Integrated Analysis of Cross-Links and Dead-End Peptides for Enhanced Interpretation of Quantitative XL-MS.

Chemical cross-linking with mass spectrometry provides low-resolution structural information on proteins in cells and tissues. Combined with quantitation, it can identify changes in the interactome between samples, for example, control and drug-treated cells or young and old mice. A difference can originate from protein conformational changes that alter the solvent-accessible distance separating the cross-linked residues. Alternatively, a difference can result from conformational changes localized to the cross-linked residues, for example, altering the solvent exposure or reactivity of those residues or post-translational modifications of the cross-linked peptides. In this manner, cross-linking is sensitive to a variety of protein conformational features. Dead-end peptides are cross-links attached only at one end to a protein with the other terminus being hydrolyzed. As a result, changes in their abundance reflect only conformational changes localized to the attached residue. For this reason, analyzing both quantified cross-links and their corresponding dead-end peptides can help elucidate the likely conformational changes giving rise to observed differences in cross-link abundance. We describe analysis of dead-end peptides in the XLinkDB public cross-link database and, with quantified mitochondrial data isolated from failing heart versus healthy mice, show how a comparison of abundance ratios between cross-links and their corresponding dead-end peptides can be leveraged to reveal possible conformational explanations.

Exploration of key ferroptosis-related genes and immune infiltration in Crohn's disease using bioinformatics.

Crohn's disease (CD) is a type of inflammatory bowel disease (IBD) that manifests mainly as chronic inflammation in different parts of the gastrointestinal tract, and its incidence has come to be increasing in recent years. Ferroptosis, a novel type of programmed cell death, it seems the role of ferroptosis-related biomarkers in CD has not been mentioned. Thus, the role of ferroptosis in CD and its relationship with immune infiltration were explored in this study. The CD dataset was downloaded from the Gene Expression Omnibus database. The validated ferroptosis genes (FRGs) were retrieved from the public FerrDb database. The gene expression matrix of the CD dataset was analyzed with the "limma" package in R language to obtain differentially expressed genes (DEGs) between diseased and healthy samples. Then, intersecting genes between DEGs and FRGs were identified as differentially expressed ferroptosis-associated genes (DE-FRGs). Protein-protein interaction (PPI) network analysis and visualization were carried out with STRING and Cytoscape, and key CD ferroptosis-related genes (CD-FRGs) were identified along with their Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways using the clusterProfiler package. Immune cell infiltration was analyzed with CIBERSORT. The correlation between key CD-FRGs and immune-infiltrated cells in CD was studied by Spearman's correlation method. A total of 37 DE-FRGs and 6 key CD-FRGs (CAV1, CD44, HIF1A, IFNG, TIMP1 and TLR4) were identified. GO and KEGG functional analysis indicated these genes enrichment in programmed cell death and apoptotic process, HIF-1 signaling pathway and IBD. Infiltration matrix analysis of immune cells showed abundant T cells CD4 memory activated, M1 macrophages, M2 macrophages, Mast cells activated and Neutrophils in CD intestinal tissues. The 6 key CD-FRGs were correlated with immune-infiltrated cells in CD based on correlation analysis. Taken together, immune cells with abnormal infiltration can be implicated in CD due to ferroptosis. This study identified 6 key CD-FRGs that may be key biomarkers of ferroptosis in CD; they include CAV1, CD44, HIF1A, IFNG, TIMP1 and TLR4. These findings suggest that the immune response is critical in CD caused by ferroptosis through the interaction between key CD-FRGs and immune infiltrating cells.

Design for a Multicentre Prospective Cohort for the Assessment of Platelet Function in Patients with Hepatitis-B-Virus-Related Acute-on-Chronic Liver Failure.

