Proteomic Identification Research Articles

Proteomic investigation and biomarker identification of lung and spleen deficiency syndrome in HIV/AIDS immunological nonresponders

Yi-Ting Wang, Shao-Xiu Ji, Xin-Yi Xia, Ting-Jun Wan, Jia-Xi Zou, Bai-Xue Li, Qian-Nan Hu, Xin Chen, Jie Mu, Quan-Sheng Feng, Li Wen

Protocol for Plasma Extracellular Vesicle and Particle Isolation and Mass Spectrometry-Based Proteomic Identification.

Plasma extracellular vesicles and particles (EVPs) are enriched in biomolecules that reflect individuals' physiological and pathological states. Several studies have demonstrated the potential of human plasma EVPs as a novel liquid biopsy. Here we describe a protocol for human plasma EVPs isolation and proteomic characterization. We isolated human plasma EVPs by the classical ultracentrifugation method and performed mass spectrometry-based proteomic profiling. Using this protocol, researchers can reveal the plasma EVPs proteome and explore the clinical application of plasma EVPs proteins for developing disease biomarkers.

Proteomics to Metabolomics: A New Insight into the Pathogenesis of Hypertensive Nephropathy

Background: Hypertensive nephropathy (HN) is a high-burden disorder and a leading cause of end-stage renal disease. Despite huge investigations, the underlying mechanisms are yet largely unknown. Systems biology is a promising approach to providing a comprehensive insight into this complex disorder. Methods: Proteome profiles of kidney tubulointerstitium and outer and inner cortex from a rat model of HN were retrieved from the proteomics identification database, and the quality of the datasets was assessed. Proteins that exhibited differential expression were detected and their interactions were analyzed in the kidney sub-compartments. Furthermore, enzymes were linked to the attributed metabolites. Functional enrichment analyses were performed to identify key pathways and processes based on the differentially expressed proteins and predicted metabolites. Results: Proteasome-mediated protein degradation, actin cytoskeleton organization, and Rho GTPase signaling pathway are involved in the pathogenesis of HN. Furthermore, tissue hypoxia and dysregulated energy homeostasis are among the key underlying events. The metabolism of purine and amino acids is also affected in HN. Conclusion: Although the proposed pathogenic mechanisms remain to be further validated in experimental studies, this study contributes to the understanding of the molecular mechanisms of HN through a systematic unsupervised approach. Considering the significant alterations of metabolic pathways, HN can be viewed as an “acquired error of metabolism.”

Quantitative changes of brain isatin-binding proteins of rats with the rotenone-induced experimental parkinsonism.

Isatin (indoldione-2,3) is an endogenous regulator found in humans and animals. It exhibits a broad range of biological activity mediated by numerous isatin-binding proteins. Isatin produces neuroprotective effects in several experimental models of diseases, including Parkinsonism induced by the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine).Rotenone (a neurotoxin used to modeling Parkinson's disease in rodents) causes significant changes in the profile of isatin-binding proteins of rat brain. Comparative proteomic identification of brain proteins of control rats and the rats with the rotenone-induced Parkinsonian syndrome (PS) revealed significant quantitative changes of 86 proteins under the influence of rotenone. This neurotoxin mainly caused the increase of the quantity of proteins involved in signal transduction and regulation of enzyme activity (24), proteins involved in cytoskeleton formation and exocytosis (23), and enzymes involved in energy generation and carbohydrate metabolism (19). However, only 11 of these proteins referred to isatin-binding proteins; the content of eight of them increased while the content of three proteins decreased. This suggests that the dramatic change of the profile of isatin-binding proteins, found in the development of the rotenone-induced PS, comes from changes in the state of the pre-existing molecules of proteins, rather than altered expression of corresponding genes.

