Intrahepatic homeobox protein MSX-1 is a novel host restriction factor of hepatitis B virus.

Chronic hepatitis B virus (HBV) infection (CHB) is a risk factor for the development of liver fibrosis, cirrhosis, and hepatocellular carcinoma. Covalently closed circular DNA serves as the sole transcription template for all viral RNAs and viral transcription is driven and enhanced by viral promoter and enhancer elements, respectively. Interactions between transcription factors and these cis-elements regulate their activities and change the production levels of viral RNAs. Here, we report the identification of homeobox protein MSX-1 (MSX1) as a novel host restriction factor of HBV in liver. In both HBV-transfected and HBV-infected cells, MSX1 suppresses viral gene expression and genome replication. Mechanistically, MSX1 downregulates enhancer II/core promoter (EnII/Cp) activity via direct binding to an MSX1 responsive element within EnII/Cp, and such binding competes with hepatocyte nuclear factor 4α binding to EnII/Cp due to partial overlap between their respective binding sites. Furthermore, CHB patients in immune active phase express higher levels of intrahepatic MSX1 but relatively lower levels of serum and intrahepatic HBV markers compared to those in immune tolerant phase. Finally, MSX1 was demonstrated to induce viral clearance in two mouse models of HBV persistence, suggesting possible therapeutic potential for CHB.IMPORTANCECovalently closed circular DNA plays a key role for the persistence of hepatitis B virus (HBV) since it serves as the template for viral transcription. Identification of transcription factors that regulate HBV transcription not only provides insights into molecular mechanisms of viral life cycle regulation but may also provide potential antiviral targets. In this work, we identified host MSX1 as a novel restriction factor of HBV transcription. Meanwhile, we observed higher intrahepatic MSX1 expression in chronic hepatitis B virus (CHB) patients in immune active phase compared to those in immune tolerant phase, suggesting possible involvement of MSX1 in the regulation of HBV activity by the host. Lastly, intrahepatic overexpression of MSX1 delivered by recombinant adenoviruses into two mouse models of HBV persistence demonstrated MSX1-mediated repression of HBV in vivo, and MSX1-induced clearance of intrahepatic HBV DNA in treated mice suggested its potential as a therapeutic target for the treatment of CHB.

Immunomodulation of cuproptosis and ferroptosis in liver cancer.

According to statistics, the incidence of liver cancer is increasing yearly, and effective treatment of liver cancer is imminent. For early liver cancer, resection surgery is currently the most effective treatment. However, resection does not treat the disease in advanced patients, so finding a method with a better prognosis is necessary. In recent years, ferroptosis and cuproptosis have been gradually defined, and related studies have proved that they show excellent results in the therapy of liver cancer. Cuproptosis is a new form of cell death, and the use of cuproptosis combined with ferroptosis to inhibit the production of hepatocellular carcinoma cells has good development prospects and is worthy of in-depth discussion by researchers. In this review, we summarize the research progress on cuproptosis combined with ferroptosis in treating liver cancer, analyze the value of cuproptosis and ferroptosis in the immune of liver cancer, and propose potential pathways in oncotherapy with the combination of cuproptosis and ferroptosis, which can provide background knowledge for subsequent related research.

Oncological risk of proximal gastrectomy for proximal advanced gastric cancer after neoadjuvant chemotherapy.

PURPOSE: This study assesses the metastasis rate of the key distal lymph nodes (KDLN) that are not routinely dissected in proximal gastrectomy, aiming to explore the oncological safety of proximal gastrectomy for upper gastric cancer who underwent neoadjuvant chemotherapy. METHODS: We analyzed a cohort of 150 patients with proximal locally advanced gastric cancer (cT3/4 before chemotherapy) from two high-volume cancer centers in China who received preoperative neoadjuvant chemotherapy (NAC) and total gastrectomy with lymph node dissection. Metastasis rate of the KDLN (No.5/6/12a) and the risk factors were analyzed. RESULTS: Key distal lymph node metastasis was detected in 10% (15/150) of patients, with a metastasis rate of 6% (9/150) in No. 5 lymph nodes, 6.7% (10/150) in No. 6 lymph nodes, and 2.7% (2/75) in No. 12a lymph nodes. The therapeutic value index of KDLN as one entity is 5.8. Tumor length showed no correlation with KDLN metastasis, while tumor regression grade (TRG) emerged as an independent risk factor (OR: 1.47; p-value: 0.04). Of those with TRG3 (no response to NAC), 80% (12/15) was found with KDLN metastasis. CONCLUSION: For cT3/4 proximal locally advanced gastric cancer patients, the risk of KDLN metastasis remains notably high even after NAC. Therefore, proximal gastrectomy is not recommended; instead, total gastrectomy with thorough distal lymphadenectomy is the preferred surgical approach.