Background: Acute-on-chronic liver failure (ACLF) has high short-term mortality and lacks sufficient medical therapy. Available algorithms are unable to precisely predict short-term outcomes or safely stratify patients with ACLF as emergent liver transplantation candidates. Therefore, a personalized prognostic tool is urgently needed. Purpose: Platelet function and its clinical significance in ACLF patients with chronic hepatitis B virus (HBV) infection have not been investigated. This study aimed to assess changes in platelet function using thromboelastography (TEG) and platelet mapping (TEG-PM) in HBV-related ACLF patients. Methods: Chronic liver disease patients with acute decompensation or acute hepatic injury were recruited. The derivation cohort enrolled HBV-related patients at Nanfang Hospital. HBV-related and non-HBV-related patients were both enrolled in internal and external validation cohorts at seven university hospitals. TEG and TEG-PM were performed at baseline in the derivation cohort and baseline, day 7, and day 14 in the validation cohorts. The primary outcome was all-cause 28-day mortality. Status check and new-onset complications were recorded during the 3-month follow-up, but status check will extend to 5 years. Conclusion and Future Plans: In this study, 586 participants were enrolled, including 100 in derivation cohort, 133 in internal validation cohort, and 353 in external validation cohort. Biomaterials, including plasma, serum, urine, and some explanted liver tissues, were collected from these patients. A 3-month follow-up with survival status was completed. The baseline characteristics indicated that 51% of the patients had adenosine diphosphate (ADP)-hyporesponsive circulating platelets. The prognostic potential of platelet function will be explored in the derivation cohort (HBV-related ACLF patients) and further substantiated in the validation cohorts (HBV-related and non-HBV-related ACLF patients). Biosamples are currently used to explore the underlying mechanisms related to ADP-hyporesponsive platelets. The ongoing proteomic and metabolic analyses will provide new insights into the pathogenesis of extrahepatic organ failures in ACLF patients.

Upregulation of mitochondrial ATPase inhibitory factor 1 (ATPIF1) mediates increased glycolysis in mouse hearts.

In hypertrophied and failing hearts, fuel metabolism is reprogrammed to increase glucose metabolism, especially glycolysis. This metabolic shift favors biosynthetic function at the expense of ATP production. Mechanisms responsible for the switch are poorly understood. We found that inhibitory factor 1 of the mitochondrial FoF1-ATP synthase (ATPIF1), a protein known to inhibit ATP hydrolysis by the reverse function of ATP synthase during ischemia, was significantly upregulated in pathological cardiac hypertrophy induced by pressure overload, myocardial infarction, or α-adrenergic stimulation. Chemical cross-linking mass spectrometry analysis of hearts hypertrophied by pressure overload suggested that increased expression of ATPIF1 promoted the formation of FoF1-ATP synthase nonproductive tetramer. Using ATPIF1 gain- and loss-of-function cell models, we demonstrated that stalled electron flow due to impaired ATP synthase activity triggered mitochondrial ROS generation, which stabilized HIF1α, leading to transcriptional activation of glycolysis. Cardiac-specific deletion of ATPIF1 in mice prevented the metabolic switch and protected against the pathological remodeling during chronic stress. These results uncover a function of ATPIF1 in nonischemic hearts, which gives FoF1-ATP synthase a critical role in metabolic rewiring during the pathological remodeling of the heart.

The Effect of Medial Branch Block on Postoperative Residual Pain Relieve After Percutaneous Kyphoplasty: A Randomized Controlled Trial With 12-Month Follow-up.