IS711 sequencing of Brucella melitensis and Brucella abortus strains, and use of microchip-based real-time PCR for rapid monitoring

In animal production systems around the world, brucellosis is a serious zoonotic disease that creates public health hazards and losses in economic terms. The aim of the study is to genotype and molecularly characterize Brucella melitensis (B. melitensis) and Brucella abortus (B. abortus) collected from different animal species and humans. A total of 50 isolates of Brucella species (16B. melitensis and 34B. abortus) were isolated from 1081 animal and human samples using a culture technique, followed by biochemical identification using the Vitek 2 compact system and proteomic identification using mass spectrometry technology. Molecular genotyping was performed on all isolates using multiplex real-time PCR. Six isolates from each genotype of Brucella species were selected and genetically evaluated by IS711 insertion sequences. Microchips-based real-time PCR for Brucella species identification was performed on twelve genetically characterized isolates as a first attempt. Forty-four (88%) isolates of Brucella species were detected using multiplex real-time PCR. Based on IS711 nucleotide sequencing, twelve isolates were phylogenetically clustered into their specific clusters. The results of the comparative analysis of conventional real time and microchips-based real time indicated that the later is faster and qualitatively more sensitive than conventional real time; however, further studies are needed to ensure that it is capable of serving as a gold standard alternative for Brucella species monitoring.

Isolation and Mass Spectrometry Identification of K48 and K63 Ubiquitin Proteome Using Chain-Specific Nanobodies.

Protein ubiquitylation is an essential mechanism regulating almost all cellular functions in eukaryotes. The understanding of the role of distinct ubiquitin chains in different cellular processes is essential to identify biomarkers for disease diagnosis and prognosis but also to open new therapeutic possibilities. The high complexity of ubiquitin chains complicates this analysis, and multiple strategies have been developed over the last decades. Here, we report a protocol for the isolation and identification of K48 and K63 ubiquitin chains using chain-specific nanobodies associated to mass spectrometry. Different steps were optimized to increase the purification yield and reduce the binding on nonspecific proteins. The resulting protocol allows the enrichment of ubiquitin chain-specific targets from mammalian cells.

Proteomic Identification of Saliva Proteins as Noninvasive Diagnostic Biomarkers.

Many biomedically relevant biomarkers are proteins with characteristic biochemical properties and a relatively restricted subcellular distribution. The comparative and mass spectrometry-based proteomic analysis of body fluids can be particularly instrumental for the targeted identification of novel protein biomarkers with pathological relevance. In this respect, new research efforts in biomarker discovery focus on the systematic mapping of the human saliva proteome, as well as the pathobiochemical identification of disease-related modifications or concentration changes in specific saliva proteins. As a product of exocrine secretion, saliva can be considered an ideal source for the biochemical identification of new disease indicators. Importantly, saliva represents a body fluid that is continuously available for diagnostic and prognostic assessments. This chapter gives an overview of saliva proteomics, including a discussion of the usefulness of both liquid chromatography and two-dimensional gel electrophoresis for efficient protein separation in saliva proteomics.

Activity-Based Proteomic Identification of the S-Thiolation Targets of Ajoene in MDA-MB-231 Breast Cancer Cells.

Garlic is a medicinal plant and spice that has been used for millennia for its health-promoting effects. These medicinal properties are associated with low molecular weight organosulfur compounds, produced following the crushing of garlic cloves. One of these compounds, ajoene, is proposed to act by S-thioallylating cysteine residues on target proteins whose identification in cancer cells holds great promise for understanding mechanistic aspects of ajoene's cancer cell cytotoxicity. To this end, an ajoene analogue (called biotin-ajoene, BA), containing a biotin affinity tag, was designed as an activity-based probe specific for the protein targets of ajoene in MDA-MB-231 breast cancer cells. BA was synthesized via a convergent "click" strategy and found to retain its cytotoxicity against MDA-MB-231 cells compared to ajoene. Widespread biotinylation of proteins was found to occur via disulfide bond formation in a dose-dependent manner, and the biotin-ajoene probe was found to share the same protein targets as its parent compound, ajoene. The biotinylated proteins were affinity-purified from the treated MDA-MB-231 cell lysate using streptavidin-coated magnetic beads followed by an on-bead reduction, alkylation, and digestion to liberate the peptide fragments, which were analyzed by liquid chromatography tandem mass chromatography. A total of 600 protein targets were identified, among which 91% overlapped with proteins with known protein cysteine modification (PCM) sites. The specific sites were enriched for those susceptible to S-glutathionylation (-SSG) (16%), S-sulfhydration (-SSH) (20%), S-sulfenylation (-SOH) (22%), and S-nitrosylation (-SNO) (31%). As target validation, both ajoene and a dansylated ajoene (DP) were found to S-thiolate the pure recombinant forms of glutathione S-transferase pi 1 (GSTP1) and protein disulfide isomerase (PDI), and the ajoene analogue DP was found to be a more potent inhibitor than 5,5-dithio-bis-(2-nitrobenzoic acid) (DTNB). Pathway analysis elucidated that ajoene targets functional and signaling pathways that are implicated in cancer cell survival, specifically cellular processes, metabolism, and genetic information processing pathways. The results of this study provide mechanistic insights into the character of the anti-cancer activity of the natural dietary compound ajoene.