High mobility group AT-hook 1 (HMGA1) is an important positive regulator of hepatitis B virus (HBV) that is reciprocally upregulated by HBV X protein.

Chronic infection with hepatitis B virus (HBV) is associated with liver cirrhosis and hepatocellular carcinoma. Upon infection of hepatocytes, HBV covalently closed circular DNA (cccDNA) exists as histone-bound mini-chromosome, subjected to transcriptional regulation similar to chromosomal DNA. Here we identify high mobility group AT-hook 1 (HMGA1) protein as a positive regulator of HBV transcription that binds to a conserved ATTGG site within enhancer II/core promoter (EII/Cp) and recruits transcription factors FOXO3α and PGC1α. HMGA1-mediated upregulation of EII/Cp results in enhanced viral gene expression and genome replication. Notably, expression of endogenous HMGA1 was also demonstrated to be upregulated by HBV, which involves HBV X protein (HBx) interacting with SP1 transcription factor to activate HMGA1 promoter. Consistent with these in vitro results, chronic hepatitis B patients in immune tolerant phase display both higher intrahepatic HMGA1 protein levels and higher serum HBV markers compared to patients in inactive carrier phase. Finally, using a mouse model of HBV persistence, we show that targeting endogenous HMGA1 through RNA interference facilitated HBV clearance. These data establish HMGA1 as an important positive regulator of HBV that is reciprocally upregulated by HBV via HBx and also suggest the HMGA1-HBV positive feedback loop as a potential therapeutic target.

Lipid nanoparticle-mediated delivery of IL-21-encoding mRNA induces viral clearance in mouse models of hepatitis B virus persistence.

Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), the transcription template for all viral mRNAs, is highly stable and current treatment options cannot effectively induce its clearance. Previously, we established an HBV persistence mouse model based on a clinical isolate (termed BPS) and identified interleukin-21 (IL-21) as a potent inducer of HBV clearance. Lipid nanoparticle (LNP) mediated delivery of mRNA has proven to be a highly safe and effective delivery platform. This work explored the applicability and effectiveness of the mRNA-LNP platform in IL-21-based HBV therapies. First, LNP-encapsulated murine IL-21 mRNA (LNP-IL-21) was prepared, characterized, and demonstrated to engender IL-21 expression in vitro and in vivo. Next, LNP-IL-21 was shown to induce clearance of both serum and intrahepatic HBV antigen and DNA in two HBV persistence mouse models based on BPS and recombinant cccDNA (rcccDNA), respectively, which was associated with HBV-specific humoral and cellular immune responses. Furthermore, peripheral blood mononuclear cells from BPS persistence mice treated ex vivo with LNP-IL-21 and HBV surface antigen (HBsAg) could induce similar HBV clearance upon infusion into recipient mice. These findings indicated that IL-21 combined with mRNA-LNP platform represents a valid and promising strategy for developing novel therapeutics against chronic HBV infection.

A novel synthetic method for backbone-cyclized polypeptide POL7080 with the help of hydrophobic-support materials.

Murepavadin (POL7080) in phase III clinical trials, a backbone-cyclized polypeptide composed of 14 amino acids, has a novel mode of action and shows a specific and efficient bactericidal effect against multidrug-resistant Pseudomonas aeruginosa. It is a potential candidate to treat severe P. aeruginosa infections in the future and still has significant commercial value for further research and development. In this paper, we report a liquid-phase peptide synthetic route for this valuable candidate polypeptide assisted by hydrophobic-support materials (tags), which overcomes the difficulties of high cost and poor yield in the traditional solid-phase synthesis of macrocyclic peptides. Through the careful optimization of reaction conditions and the innovative strategy of synthetic post-treatment, we established a simple and efficient liquid-phase synthetic route suitable for POL7080 and other similar structures, with satisfactory yield, high purity and a production process not being controlled by scale.

