 knockdown HaCaT keratinocytes. As a protocol for proteomic profiling of cells, we used the approach of obtaining LC-MS/MS measurements followed by their processing with MaxQuant software (version 1.6.3.4). The "RAW" files were deposited to the ProteomeXchange with identifier PXD033538.

Deep proteomic dataset of human liver samples obtained by two-dimensional sample fractionation coupled with tandem mass spectrometry.

The data was acquired from 3 normal human liver tissues by LC-MS methods. The tissue liver samples from male subjects post mortem were obtained from ILSBio LLC (https://bioivt.com/). Liver tissue was frozen in liquid nitrogen, transported and shipped on dry ice. The proteins were extracted and purified followed up by trypsin hydrolysis. The peptide mixture was aliquoted and analyzed by different LC-MS approaches: one-dimensional shotgun LC-MS, two-dimensional LC-MS, two-dimensional SRM SIS (Selected Reaction Monitoring with Stable Isotope-labeled peptide Standards). The Shotgun assay resulted in a qualitative in-depth human liver proteome, and a semi-quantitative iBAQ (intensity-based absolute quantification) value was calculated to show the relative protein content of the sample. Absolute quantitative concentrations of proteins encoded by human chromosome 18 using SRM SIS were obtained.

Exploring the Interactome of Cytochrome P450 2E1 in Human Liver Microsomes with Chemical Crosslinking Mass Spectrometry.

Aiming to elucidate the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) on drug metabolism, we explored the array of its protein-protein interactions (interactome) in human liver microsomes (HLM) with chemical crosslinking mass spectrometry (CXMS). Our strategy employs membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide and 4-(N-succinimidylcarboxy)benzophenone. Exposure of bait-incorporated HLM samples to light was followed by isolating the His-tagged bait protein and its crosslinked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the crosslinked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively crosslinked partners of CYP2E1 are the cytochromes P450 2A6, 2C8, 3A4, 4A11, and 4F2, UDP-glucuronosyltransferases (UGTs) 1A and 2B, fatty aldehyde dehydrogenase (ALDH3A2), epoxide hydrolase 1 (EPHX1), disulfide oxidase 1α (ERO1L), and ribophorin II (RPN2). These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes.

Dataset on the effects of environmentally relevant humic acid concentrations on the liver protein profile in Japanese medaka (Oryzias latipes).

The data were obtained by a label-free quantification approach from a shotgun proteomics experiment, using STrap sample processing technique for protein digestion and high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS) for peptide analysis. MaxQuant data processing was used to obtain proteomics data. The dataset reflects changes in the liver protein profile of Japanese medaka exposed to 0, 5, 40 and 80 mg/L nominal concentrations of Sigma-Aldrich humic acid for 96 h. Actual concentrations of humic acid were measured using the potassium dichromate photometric method and reported in mg organic carbon/L. These proteomics data are relevant for further insights into fish stress responses to humic substances-related challenge.

Temperature-induced reorganisation of Schistocephalus solidus (Cestoda) proteome during the transition to the warm-blooded host.

The protein composition of the cestode Schistocephalus solidus was measured in an experiment simulating the trophic transmission of the parasite from a cold-blooded to a warm-blooded host. The first hour of host colonisation was studied in a model experiment, in which sticklebacks Gasterosteus aculeatus infected with S. solidus were heated at 40°C for 1 h. As a result, a decrease in the content of one tegument protein was detected in the plerocercoids of S. solidus. Sexual maturation of the parasites was initiated in an experiment where S. solidus larvae were taken from fish and cultured in vitro at 40°C for 48 h. Temperature-independent changes in the parasite proteome were investigated by incubating plerocercoids at 22°C for 48 h in culture medium. Analysis of the proteome allowed us to distinguish the temperature-induced genes of S. solidus, as well as to specify the molecular markers of the plerocercoid and adult worms. The main conclusion of the study is that the key enzymes of long-term metabolic changes (glycogen consumption, protein production, etc.) in parasites during colonisation of a warm-blooded host are induced by temperature.

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.

Proteome Profiling of PMJ2-R and Primary Peritoneal Macrophages.

In vitro models are often used for studying macrophage functions, including the process of phagocytosis. The application of primary macrophages has limitations associated with the individual characteristics of animals, which can lead to insufficient standardization and higher variability of the obtained results. Immortalized cell lines do not have these disadvantages, but their responses to various signals can differ from those of the living organism. In the present study, a comparative proteomic analysis of immortalized PMJ2-R cell line and primary peritoneal macrophages isolated from C57BL/6 mice was performed. A total of 4005 proteins were identified, of which 797 were quantified. Obtained results indicate significant differences in the abundances of many proteins, including essential proteins associated with the process of phagocytosis, such as Elmo1, Gsn, Hspa8, Itgb1, Ncf2, Rac2, Rack1, Sirpa, Sod1, C3, and Msr1. These findings indicate that outcomes of studies utilizing PMJ2-R cells as a model of peritoneal macrophages should be carefully validated. All MS data are deposited in ProteomeXchange with the identifier PXD022133.

Does Proteomic Mirror Reflect Clinical Characteristics of Obesity?