Proteomic Identification of Urine Biomarkers of Pediatric Nephrotic Syndrome

Proteomic Identification of Urine Biomarkers of Pediatric Nephrotic Syndrome

Proteomic identification and structural basis for the interaction between sorting nexin SNX17 and PDLIM family proteins

The sorting nexin SNX17 controls endosomal recycling of transmembrane cargo proteins including integrins, the amyloid precursor protein, and lipoprotein receptors. This requires association with the Commander trafficking complex and depends on the C terminus of SNX17 through unknown mechanisms. Using proteomics, we find that the SNX17 C terminus is sufficient for Commander interaction and also associates with members of the PDZ and LIM domain (PDLIM) family. SNX17 contains a type III PDZ binding motif that binds specifically to the PDLIM proteins. The structure of the PDLIM7 PDZ domain bound to the SNX17 C terminus reveals an unconventional perpendicular peptide interaction mediated by electrostatic contacts and a uniquely conserved proline-containing loop sequence in the PDLIM protein family. Our results define the mechanism of SNX17-PDLIM interaction and suggest that the PDLIM proteins may play a role in regulating the activity of SNX17 in conjunction with Commander and actin-rich endosomal trafficking domains.

Proteomic Identification of Markers of Membrane Repair, Regeneration and Fibrosis in the Aged and Dystrophic Diaphragm.

Deficiency in the membrane cytoskeletal protein dystrophin is the underlying cause of the progressive muscle wasting disease named Duchenne muscular dystrophy. In order to detect novel disease marker candidates and confirm the complexity of the pathobiochemical signature of dystrophinopathy, mass spectrometric screening approaches represent ideal tools for comprehensive biomarker discovery studies. In this report, we describe the comparative proteomic analysis of young versus aged diaphragm muscles from wild type versus the dystrophic mdx-4cv mouse model of X-linked muscular dystrophy. The survey confirmed the drastic reduction of the dystrophin-glycoprotein complex in the mdx-4cv diaphragm muscle and concomitant age-dependent changes in key markers of muscular dystrophy, including proteins involved in cytoskeletal organization, metabolite transportation, the cellular stress response and excitation-contraction coupling. Importantly, proteomic markers of the regulation of membrane repair, tissue regeneration and reactive myofibrosis were detected by mass spectrometry and changes in key proteins were confirmed by immunoblotting. Potential disease marker candidates include various isoforms of annexin, the matricellular protein periostin and a large number of collagens. Alterations in these proteoforms can be useful to evaluate adaptive, compensatory and pathobiochemical changes in the intracellular cytoskeleton, myofiber membrane integrity and the extracellular matrix in dystrophin-deficient skeletal muscle tissues.

Integrating ZooMS and zooarchaeology: New data from the Uluzzian levels of Uluzzo C Rock Shelter, Roccia San Sebastiano cave and Riparo del Broion.