BACKGROUND: Percutaneous kyphoplasty (PKP) is a minimally invasive technique, and effective treatment, for an osteoporotic vertebral compression fracture (OVCF). Residual back pain is the most common complication of PKP. Medial branch block (MBB) is a treatment option for painful OVCF, it can break the vicious cycle to release short- or long-term pain. OBJECTIVES: We aimed to determine the effects of MBB on postoperative residual back pain in OVCF patients after PKP surgery. STUDY DESIGN: A randomized, controlled, single-center trial. SETTING: Medical university center and local hospitals. METHODS: A total of 198 patients were recruited and randomly assigned to either the MBB or Non-MBB group. In the MBB group, patients received MBB during PKP surgery, the injection contained a mixture of lidocaine and budesonide. The Non-MBB group was injected with normal saline in the target nerve area during PKP surgery. The primary outcome was back pain assessed by the Visual Analog Scale (VAS), and residual back pain was defined as a VAS score greater than or equal to 4. The secondary outcomes included physical function assessed by Patient-Reported Outcome Measurement Information System Physical Function (PROMIS PF) and satisfaction with surgery was assessed using the S6 satisfaction scale. All parameters were measured at baseline, 1 day, 1 week, 1 month, 3, 6, and 12 months after the intervention. RESULTS: A total of 179 patients, including 91 patients in the MBB group and 88 patients in the Non-MBB group, were included for a comprehensive assessment. The VAS score in the MBB group was significantly lower than in the Non-MBB group within a one-month follow-up. PROMIS PF score in the MBB group was significantly higher than in the Non-MBB group within a one-month follow-up. The incidence of residual back pain in the MBB group was lower than the Non-MBB group within a one-month follow-up. The MBB group had a significantly higher satisfaction rate compared with the Non-MBB group at final follow-up. LIMITATIONS: Firstly, patients are from a single institution and the sample size is small. Secondly, some of the potential factors which may lead to back pain, such as infection, new symptomatic compression fracture, and serious cement leakage, did not occur. Thirdly, the conservative treatment group is not included. Finally, we were unable to determine individual differences in pain tolerance. CONCLUSIONS: MBB can effectively relieve back pain and reduce the incidence of residual back pain in OVCF patients after PKP surgery. Besides, it can also significantly improve postoperative physical function and patients' satisfaction with treatment.

Mitochondrial protein interaction landscape of SS-31.

Mitochondrial dysfunction underlies the etiology of a broad spectrum of diseases including heart disease, cancer, neurodegenerative diseases, and the general aging process. Therapeutics that restore healthy mitochondrial function hold promise for treatment of these conditions. The synthetic tetrapeptide, elamipretide (SS-31), improves mitochondrial function, but mechanistic details of its pharmacological effects are unknown. Reportedly, SS-31 primarily interacts with the phospholipid cardiolipin in the inner mitochondrial membrane. Here we utilize chemical cross-linking with mass spectrometry to identify protein interactors of SS-31 in mitochondria. The SS-31-interacting proteins, all known cardiolipin binders, fall into two groups, those involved in ATP production through the oxidative phosphorylation pathway and those involved in 2-oxoglutarate metabolic processes. Residues cross-linked with SS-31 reveal binding regions that in many cases, are proximal to cardiolipin-protein interacting regions. These results offer a glimpse of the protein interaction landscape of SS-31 and provide mechanistic insight relevant to SS-31 mitochondrial therapy.

Impacts of cigarette smoking on liver fibrosis and its regression under therapy in male patients with chronic hepatitis B.

BACKGROUND & AIMS: The role of cigarette smoking in the development of chronic hepatitis B (CHB) remains poorly understood. We assessed the potential contributions of cigarette smoking to liver fibrosis and its regression after starting antiviral therapy in CHB patients. METHODS: In this cohort study, 2144 consecutive male CHB patients under no antiviral therapy were evaluated and 206 patients with significant liver fibrosis (≥F2) initiating antiviral therapy had longitudinal follow-up. Liver fibrosis was measured by liver stiffness measurement using transient elastography. To adjust for imbalances between smoking history and never smoking groups, propensity score (PS) matching model with 1:1 ratios were performed. Cigarette smoking history and intensity (pack-years) were collected and documented using a standardized questionnaire. RESULTS: Before PS matching, 432/2144 patients had advanced fibrosis in prevalence cohort. Patients with smoking history (n = 1002) had a greater prevalence of advanced fibrosis than those without (n = 1142) (24.4% vs 16.5%, P = 0.001). Multivariate logistic regression analysis demonstrated that smoking contributed to advanced fibrosis (OR, 1.458; 95% CI, 1.114-1.908). In longitudinal cohort, multivariate logistic regression analysis demonstrated retarded fibrosis regression in patients with history of smoking ≥10 pack-years (OR, 0.288; 95% CI, 0.1-0.825). After PS matching, patients with smoking history had higher prevalence of advanced fibrosis (22.8% vs 18%, P = 0.024) than those non-smokers. In post-PS-matching logistic regression, the effect of smoking on advanced fibrosis persisted (OR, 1.415; 95% CI, 1.047-1.912; P = 0.024). CONCLUSIONS: Cigarette smoking in male CHB patients aggravated liver fibrosis prior to and delayed fibrosis regression under antiviral therapy.