Obesity is a frightening chronic disease, which has tripled since 1975. It is not expected to slow down staying one of the leading cases of preventable death and resulting in an increased clinical and economic burden. Poor lifestyle choices and excessive intake of "cheap calories" are major contributors to obesity, triggering type 2 diabetes, cardiovascular diseases, and other comorbidities. Understanding the molecular mechanisms responsible for development of obesity is essential as it might result in the introducing of anti-obesity targets and early-stage obesity biomarkers, allowing the distinction between metabolic syndromes. The complex nature of this disease, coupled with the phenomenon of metabolically healthy obesity, inspired us to perform data-centric, hypothesis-generating pilot research, aimed to find correlations between parameters of classic clinical blood tests and proteomic profiles of 104 lean and obese subjects. As the result, we assembled patterns of proteins, which presence or absence allows predicting the weight of the patient fairly well. We believe that such proteomic patterns with high prediction power should facilitate the translation of potential candidates into biomarkers of clinical use for early-stage stratification of obesity therapy.

Effects of alcohol-induced increase in CYP2E1 content in human liver microsomes on the activity and cooperativity of CYP3A4.

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4.

Cysteine alkylation methods in shotgun proteomics and their possible effects on methionine residues.

In order to optimize sample preparation for shotgun proteomics, we compared four cysteine alkylating agents: iodoacetamide, chloroacetamide, 4-vinylpyridine and methyl methanethiosulfonate, and estimated their effects on the results of proteome analysis. Because alkylation may result in methionine modification in vitro, proteomics data were searched for methionine to isothreonine conversions, which may mimic genomic methionine to threonine substitutions found in proteogenomic analyses. We found that chloroacetamide was superior to the other reagents in terms of the number of identified peptides and undesirable off-site reactions. Among the reagents evaluated, iodoacetamide increased the rate of methionine-to-isothreonine conversion, especially if the sample was prepared in gel. The presence of proline following methionine in a protein sequence increased the modification rate as well. Generally, the methionine-to-isothreonine conversion events were relatively rare, but should be taken into account in proteogenomic studies when searching for single nucleotide polymorphism events at the protein level. Additionally, we have evaluated other methionine modifications, such as oxidation and carbamidomethylation. We found that carbamidomethylation may affect up to 80% of peptides containing methionine under the condition of iodoacetamide alkylation. In this case, carbamidomethylation of methionine is more common than oxidation and should be accounted for as a variable modification during proteomic search. SIGNIFICANCE: One of the most trending questions in bottom-up proteomics is the depth of proteome profiling, in other words, the coverage of proteins by identified tryptic peptides. In proteogenomics, where the identification of a single peptide, e.g. bearing an amino acid substitution, may be of interest, high sequence coverage is especially important. Chemical modifications during sample preparation may mimic biologically significant coding mutations at the proteome level. A typical example of such modification is methionine to isothreonine conversion during alkylation, which mimics methionine to threonine substitution in protein sequences due to respective genomic mutations. Therefore, the studies on the proper selection of alkylating reagents which balance the cysteine alkylation efficiency and the extent of methionine conversion upon conventional proteomic sample preparation workflow are crucial for the outcome of proteogenomic analyses and should present a general interest for the proteomic community.

Proteomic Analysis of Chr 18 Proteins Using 2D Fractionation.

One of the main goals of the Chromosome-Centric Human Proteome Project (C-HPP) is detection of "missing proteins" (PE2-PE4). Using the UPS2 (Universal proteomics standard 2) set as a model to simulate the range of protein concentrations in the cell, we have previously shown that 2D fractionation enables the detection of more than 95% of UPS2 proteins in a complex biological mixture. In this study, we propose a novel experimental workflow for protein detection during the analysis of biological samples. This approach is extremely important in the context of the C-HPP and the neXt-MP50 Challenge, which can be solved by increasing the sensitivity and the coverage of the proteome encoded by a particular human chromosome. In this study, we used 2D fractionation for in-depth analysis of the proteins encoded by human chromosome 18 (Chr 18) in the HepG2 cell line. Use of 2D fractionation increased the sensitivity of the SRM SIS method by 1.3-fold (68 and 88 proteins were identified by 1D fractionation and 2D fractionation, respectively) and the shotgun MS/MS method by 2.5-fold (21 and 53 proteins encoded by Chr 18 were detected by 1D fractionation and 2D fractionation, respectively). The results of all experiments indicate that 111 proteins encoded by human Chr 18 have been identified; this list includes 42% of the Chr 18 protein-coding genes and 67% of the Chr 18 transcriptome species (Illumina RNaseq) in the HepG2 cell line obtained using a single sample. Corresponding mRNAs were not registered for 13 of the detected proteins. The combination of 2D fractionation technology with SRM SIS and shotgun mass spectrometric analysis did not achieve full coverage, i.e., identification of at least one protein product for each of the 265 protein-coding genes of the selected chromosome. To further increase the sensitivity of the method, we plan to use 5-10 crude synthetic peptides for each protein to identify the proteins and select one of the peptides based on the obtained mass spectra for the synthesis of an isotopically labeled standard for subsequent quantitative analysis. Data are available via ProteomeXchange with the identifier PXD019263.