In this study we explore the potential of combining traditional zooarchaeological determination and proteomic identification of morphologically non-diagnostic bone fragments (ZooMS) collected from the Uluzzian levels of three Italian sites: Uluzzo C Rock Shelter, Roccia San Sebastiano cave, and Riparo del Broion. Moreover, we obtained glutamine deamidation ratios for all the contexts analysed during routine ZooMS screening of faunal samples, giving information on collagen preservation. We designed a selection protocol that maximizes the efficiency of the proteomics analyses by excluding particularly compromised fragments (e.g. from taphonomic processes), and that aims to identify new human fragments by favouring bones showing morphological traits more similar to Homo. ZooMS consistently provided taxonomic information in agreement with the faunal spectra outlined by traditional zooarchaeology. Our approach allows us to delineate and appreciate differences between the analysed contexts, particularly between the northern and southern sites, related to faunal, environmental, and climate composition, although no human remains were identified. We reconstructed the faunal assemblage of the different sites, giving voice to morphologically undiagnostic bone fragments. Thus, the combination of these analyses provides a more complete picture of the faunal assemblage and of the paleoenvironment during the Middle-Upper Palaeolithic transition in Italy.

Diverse susceptibilities and responses of human and rodent cells to orthohantavirus infection reveal different levels of cellular restriction.

Orthohantaviruses are rodent-borne emerging viruses that may cause severe diseases in humans but no apparent pathology in their small mammal reservoirs. However, the mechanisms leading to tolerance or pathogenicity in humans and persistence in rodent reservoirs are poorly understood, as is the manner in which they spread within and between organisms. Here, we used a range of cellular and molecular approaches to investigate the interactions of three different orthohantaviruses–Puumala virus (PUUV), responsible for a mild to moderate form of hemorrhagic fever with renal syndrome in humans, Tula virus (TULV) with low pathogenicity, and non-pathogenic Prospect Hill virus (PHV)–with human and rodent host cell lines. Besides the fact that cell susceptibility to virus infection was shown to depend on the cell type and virus strain, the three orthohantaviruses were able to infect Vero E6 and HuH7 human cells, but only the former secreted infectious particles. In cells derived from PUUV reservoir, the bank vole (Myodes glareolus), PUUV achieved a complete viral cycle, while TULV did not enter the cells and PHV infected them but did not produce infectious particles, reflecting differences in host specificity. A search for mature virions by electron microscopy (EM) revealed that TULV assembly occurred in part at the plasma membrane, whereas PHV particles were trapped in autophagic vacuoles in cells of the heterologous rodent host. We described differential interactions of orthohantaviruses with cellular factors, as supported by the cellular distribution of viral nucleocapsid protein with cell compartments, and proteomics identification of cellular partners. Our results also showed that interferon (IFN) dependent gene expression was regulated in a cell and virus species dependent manner. Overall, our study highlighted the complexity of the host-virus relationship and demonstrated that orthohantaviruses are restricted at different levels of the viral cycle. In addition, the study opens new avenues to further investigate how these viruses differ in their interactions with cells to evade innate immunity and how it depends on tissue type and host species.

Proteomic identification of MHC class I-associated peptidome derived from non-obese diabetic mouse thymus and pancreas

