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Aging is a major risk for developing cardiac and skeletal muscle dysfunction, yet the underlying mechanism remains elusive. Here we demonstrated that the mitochondria-associated endoplasmic reticulum membranes (MAMs) in the rat heart and skeletal muscle were disrupted during aging. Using quantitative morphological analysis, we showed that the mitochondria-endoplasmic reticulum contacts (MERCs) were reduced by half over the lifespan with an early onset of accelerated thickening in the clefts. The ultrastructural changes were further validated by proteomic profiling of the MAM fractions. A combination of subcellular fractionation and quantitative mass spectrometry identified 1306 MAM-enriched proteins in both heart and skeletal muscle, with a catalog of proteins dysregulated with aging. Functional mapping of the MAM proteome suggested several aging signatures to be closely associated with the ER-mitochondria crosstalk, including local metabolic rewiring, calcium homeostasis imbalance, and impaired organelle dynamics and autophagy. Moreover, we identified a subset of highly interconnected proteins in an ER-mitochondria organization network, which were consistently down-regulated with aging. These decreased proteins, including VDAC1, SAMM50, MTX1 and MIC60, were considered as potential contributors to the age-related MAM dysfunction. This study highlights the perturbation in MAM integrity during the striated muscle aging process, and provides a framework for understanding aging biology from the perspective of organelle interactions., (© 2022. The Author(s).)

The differential diagnosis of Crohn disease (CD) from intestinal tuberculosis (ITB) and primary intestinal lymphoma (PIL) is challenging in patients who exhibit atypical clinical characteristics. The aim of the present study was to explore the serum proteome profiles of CD, PIL and ITB and to identify their differentiations.Treatment-naïve patients with CD (n = 10), PIL (n = 10) and ITB (n = 10) were enrolled in the present study. Differentially expressed proteins (DEPs) in patient serum samples were compared between groups using tandem mass tag labeled proteomic technology. A principal component analysis (PCA) plot and volcano maps were also visualized. Functional pathway analysis was performed using Reactome. The Area under the Curve (AUC) was calculated for each DEP.A total of 818 proteins were identified through proteomic quantification. Among them, 108 DEPs were identified to be differentiated between CD and ITB, 105 proteins between CD and PIL and 55 proteins between ITB and PIL. The proteome from the three groups was distinguishable in the PCA plot. The results revealed that 19, 12, and 10 proteins (AUC ≥ 0.95) were differentially expressed between CD and PIL, CD and ITB, and PIL and ITB, respectively. Among these DEPs, tumor necrosis factor ligand superfamily member 13 was higher in CD than in ITB and PIL. Peroxiredoxin-5, T-complex protein 1 subunit Gamma, CutA, and Fibulin-5 were increased in CD and PIL when compared with ITB. The levels of fibrinogen chains were also significantly higher in patients with PIL compared with CD.The current study demonstrated that serum proteome was distinguishable among patients with CD, PIL, and ITB. The identified proteins may assist in the clinical differentiation among them.

Inflammatory bowel disease (IBD) has become a major health challenge worldwide. However, the precise etiological and pathophysiological factors involved in IBD remain unclear. Proteomics can be used for large-scale protein identification analysis. In the current study, using tandem mass tag- (TMT-) based shotgun proteomics, proteomic differences between intestinal tissue from health controls, patients with Crohn's disease (CD), and patients with ulcerative colitis (UC) were compared. Proteins with fold change >2 or <0.5 and P value < 0.05 between groups were considered differentially expressed. ProteinAtlas was used to analyze the tissue specificity of differentially expressed proteins (DEPs). Reactome pathway analysis was applied to cluster functional pathways. A total of 4786 proteins were identified, with 59 proteins showing higher levels and 43 showing lower levels in patients with IBD than in controls. Seventeen proteins, including angiotensin converting enzyme 2 (ACE2) and angiotensin converting enzyme 1 (ACE), showed higher levels in CD than in UC. Several novel proteins such as CD38, chitinase 3-like 1 (CHI3L1), olfactomedin 4 (OLFM4), and intelectin 1 were screened out between patients with IBD and controls. When proteins with fold change >1.2 or <0.84 and P value < 0.05 between groups were considered differentially expressed, the expression of 10 proteins, including CD38, involved in the nicotinamide adenine dinucleotide (NAD) metabolism and signaling pathway showed significant changes in IBD. Using the NCBI GEO database, we confirmed increased CD38 mRNA expression in patients with UC and in mouse colitis models. Protein CD38 expression was higher in CD and UC than in normal controls. CD38 expression was higher in inflamed tissues than in noninflamed tissues, and CD38 was located in F4/80-positive cells. Our study may provide novel insights into the molecular pathogenesis of IBD. Further studies are required on the role of NAD metabolism and CD38 in intestinal inflammation.