In-hospital adverse outcomes and risk factors among chronic kidney disease patients infected with the omicron variant of SARS-CoV-2: a single-center retrospective study.

INTRODUCTION: The SARS-CoV-2 Omicron variant has decreased virulence and pathogenicity, yet the number of Omicron infections worldwide is unprecedentedly high, with rather high mortality and severe disease rate. Chronic kidney disease (CKD) patients are particularly vulnerable to the SARS-CoV-2 Omicron variant and have unique clinical outcomes. METHODS: We retrospectively collected data from 2140 hospitalized patients with SARS-CoV-2 Omicron variant infection from March 29, 2022, to May 17, 2022. Demographic characteristics, ancillary examination results, and clinical treatments were described. Occurrence of critical COVID-19 or death and time of positive-to-negative conversion was defined as primary outcomes. The presence of COVID-19 pneumonia and the usage of respiratory or circulatory support was defined as secondary outcomes. Univariate or multivariate logistic regression analyses were performed to identify risk factors for primary outcomes. RESULTS: 15.74% of CKD patients infected with the SARS-CoV-2 Omicron variant ended up with critical COVID-19 or death. Pre-existing CKD was a risk factor for critical COVID-19 or death and prolonged time of positive-to-negative conversion of SARS-CoV-2. Nirmatrelvir-ritonavir facilitated viral clearance among COVID-19 patients with non-severe CKD. CONCLUSION: We found patients with CKD and COVID-19 due to Omicron experienced worse clinical outcomes and prolonged time of positive-to-negative conversion of SARS-CoV-2 compared to patients without CKD, which helps rationalize limited medical resources and offers guidance for appropriate clinical treatments.

Design, synthesis and evaluation of oxazolopyridinone derivatives as quorum sensing inhibitors.

The antibiotic crisis is associated with the appearance of multidrug resistant (MDR) pathogens, which has caused severe bacterial infections and imposed a huge burden on modern society. Therefore, there is an urgent need to develop new antibacterial drugs with novel mechanism of action. Here we designed and synthesized three series of benzoxazolone, oxazolopyridinone and 3-(2-hydroxyphenyl)hydantoin derivatives and evaluated their activity as novel quorum sensing (QS) inhibitors. We found that benzoxazolone and oxazolopyridinone derivatives had promising QS inhibitory activity in the minimum inhibitory concentration, pyocyanin and rhamnolipid inhibition assays. In particular, A10 and B20 at 256 µg/mL not only suppressed pyocyanin production regulated by QS in P. aeruginosa PAO1 by 36.55% and 46.90%, respectively, but also showed the strongest rhamnolipid inhibitory activity with the IC50 values of 66.35 and 56.75 µg/mL, respectively. Further studies demonstrated that B20 at 64 µg/mL inhibited biofilm formation in P. aeruginosa PAO1 by 40%, and weakened its swarming motility. More importantly, the bacterial mortality of B20 combined with ciprofloxacin and clarithromycin against P. aeruginosa were 48.27% and 49.79%, respectively, while ciprofloxacin and clarithromycin had only 16.99% and 29.11% of bacterial mortality against P. aeruginosa when used alone. Mechanistic studies indicated that B20 directly inhibited the QS pathway based on the GFP reporter strain assay. Overall, this compound with oxazolopyridinone core could serve as an antibacterial lead of QS inhibitor for further evaluation of its drug-likeness.

Harmonization of distributed multi-center analysis based on dried blood spot reference materials supporting the screening of neonatal inherited metabolic disorders.

BACKGROUND: The standardization of quantification data is critical for ensuring the reliability and measurement traceability in the screening of neonatal inherited metabolic disorders. However, the availability of national certified reference materials is limited in China. METHODS: In this study, we developed a series of dried blood spot (DBS) reference materials containing 9 amino acids (AA) and 10 acylcarnitines (AC) for neonatal screening. Four levels of the reference materials were measured with tandem mass spectrometry (MS/MS) by seven laboratories using different commercial In Vitro Diagnostic Device (IVD) kits. Then, 100 clinical samples were measured using both derivatization and non-derivatization methods by the same laboratory. RESULTS: We found high homogeneity and stability at all levels of the reference materials, with the coefficient of variation (CV) of the analytes less than 15%. These reference materials can be used to assess the testing capabilities of different laboratories. Our test also revealed that the correction factors (CF) calculated by the reference materials, along with clinical samples, could increase the consistency for different kits. CONCLUSION: The DBS reference materials proposed in this study provide reliability for the harmonization in multi-center analysis for the screening of neonatal inherited metabolic disorders. And applying our correction method for the screening could improve the data consistency of the DBS samples prepared by different methods.