The peptides repertoire presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC I-associated peptidome (MIP), which regulates thymus development, peripheral survival and function during lifetime of CD8+ T cells. Type 1 diabetes (T1D) is an organ-specific autoimmune disease caused by pancreatic β cells destruction mediated primarily by autoreactive CD8+ T cells. Non-obese diabetic (NOD) mouse is an important animal model of T1D. Here, we deeply analyzed the MIP derived from NOD mice thymus and pancreas, and demonstrated that the thymus MIP source proteins partially shared with the MIP source proteins derived from NOD mice pancreas and β cell line. One H-2Kd restricted peptide SLC35B126–34 which was shared by MIP derived from both NOD mice pancreatic tissues and islet β-cell line, but absent in MIP from NOD thymus tissues, showed ability to stimulate IFN-γ secretion and proliferation of NOD mice splenic CD8+ T cells. The global view of the MHC I-associated self-peptides repertoire in the thymus and pancreas of NOD mice may serve as a biological reference to identify potential autoantigens targeted by autoreactive CD8+ T cells in T1D. Data are available via ProteomeXchange with identifier PXD031966. SignificanceThe peptides repertoire presented to CD8+ T cells by major histocompatibility complex (MHC) class I molecules is referred to as the MHC I-associated peptidome (MIP). The MIP presented by thymic antigen presenting cells (APCs) is crucial for shaping CD8+ T cell repertoire and self-tolerance, while the MIP presented by peripheral tissues and organs is not only involved in maintaining periphery CD8+ T cell survival and homeostasis, but also mediates immune surveillance and autoimmune responses of CD8+ T cells under pathological conditions. Type 1 diabetes (T1D) is an organ-specific autoimmune disease caused by the destruction of pancreatic β cells, mediated primarily by autoreactive CD8+ T cells. Non-obese diabetic (NOD) mouse is one of important animal models of spontaneous autoimmune diabetes that shares several key features with human T1D. The global view of the MHC I-associated self-peptides repertoire in the thymus and pancreas of NOD mice may serve as a good biological reference to identify potential autoantigens targeted by autoreactive CD8+ T cells in T1D. It has great significance for further clarifying the immune recognition and effect mechanism of autoreactive CD8+ T cells in the pathogenesis of T1D, and then developing antigen-specific immune intervention strategies.

Enrichment and proteomic identification of Cryptosporidium parvum oocyst wall

BackgroundCryptosporidium parvum is a zoonotic parasitic protozoan that can infect a variety of animals and humans and is transmitted between hosts via oocysts. The oocyst wall provides strong protection against hostile environmental factors; however, research is limited concerning the oocyst wall at the proteomic level.MethodsA comprehensive analysis of the proteome of oocyst wall of C. parvum was performed using label-free qualitative high-performance liquid chromatography (HPLC) fractionation and mass spectrometry-based qualitative proteomics technologies. Among the identified proteins, a surface protein (CpSP1) encoded by the C. parvum cgd7_5140 (Cpcgd7_5140) gene was predicted to be located on the surface of the oocyst wall. We preliminarily characterized the sequence and subcellular localization of CpSP1.ResultsA total of 798 proteins were identified, accounting for about 20% of the CryptoDB proteome. By using bioinformatic analysis, functional annotation and subcellular localization of the identified proteins were examined for better understanding of the characteristics of the oocyst wall. To verify the localization of CpSP1, an indirect immunofluorescent antibody assay demonstrated that the protein was localized on the surface of the oocyst wall, illustrating the potential usage as a marker for C. parvum detection in vitro.ConclusionThe results provide a global framework about the proteomic composition of the Cryptosporidium oocyst wall, thereby providing a theoretical basis for further study of Cryptosporidium oocyst wall formation as well as the selection of targets for Cryptosporidium detection.Graphical

Tandem Mass Tags Quantitative Proteome Identification and Function Analysis of ABC Transporters in Neofusicoccum parvum.

Neofusicoccum parvum can cause twig blight of the walnut (Juglans spp.), resulting in great economic losses and ecological damage. We performed proteomic tandem mass tags (TMT) quantification of two Neofusicoccum parvum strains with different substrates, BH01 in walnut substrate (SW) and sterile water (SK), and BH03 in walnut substrate (WW) and sterile water (WK), in order to identify differentially expressed proteins. We identified 998, 95, and 489 differentially expressed proteins (DEPs) between the SK vs. WK, SW vs. SK, and WW vs. WK comparison groups, respectively. A phylogenetic analysis was performed to classify the ABC transporter proteins annotated in the TMT protein quantification into eight groups. Physicochemical and structural analyses of the 24 ATP-binding cassette (ABC) transporter proteins revealed that 14 of them had transmembrane structures. To elucidate the functions of these transmembrane proteins, we determined the relative expression levels of ABC transporter genes in strains cultured in sodium chloride, hydrogen peroxide, copper sulfate, and carbendazim mediums, in comparison with pure medium; analysis revealed differential upregulation. To verify the expression results, we knocked out the NpABC2 gene and compared the wild-type and knockout mutant strains. The knockout mutant strains exhibited a higher sensitivity to antifungal drugs. Furthermore, the virulence of the knockout mutant strains was significantly lower than the wild-type strains, thus implying that NpABC2 plays a role in the drug resistance of N. parvum and affects its virulence.