Chronic HBV infection (CHB) can lead to acute-on-chronic liver failure (HBV-ACLF) characterized by high mortality. This study aimed to reveal ACLF-related proteomic alterations, from which protein based diagnostic and prognostic scores for HBV-ACLF were developed. Methods : Ten healthy controls, 16 CHB, and 19 HBV-ACLF according to COSSH (Chinese group on the study of severe hepatitis B) criteria were enrolled to obtain the comprehensive proteomic portrait related to HBV-ACLF initiation and progression. Potential markers of HBV-ACLF were further selected based on organ specificity and functionality. An additional cohort included 77 healthy controls, 92 CHB and 71 HBV-ACLF was used to validate the proteomic signatures via targeted proteomic assays. Results : Significant losses of plasma proteins related to multiple functional clusters, including fatty acid metabolism/transport, immuno-response, complement and coagulation systems, were observed in ACLF patients. In the validation study, 28 proteins were confirmed able to separate ACLF, CHB patients. A diagnostic classifier P4 (APOC3, HRG, TF, KLKB1) was built to differentiate ACLF from CHB with high accuracy (auROC = 0.956). A prognostic model P8 (GC, HRG, HPR, SERPINA6, age, NEU, INR and total protein) was built to distinguish survivors from non-survivors in 28 and 90-days follow-up (auROC = 0.882, 0.871), and to stratify ACLF patients into risk subgroups showing significant difference in 28 and 90-days mortality (HR=7.77, 7.45, both P<0.0001). In addition, P8 score correlated with ACLF grades and numbers of extra-hepatic organ failures in ACLF patients, and was able to predict ACLF-associated coagulation and brain failure within 90 days (auROC = 0.815, 0.842). Conclusions : Proteomic signatures developed in this study reflected the deficiency of key hematological functions in HBV-ACLF patients, and show potential for HBV-ACLF diagnosis and risk prediction in complementary to current clinical based parameters., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Despite being orthogonal to reverse-phase separation and valuable for posttranslational modification (PTM) pre-enrichment, hydrophilic interaction liquid chromatography (HILIC) has not been widely adopted for large-scale proteomic applications. Here, we first evaluated the performance of HILIC in comparison with the popular high-pH reverse-phase (HPRP) separation, as the first dimension for tryptic peptide fractionation in a shotgun workflow to characterize the complex 293T cell proteome. The data indicated that the complementary nature of HILIC and HPRP for peptide separation was mainly due to different hydrophobicity preferences. Realizing that uncaptured components from one mode can be resolved in the other mode, we then designed and compared two multidimensional separation schemes using HILIC and HPRP in tandem for peptide prefractionation, in terms of identification efficiency and coverage at peptide, protein, and PTM levels. A total of 22,604 and 23,566 peptides corresponding to 4481 and 4436 proteins from 293T cell lysate were detected using HILIC-HPRP- and HPRP-HILIC-based shotgun proteomics workflow, respectively. In addition, without assistance of enrichment techniques, the tandem fractionation methods aided to identify 46 different PTMs from over 10,000 of spectra using blind modification search algorithm. We concluded that HILIC is a valuable alternative option for peptide prefractionation in a large-scale proteomic study, but can be further augmented with the use of a secondary HPRP separation.

