Journal of Proteome Research最新文献

Spontaneous Achilles tendon rupture (SATR) predominantly affects middle-aged and elderly individuals with chronic injuries. However, the exact cause and mechanism of SATR remain elusive, and potential therapeutic intervention or prevention is still insufficient. The present study aimed to uncover the key pathological molecules by using iTRAQ proteomics. The results identified 2432 candidate proteins in SATR patients using iTRAQ proteomic analysis. A total of 307 differentially expressed proteins (DEPs) were identified and linked to 211 KEGG signaling pathways including Coronavirus disease (COVID-19), focal adhesion, and ribosomes. GO enrichment analysis highlighted significant enrichment in processes such as biological adhesion, ossification, lipid (APOA4) processes, and extracellular matrix (ECM) organization (collagen). PPI network analysis identified hub genes such as serum albumin (ALB), fibronectin (FN1), and actin cytoplasmic 1. The WB analysis confirmed that FN1 and the receptor for activated C kinase (RACK1) were downregulated in the SATR tendon. Immunohistochemical staining revealed that collagen I and III were suppressed, while collagen II and APOA4 expression were higher in the SATR pathological tissue (P < 0.05). However, the primary cultured tenocytes (PCTs) from SATR patients showed enhanced proliferation and, consistent with tissue staining, reduced collagen I and III and increased collagen II. Our findings reveal vital targets and pathways in SATR's etiological progression, offering a new perspective on the diagnosis, treatment, and prognosis of this complex disorder.

The outbreak of COVID-19, led to an ongoing pandemic with devastating consequences for the global economy and human health. With the global spread of SARS-CoV-2, multidisciplinary initiatives were launched to explore new diagnostic, therapeutic, and vaccination strategies. From this perspective, proteomics could help to understand the mechanisms associated with SARS-CoV-2 infection and to identify new therapeutic options. A TMT-based quantitative proteomics and phosphoproteomics analysis was performed to study the proteome remodeling of human lung alveolar cells expressing human ACE2 (A549-ACE2) after infection with SARS-CoV-2. Detectability and the prognostic value of selected proteins was analyzed by targeted PRM. A total of 6802 proteins and 6428 phospho-sites were identified in A549-ACE2 cells after infection with SARS-CoV-2. The differential proteins here identified revealed that A549-ACE2 cells undergo a time-dependent regulation of essential processes, delineating the precise intervention of the cellular machinery by the viral proteins. From this mechanistic background and by applying machine learning modeling, 29 differential proteins were selected and detected in the serum of COVID-19 patients, 14 of which showed promising prognostic capacity. Targeting these proteins and the protein kinases responsible for the reported phosphorylation changes may provide efficient alternative strategies for the clinical management of COVID-19.

Crossbreeding of zebu cattle (Bos indicus) with European breeds (Bos taurus) producing crossbred cattle was performed to overcome the low growth rates and milk production of indigenous tropical cattle breeds. However, zebu cattle fertility is higher than those of crossbred cattle and European breeds under warm conditions. Combination study of proteomics and metabolomics toward Malaysian indigenous breed Kedah × Kelantan-KK (B. indicus) and crossbreed Mafriwal-M (B. taurus × B. indicus) to understand physiological reasons for higher thermotolerance and fertility in Zebu cattle sperm. 161 regulated metabolites and 96 regulated proteins in KK and M (p < 0.05) showed more efficient carbohydrate and energy metabolism, higher integrity of the DNA and plasma membrane, a lower level of reactive oxygen species, and higher levels of phospholipids, which confirmed higher sperm plasma membrane integrity in KK. A stronger antioxidant system and lower polyunsaturated fatty acids help KK sperm cope with oxidative stress under warm conditions. The higher abundance of flagella structural proteins in KK provides a stronger structure that supports sperm motility. Abnormality of flagella, plasma membrane disruption, and DNA fragmentation were higher in M. These findings provide selective molecular markers for developing high-producing and more thermotolerant cattle breeds in tropical areas (197 words).