Proteomic Identification and Clinicopathologic Characterization of Splenic Amyloidosis.

The spleen is a commonly encountered specimen in surgical pathology. However, little is known about the incidence, morphologic pattern, and clinical features of spleens involved by amyloidosis. We retrospectively identified 69 spleen amyloid cases typed using a proteomics-based method between 2008 and 2020. The frequency of amyloid types, clinicopathologic features, and distribution of amyloid deposits were assessed. Four amyloid types were detected: immunoglobulin light chain (AL) (N=30; 43.5%); leukocyte chemotactic factor 2 amyloidosis (ALECT2) (N=30; 43.5%); amyloid A (AA) (N=8; 11.6%); and fibrinogen alpha (AFib) (N=1; 1.4%). The splenic amyloid showed 5 distinct distribution patterns: (1) diffuse pattern, exhibited by most AL cases; (2) red pulp pattern, exhibited by most ALECT2 cases; (3) multinodular pattern, seen in subsets of AA and AL-kappa cases; (4) mass-forming pattern, seen in the AFib case; and (5) vascular only, seen in a subset of AA cases. Atraumatic splenic rupture was the most common reason for splenectomy in AL cases, while most ALECT2 spleens were removed incidentally during an unrelated abdominal surgery. Splenomegaly was significantly more common in AA spleens than in AL or ALECT2 spleens and was often the reason for splenectomy in this group. In conclusion, splenic amyloid may be underrecognized as it is often an incidental finding. Although, as expected, many of the spleens were involved by AL amyloidosis, ALECT2 emerged as another common spleen amyloid type. Although the spleen amyloid types exhibited characteristic distribution patterns, proteomics-based typing is warranted as some morphologic overlap still exists. Awareness of ALECT2 as a major spleen amyloid type is important for appropriate diagnostic workup and patient management.

Proteomic analysis and identification reveal the anti-inflammatory mechanism of clofazimine on lipopolysaccharide-induced acute lung injury in mice.

Background and objectiveAcute lung injury (ALI)/ acute respiratory distress syndrome (ARDS) was increasingly recognized as one of the most severe acute hyperimmune response of coronavirus disease 2019 (COVID-19). Clofazimine (CFZ) has attracted attention due to its anti-inflammatory property in immune diseases as well as infectious diseases. However, the role and potential molecular mechanism of CFZ in anti-inflammatory responses remain unclear.MethodsWe analyze the protein expression profiles of CFZ and LPS from Raw264.7 macrophages using quantitative proteomics. Next, the protective effect of CFZ on LPS-induced inflammatory model is assessed, and its underlying mechanism is validated by molecular biology analysis.ResultsLC–MS/MS-based shotgun proteomics analysis identified 4746 (LPS) and 4766 (CFZ) proteins with quantitative information. The key proteins and their critical signal transduction pathways including TLR4/NF-κB/HIF-1α signaling was highlighted, which was involved in multiple inflammatory processes. A further analysis of molecular biology revealed that CFZ could significantly inhibit the proliferation of Raw264.7 macrophages, decrease the levels of TNF-α and IL-1β, alleviate lung histological changes and pulmonary edema, improve the survival rate, and down-regulate TLR4/NF-κB/HIF-1α signaling in LPS model.ConclusionThis study can provide significant insight into the proteomics-guided pharmacological mechanism study of CFZ and suggest potential therapeutic strategies for infectious disease.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00011-022-01623-w.

Spectral Library-Based Single-Cell Proteomics Resolves Cellular Heterogeneity.