Acute liver failure (ALF) is a fatal condition hallmarked by rapid development. The present study aimed to describe the dynamic alterations of serum proteins associated with ALF development, and to seek for novel biomarkers of ALF. Miniature pigs ( n = 38) were employed to establish ALF models by infusing d-galactosamine (GALN, 1.3 g/kg). A total of 1310 serum proteins were compared in pooled serum samples ( n = 10) before and 36 h after GALN administration through label-free quantitation (LFQ) based shotgun proteomics. Functional analysis suggested a significant enrichment of ALF-related proteins involved in energy metabolism. Temporal changes of 20 energy metabolism related proteins were investigated in individual pigs ( n = 8) via parallel reaction monitoring (PRM) based targeted proteomics. In addition, mitochondrion degeneration and gene expression alteration of aerobic metabolism genes were confirmed in GALN-insulted pig liver. In clinical validation study enrolled 34 ALF patients and 40 healthy controls, fructose-1,6-bisphosphatase 1 (FBP1) showed a prognostic value for short-term survival (30 days) equal to that of the Model of End-stage Liver Disease score (ROC-AUC = 0.778). Survival analysis suggested significantly higher death-related hazard in ALF patients with higher FBP1 levels (>16.89 μg/dL) than in those with lower FBP1 levels ( p = 0.002). Additionally, serum retinol binding protein 4 (RBP4) level was found decreased prior to ALT elevation in GALN-insulted pig model. We also confirmed that serum level of RBP4 is significantly lower in ALF patients ( p < 0.001) as compared with healthy controls. In summary, this translational study, displayed by multistaged proteomics techniques, unveiled underlying functional changes related to the development of ALF and facilitated the discovery of novel ALF markers., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

We describe a cheap, robust, fast, high-throughput, and flexible proteomic sample processing method based on a regular 96-well plate by acetone precipitation under low centrifuge speed (96PACS), which enables predigestion processing of 96 samples within 2 h. Tested on a complex Huh-7 total lysate, 96PACS produced comparable proteome coverage and even showed better reproducibility than FASP. Quantitative performance of 96PACS was further tested using data-independent acquisition and parallel reaction monitoring quantitation in a set of 6 benchmark samples consisting of 6 serial dilutions of BSA spiked in complex E. coli proteome background. The protocol was also successfully modified for automation and was validated in a comparative label-free proteomic study to develop serum markers for early detection of liver fibrosis and necroinflammation in patients chronically infected with hepatitis B virus.

BACKGROUND. HBV-related acute-on-chronic liver failure (HBV-ACLF) is hallmarked by high short-term mortality rates, calling for accurate prognostic biomarkers for initial risk stratification. METHODS. Three tandem mass tag-labeled (TMT-labeled) quantitative proteomic studies were performed on 10 patients with HBV-related acute hepatic decompensation and on 20 patients with HBV-ACLF. Candidate biomarkers were preliminarily verified in a cross-sectional cohort (n = 144) and further confirmed in 2 prospective cohorts (n = 207 and n = 148). RESULTS. Plasminogen, a potential prognostic biomarker for HBV-ACLF, was identified by TMT quantitative proteomics and preliminarily verified in the cross-sectional cohort. Further validation with a prospective cohort (n = 207) showed that plasminogen levels at admission were significantly lower (P < 0.001) in HBV-ACLF nonsurvivors than in survivors. The cumulative survival duration of patients with high plasminogen levels was significantly longer (P < 0.001) than that of patients with low plasminogen levels. During hospitalization, plasminogen levels significantly decreased (P = 0.008) in the deterioration group but significantly increased (P < 0.001) in the improvement group. Additionally, plasminogen levels gradually increased in survivors but gradually decreased in nonsurvivors. The P5 score, a prognostic panel incorporating plasminogen levels, hepatic encephalopathy occurrence, age, international normalized ratio (INR), and total bilirubin, was significantly superior to the Child-Pugh, Model for End-stage Liver Disease (MELD), Chronic Liver Failure Consortium ACLF (CLIF-C ACLF), Chinese Group on the Study of Severe Hepatitis B (COSSH), and HINT (a prognostic score based on hepatic encephalopathy occurrence, INR, neutrophil count, and thyroid-stimulating hormone) scores (all P < 0.05). The performances of the plasminogen level and P5 score were validated in a second multicenter, prospective cohort (n = 148). CONCLUSIONS. Plasminogen is a promising prognostic biomarker for HBV-ACLF, and sequential plasminogen measurements could profile the clinical course of HBV-ACLF. P5 is a high-performance prognostic score for HBV-ACLF. FUNDING. The National Key Research and Development Program (2017YFC1200204); the National Natural Science Foundation of China (81400589, 81600497); the Foundation for Innovative Research Groups of the National Natural Science Foundation of China (81121002); the Chinese High-Tech Research and Development Programs (2012AA020204); the National S&T Major Project (2012ZX10002004); and the Zhejiang Provincial Medicine and Health Science and Technology Project (2016147735)., Introduction Acute-on-chronic liver failure (ACLF) is a life-threatening clinical condition that occurs in patients with preexisting chronic liver disease (CLD). It can progress rapidly and has a mortality rate of [...]