Cross-linking measurements of in vivo protein complex topologies.

Identification and measurement of in vivo protein interactions pose critical challenges in the goal to understand biological systems. The measurement of structures and topologies of proteins and protein complexes as they exist in cells is particularly challenging, yet critically important to improve understanding of biological function because proteins exert their intended function only through the structures and interactions they exhibit in vivo. In the present study, protein interactions in E. coli cells were identified in our unbiased cross-linking approach, yielding the first in vivo topological data on many interactions and the largest set of identified in vivo cross-linked peptides produced to date. These data show excellent agreement with protein and complex crystal structures where available. Furthermore, our unbiased data provide novel in vivo topological information that can impact understanding of biological function, even for cases where high resolution structures are not yet available.

Identification of protein-protein interactions and topologies in living cells with chemical cross-linking and mass spectrometry.

We present results from a novel strategy that enables concurrent identification of protein-protein interactions and topologies in living cells without specific antibodies or genetic manipulations for immuno-/affinity purifications. The strategy consists of (i) a chemical cross-linking reaction: intact cell labeling with a novel class of chemical cross-linkers, protein interaction reporters (PIRs); (ii) two-stage mass spectrometric analysis: stage 1 identification of PIR-labeled proteins and construction of a restricted database by two-dimensional LC/MSMS and stage 2 analysis of PIR-labeled peptides by multiplexed LC/FTICR-MS; and (iii) data analysis: identification of cross-linked peptides and proteins of origin using accurate mass and other constraints. The primary advantage of the PIR approach and distinction from current technology is that protein interactions together with topologies are detected in native biological systems by stabilizing protein complexes with new covalent bonds while the proteins are present in the original cellular environment. Thus, weak or transient interactions or interactions that require properly folded, localized, or membrane-bound proteins can be labeled and identified through the PIR approach. This strategy was applied to Shewanella oneidensis bacterial cells, and initial studies resulted in identification of a set of protein-protein interactions and their contact/binding regions. Furthermore most identified interactions involved membrane proteins, suggesting that the PIR approach is particularly suited for studies of membrane protein-protein interactions, an area under-represented with current widely used approaches.

Informatics strategies for large-scale novel cross-linking analysis.

The detection of protein interactions in biological systems represents a significant challenge for today's technology. Chemical cross-linking provides the potential to impart new chemical bonds in a complex system that result in mass changes in a set of tryptic peptides detected by mass spectrometry. However, system complexity and cross-linking product heterogeneity have precluded widespread chemical cross-linking use for large-scale identification of protein-protein interactions. The development of mass spectrometry identifiable cross-linkers called protein interaction reporters (PIRs) has enabled on-cell chemical cross-linking experiments with product type differentiation. However, the complex datasets resultant from PIR experiments demand new informatics capabilities to allow interpretation. This manuscript details our efforts to develop such capabilities and describes the program X-links, which allows PIR product type differentiation. Furthermore, we also present the results from Monte Carlo simulation of PIR-type experiments to provide false discovery rate estimates for the PIR product type identification through observed precursor and released peptide masses. Our simulations also provide peptide identification calculations based on accurate masses and database complexity that can provide an estimation of false discovery rates for peptide identification. Overall, the calculations show a low rate of false discovery of PIR product types due to random mass matching of approximately 12% with 10 ppm mass measurement accuracy and spectral complexity resulting from 100 peptides. In addition, consideration of a reduced database resulting from stage 1 analysis of Shewanella oneidensis MR-1 containing 367 proteins resulted in a significant reduction of expected identification false discovery rate estimation compared to that from the entire Shewanella oneidensis MR-1 proteome.