Dissecting the proteome of cell types and states at single-cell resolution, while being highly challenging, has significant implications in basic science and biomedicine. Mass spectrometry (MS)-based single-cell proteomics represents an emerging technology for system-wide, unbiased profiling of proteins in single cells. However, significant challenges remain in analyzing an extremely small amount of proteins collected from a single cell, as a proteome-wide amplification of proteins is not currently feasible. Here, we report an integrated spectral library-based single-cell proteomics (SLB-SCP) platform that is ultrasensitive and well suited for a large-scale analysis. To overcome the low MS/MS signal intensity intrinsically associated with a single-cell analysis, this approach takes an alternative approach by extracting a breadth of information that specifically defines the physicochemical characteristics of a peptide from MS1 spectra, including monoisotopic mass, isotopic distribution, and retention time (hydrophobicity), and uses a spectral library for proteomic identification. This conceptually unique MS platform, coupled with the DIRECT sample preparation method, enabled identification of more than 2000 proteins in a single cell to distinguish different proteome landscapes associated with cellular types and heterogeneity. We characterized individual normal and cancerous pancreatic ductal cells (HPDE and PANC-1, respectively) and demonstrated the substantial difference in the proteomes between HPDE and PANC-1 at the single-cell level. A significant upregulation of multiple protein networks in cancer hallmarks was identified in the PANC-1 cells, functionally discriminating the PANC-1 cells from the HPDE cells. This integrated platform can be built on high-resolution MS and widely accepted proteomic software, making it possible for community-wide applications.

Comprehensive proteomic signature and identification of CDKN2A as a promising prognostic biomarker and therapeutic target of colorectal cancer.

BACKGROUNDThe carcinogenesis of colorectal cancer (CRC) involves many different molecules and multiple pathways, and the specific mechanism has not been elucidated until now. Existing studies on the proteomic signature profiles of CRC are relatively limited. Therefore, we herein aimed to provide a more comprehensive proteomic signature profile and discover new prognostic markers and therapeutic targets by performing proteomic analysis of CRC and paired normal tissues.AIMTo investigate the proteomic signature and identify novel protein prognostic biomarkers of CRC.METHODSCancer tissues and paired normal tissues were collected from 48 patients who underwent surgical removal at the China-Japan Friendship Hospital from January 2020 to June 2021. Data independent acquisition (DIA) quantitative proteomic analysis was performed using high-performance liquid chromatography–mass spectrometry/mass spectrometry (nano-UHPLC–MS/MS) to identify differentially expressed proteins, among which those with a P adj value (t test, BH correction) < 0.05 and an absolute fold change (|log2FC|) > 2 were identified as potential markers. Differentially expressed proteins were selected by bioinformatics analysis and validated by immunohistochemical tissue microarrays, and their association with prognosis was further analyzed with the Gene Expression Profiling Interactive Analysis database to identify prognostic protein biomarkers of CRC.RESULTSSignificantly differential protein expression was observed between cancer tissues and normal tissues. Compared with normal tissues, 1115 proteins were upregulated and 705 proteins were downregulated in CRC based on P adj < 0.05 and |log2FC| > 2, and bioinformatics analysis revealed that the differentially expressed proteins were involved in multiple biological processes associated with tumorigenesis, including ribosome biogenesis in eukaryotes, focal adhesion, extracellular matrix-receptor interactions and other tumor metabolism processes. Moreover, cyclin-dependent kinase inhibitor 2A (CDKN2A) expression was markedly upregulated in CRC, as validated by immunohistochemistry (0.228 vs 0.364, P = 0.0044), and was significantly enriched in tumor proliferation and signal transduction pathways such as the cell cycle and p53 signaling pathways. High CDKN2A expression was significantly correlated with poor prognosis (P = 0.021). These results demonstrated that CDKN2A functions as a driver of CRC.CONCLUSIONOur study provides a comprehensive proteomic signature of CRC and highlights CDKN2A as a potential powerful prognostic marker and precision therapeutic target.