Protein N-glycosylation is vital in the human liver and influences functions such as lipid metabolism, apoptosis, and inflammation. However, site-specific N-glycosylation patterns and variations in liver biopsy samples between healthy individuals and those with nonalcoholic fatty liver disease (NAFLD) remain incompletely characterized, primarily due to the limitations of current clinical glycoproteomic methods, including a large demand for clinical samples, low efficiency of tissue protein extraction, and a low recovery rate of intact N-glycopeptides (IGPs). To address this issue, we developed GlycoPCT, a quantitative glycoproteomic method based on pressure cycling technology. It enables efficient recovery of IGPs and accurate analysis of trace liver biopsy samples. Our research revealed a total of 4,459 unique IGPs and 361 glycans from 758 glycoproteins. High-mannose type, complex type, fucosylation type, and sialylation type N-glycans were significantly upregulated in the NAFLD group (p < 0.001, t test). Notably, we also identified 182 upregulated IGPs from 67 proteins (p < 0.05, FC > 1.50) and 108 downregulated IGPs from 44 proteins (p < 0.05, FC < 0.67) in the NAFLD group. Furthermore, we highlighted an essential acute phase glycoprotein, alpha-1-acid glycoprotein 1 (A1TA), which is synthesized in the liver and plays a significant role in NAFLD progression. These novel glyco-signatures provide crucial clues for the diagnosis and pathogenesis of NAFLD.

Psoriasis, an immune-mediated chronic inflammatory skin disease, is primarily diagnosed through clinical assessment. Currently, specific markers for the accurate diagnosis and prediction of psoriatic disease are lacking. Here, we employed a three-step designed study to perform untargeted metabolomics, with the aim of identifying candidate biomarkers for psoriasis. Through comprehensive multivariate and univariate statistical analyses, we screened eight potential biomarkers specific to psoriasis, with five structurally identified. Two dipeptide biomarkers, γ-GluSer and ThrGly, along with a lysine glycation metabolite, Nα-fructosyl-lysine (Fruc-Lys), were found to be psoriasis biomarkers for the first time. Receiver operating characteristic curve analysis revealed that the area under the curve (AUC) values of these eight metabolites/features ranged from 0.68 to 0.94. A biomarker panel comprising ThrGly and feature m/z 120.0656 (C₄H₉NO₃) demonstrated high diagnostic accuracy (AUC = 0.97) in distinguishing psoriasis patients from healthy controls. Overall, our study identified and validated a panel of plasma metabolites/features that could serve as potential biomarkers for the diagnosis of psoriasis, providing new insights into the diagnosis and pathogenesis of this disease.

Abnormal accumulation of tau protein in the brain is one pathological hallmark of Alzheimer's disease (AD). Many tau protein post-translational modifications (PTMs) are associated with the development of AD, such as phosphorylation, acetylation, and methylation. Therefore, a complete picture of the PTM landscape of tau is critical for understanding the molecular mechanisms of AD progression. Here, we offered a pilot study of combining two complementary analytical techniques, capillary zone electrophoresis (CZE)-tandem mass spectrometry (MS/MS) and reversed-phase liquid chromatography (RPLC)-MS/MS, for bottom-up proteomics of recombinant human tau-0N3R. We identified 50 phosphorylation sites of tau-0N3R in total, which is about 25% higher than that from RPLC-MS/MS alone. CZE-MS/MS provided more PTM sites (i.e., phosphorylation) and modified peptides of tau-0N3R than RPLC-MS/MS, and its predicted electrophoretic mobility helped improve the confidence of the identified modified peptides. We developed a highly efficient capillary isoelectric focusing (cIEF)-MS technique to offer a bird's-eye view of tau-0N3R proteoforms, with 11 putative tau-0N3R proteoforms carrying up to nine phosphorylation sites and lower pI values from more phosphorylated proteoforms detected. Interestingly, under native-like cIEF-MS conditions, we observed three putative tau-0N3R dimers carrying phosphate groups. The findings demonstrate that CE-MS is a valuable analytical technique for the characterization of tau PTMs, proteoforms, and even oligomerization.