Design and performance of an atmospheric pressure ion mobility Fourier transform ion cyclotron resonance mass spectrometer.

This manuscript presents our initial results and development of a novel hybrid instrument that combines atmospheric pressure ion mobility spectrometry (AP-IMS) with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). Our preliminary results obtained from atmospheric pressure mobility separation of peptide mixtures combined with high-resolution FTICR mass analysis are demonstrated. The custom IMS system was constructed in-house and was coupled to the commercial FTICR-MS instrument through a flared inlet capillary interface. Dual-gate ion filtration was adapted to allow concurrent measurement of both mobility and m/z values. The feasibility of mobility separation was demonstrated with baseline separation of the peptides bradykinin and angiotensin II and their measured reduced mobility constants which were consistent with those previously reported. Furthermore, the unique size-to-charge separation mechanism of IMS that allows isomer separation was explored and demonstrated with the partial separation of two isomeric phosphopeptides. We feel the combination of IMS and FTICR-MS holds great potential for accurate mass analysis of mobility-selected ions and these results are the first to demonstrate the feasibility of coupling these two techniques.

Proteomic identification of heat shock protein 90 as a candidate target for p53 mutation reactivation by PRIMA-1 in breast cancer cells.

INTRODUCTION: A loss of p53 function resulting from mutation is prevalent in human cancers. Thus, restoration of p53 function to mutant p53 using small compounds has been extensively studied for cancer therapy. We previously reported that PRIMA-1 (for 'p53 reactivation and induction of massive apoptosis') restored the transcriptional activity of p53 target genes in breast cancer cells with a p53 mutation. By using functional proteomics approach, we sought to identify molecular targets that are involved in the restoration of normal function to mutant p53. METHODS: PRIMA-1 treated cell lysates were subjected to immunoprecipitation with DO-1 primary antibody against p53 protein, and proteins bound to p53 were separated on a denaturing gel. Bands expressed differentially between control and PRIMA-1-treated cells were then identified by matrix-assisted laser desorption ionization-time-of-flight spectrometry. Protein expression in whole cell lysates and nuclear extracts were confirmed by Western blotting. The effect of combined treatment of PRIMA-1 and adriamycin in breast cancer cells was determined with a cytotoxicity assay in vitro. RESULTS: PRIMA-1 treated cells distinctly expressed a protein band of 90 kDa that was identified as heat shock protein 90 (Hsp90) by the analysis of the 90 kDa band tryptic digest. Immunoblotting with isoform-specific antibodies against Hsp90 identified this band as the alpha isoform of Hsp90 (Hsp90alpha). Co-immunoprecipitation with anti-Hsp90alpha antibody followed by immunoblotting with DO-1 confirmed that p53 and Hsp90alpha were interacting proteins. PRIMA-1 treatment also resulted in the translocation of Hsp90alpha to the nucleus by 8 hours. Treatment of cells with PRIMA-1 alone or in combination with adriamycin, a DNA-targeted agent, resulted in increased sensitivity of tumor cells. CONCLUSION: The studies demonstrate that PRIMA-1 restores the p53-Hsp90alpha interaction, enhances the translocation of the p53-Hsp90alpha complex and reactivates p53 transcriptional activity. Our preliminary evidence also suggests that PRIMA-1 could be considered in combination therapy with DNA-targeted agents for the treatment of breast cancer, especially for tumors with aberrant p53 function.