Objective: This study investigated the proteomic characteristics of cerebrospinal fluid (CSF) in patients with varicella zoster virus (VZV) meningitis to understanding the pathogenesis of central nervous system (CNS) infection by reactivated VZV. Method: We used data‐independent acquisition model to analyze the CSF proteomic differences of 28 patients with VZV meningitis and 11 herpes zoster (HZ) patients. According to the clinical manifestations at discharge, 28 VZV meningitis patients were divided into favorable outcome group and unfavorable outcome (UO) group and their differences in CSF proteome were also analyzed. Results: Compared with the HZ group, the proteins (CXCL10, ELANE, IL‐1RN, MPO, PRTN3, etc.) related to inflammation and immune cell activation were significantly upregulated in the VZV meningitis group (p <.01). The protein related to the nerve function and energy metabolism (CKMT1B, SLITRK3, Synaptotagmin‐3, KIF5B, etc.) were significantly downregulated (p <.05). The levels of a pro‐inflammatory factor, IL‐18, in CSF were significantly higher in patients in the UO group as compared to patients with favorable prognosis (p <.05). Conclusion: Inflammatory immune response is an important pathophysiological mechanism of CNS infection by VZV, and the CSF IL‐18 levels might be a potential prognostic indicator of the outcomes of VZV meningitis. [ABSTRACT FROM AUTHOR]

Nevertheless, we have solid evidence that the human forms of these proteins interact with ICP0 because their endogenous proteins were identified in the ICP0 interactome in both 293T and HFF cells and their transfection resulted in detectable interaction with ICP0 in both 293T and Neuro-2a cells. After the cells were lysed, the indicated proteins were immunoprecipitated by an anti-Flag antibody and analyzed by Western blots for ICP0 and the corresponding proteins using an ICP0 and Flag antibody, respectively. OTUD4 and VIM Could Be Ubiquitinated by ICP0 Comparison of the interactome and ubiquitinome data identified three viral proteins and eight host proteins, which interacted with ICP0 and exhibited elevated ubiquitination in the presence of ICP0, indicating that they were highly likely to be substrates of ICP0 (Figure 5A). Previously reported ICP0-interacting proteins are marked in red. (F) 293T (left) or Neuro-2a (right) cells in each 100-mm plate were co-transfected with 7.5 g of an empty vector (pcDNA) or a plasmid expressing the indicated protein linked to a Flag-tag and 7.5 g of a plasmid expressing untagged ICP0 for 48 h. Keywords: herpes simplex virus; ICP0; interactome; ubiquitinome; proteomics EN herpes simplex virus ICP0 interactome ubiquitinome proteomics 1 15 15 04/21/22 20220418 NES 220418 Introduction Herpes simplex virus 1 (HSV-1) is a DNA virus that widely infects humans and can cause lesions in orolabial mucosa, eyes, and genital areas during productive (lytic) infection. [Extracted from the article]