Direct detection of biotinylated proteins (DiDBiT) is a proteomic method that can enrich and detect newly synthesized proteins (NSPs) labeled with bio-orthogonal amino acids with 20-fold improved detectability compared to conventional methods. However, DiDBiT has currently been used to compare only two conditions per experiment. Here, we present DiDBiT-TMT, a method that can be used to quantify NSPs across many conditions and replicates in the same experiment by combining isobaric tandem mass tagging (TMT) with DiDBiT. We applied DiDBiT-TMT to brain slices to determine changes in the de novo proteome that occur after inducing chemical long-term potentiation (cLTP) or treatment with the neuromodulator norepinephrine. We successfully demonstrated DiDBiT-TMT's capacity to quantitatively compare up to 9 samples in parallel. We showed that there is a minimal overlap among NSPs that are differentially expressed in cLTP-treated organotypic brain slices, norepinephrine-treated organotypic brain slices, and organotypic slices undergoing combinatorial treatment with norepinephrine and cLTP. Our results point to the possible divergence of the molecular mechanisms underlying these treatments and showcase the applicability of DiDBiT-TMT for studying neurobiology.

Macrophages make up a heterogeneous population of immune cells that exhibit diverse phenotypes and functions in health and disease. Although macrophage epigenomic and transcriptomic profiles have been reported, the proteomes of distinct macrophage populations under various pathological conditions remain largely elusive. Here, we employed a label-free proteomic approach to characterize the diversity of the hepatic macrophage pool in an experimental model of CCl₄-induced liver fibrosis. We found a decrease in the proportion of liver resident embryo-derived KCs (EmKCs), and a drastic increase in the proportion of monocyte-derived KCs (MoKCs) and CLEC2^-Macs. Proteomic profiling revealed that MoKCs largely resembled EmKCs, whereas CLEC2^-Macs exhibited greater proteomic alternations compared with EmKCs, suggesting two distinct destinations for monocyte differentiation during liver fibrosis. Furthermore, CLEC2^-Macs were characterized by increased expression of proteins associated with inflammatory response, antigen processing and presentation processes, which may be involved in the pathogenesis of liver fibrosis. Collectively, our study provides insights into the considerable heterogeneity within the hepatic macrophage pool during liver fibrosis.

Glioblastoma multiforme (GBM) is the most prevalent and aggressive brain tumor found in adult humans with a poor prognosis and average survival of 14-15 months. In order to have a comprehensive understanding of proteome and identify novel therapeutic targets, this study focused mainly on the differentially abundant proteins (DAPs) of Ras^V12-induced GBM. Ras^V12 is a constitutively active Ras mutant form essential for tumor progression by continuously activating signaling pathways leading to uncontrolled tumor growth. This study used a transgenic Drosophila model with Ras^V12 overexpression using the repo-GAL4 driver line, specifically in glial cells, to study GBM. The high-resolution mass spectrometry (HRMS)-based proteomic analysis of the GBM larval central nervous system identified three novel DAPs specific to mitochondria. These DAPs, probable maleylacetoacetate isomerase 2 (Q9VHD2), bifunctional methylene tetrahydrofolate dehydrogenase (Q04448), and glutamine synthetase1 (P20477), identified through HRMS were further validated by qRT-PCR. The protein-protein interaction analysis revealed interactions between Ras^V12 and DAPs, with functional links to mitochondrial dynamics regulators such as Drp1, Marf, Parkin, and HtrA2. Notably, altered expressions of Q9VHD2, P20477, and Q04448 were observed during GBM progression, which offers new insights into the involvement of mitochondrial dynamic regulators in Ras^V12-induced GBM pathophysiology.

Distraction osteogenesis (DO) represents a highly effective method for addressing significant bone defects; however, it necessitates a long treatment period. Exosomes are key mediators of intercellular communication. To investigate their role in the angiogenesis and osteogenesis of DO, we established a canine mandibular DO model with a bone defect (BD) group as the control. Higher levels of angiogenesis were observed in the regenerating tissue from the DO group compared to those from the BD group, accompanied by earlier osteogenesis. Proteomic analysis was performed on circulating exosomes at different phases of the DO using a data-independent acquisition method. Data are available via ProteomeXchange with the identifier PXD050531. The results indicated specific alterations in circulating exosome proteins at different phases of DO, reflecting the regenerative activities in the corresponding tissues. Notably, fibronectin 1 (FN1), thrombospondin 1 (THBS1), and transferrin receptor (TFRC) emerged as potential candidate proteins related to the angiogenic response in DO. Further cellular experiments validated the potential of DO-associated circulating exosomes to promote angiogenesis in endothelial cells. Collectively, these data reveal previously unknown mechanisms that may underlie the efficacy of DO and suggest that exosome-derived proteins may be useful as therapeutic targets for strategies designed to improve DO-related angiogenesis and bone regeneration.