The Crohn's-like lymphoid reaction density: a new artificial intelligence quantified prognostic immune index in colon cancer.

BACKGROUND: The Crohn's-like lymphoid reaction (CLR) is manifested as peritumoral lymphocytes aggregation in colon cancer, which is a major component of the host immune response to cancer. However, the lack of a unified and objective CLR evaluation standard limits its clinical application. We, therefore, developed a deep learning model for the fully automated CLR density quantification on routine hematoxylin and eosin (HE)-stained whole-slide images (WSIs) and further investigated its prognostic validity for patient stratification. METHODS: The CLR density was calculated by using a deep learning method on HE-stained WSIs. A training (N = 279) and a validation (N = 194) cohorts were used to evaluate the prognostic value of CLR density for overall survival (OS). RESULT: The fully automated quantified CLR density was an independent prognostic factor, with high CLR density associated with increased OS in the discovery (HR 0.58, 95% CI 0.38-0.89, P = 0.012) and validation cohort (0.45, 0.23-0.88, 0.020). Integrating CLR density into a Cox model with other risk factors showed improved prognostic capability. CONCLUSION: We developed a new immune indicator (CLR density) quantified by a deep learning method to evaluate the lymphocytes aggregation in colon cancer. The CLR density was demonstrated its predictive value for OS in two independent cohorts. This approach allows for the objective and standardized quantification while reducing pathologists' workload. Therefore, this fully automated standardized method of CLR evaluation had potential clinical value.

Proportion of asymptomatic infection and nonsevere disease caused by SARS-CoV-2 Omicron variant: A systematic review and analysis.

SARS-CoV-2 Omicron variant seemed to cause milder disease compared to previous predominated variants. We aimed to conduct a meta-analysis to assess the pooled proportion of nonsevere disease and asymptomatic infection among COVID-19 patients infected with Omicron and Delta. We searched PubMed, Embase, Web of Science, and China National Knowledge Infrastructure (CNKI) databases. We included studies of SARS-CoV-2 Omicron infection from November 1, 2021, to April 18, 2022, and studies of Delta infection from October 1, 2020, to June 30, 2022. Studies without corresponding data, with less than 50 patients, or obviously biased concerning main outcome were excluded. Meta-analysis was performed in R 4.2.0 with the "meta" package. Subgroup analyses were conducted by study group and vaccination status. The pooled proportion of asymptomatic infection and nonsevere disease with Omicron were 25.5% (95% confidence interval [CI] 17.0%-38.2%) and 97.9% (95% CI 97.1%-98.7%), significantly higher than those of Delta with 8.4% (95% CI 4.4%-16.2%) and 91.4% (95% CI 87.0%-96.0%). During Omicron wave, children and adolescents had higher proportion of asymptomatic infection, SOTR and the elderly had lower proportion of nonsevere disease, vaccination of a booster dose contributed to higher proportion of both asymptomatic infection and nonsevere disease. This study estimates the pooled proportion of asymptomatic infection and nonsevere disease caused by SARS-CoV-2 Omicron compared to other predominant variants. The result has important implications for future policy making.

An automated magnetic bead extraction method for measuring plasma metanephrines and 3-methoxytyramine using liquid chromatography tandem mass spectrometry.

Liquid chromatography tandem mass spectrometry (LC-MS/MS) is used routinely in clinical diagnostics; however, automating the sample pretreatment is challenging. We established and evaluated an automated method based on the magnetic bead extraction principle (MBE) to measure normetanephrine (NMN), metanephrine (MN), and 3-methoxytyramine (3-MT). The target analytes were extracted, purified, and concentrated using different solvents and chemical bond-modified magnetic beads transferred via a magnetic bar. The linearity, recovery, matrix effect, and precision of MBE were evaluated thoroughly, and compared with traditional solid-phase extraction (SPE) using 131 plasma samples. The chromatography peaks of metanephrines and 3-MT, extracted via MBE, are symmetrical, without interfering peaks. The linearity was excellent with correlation coefficient (r) > 0.99. The MBE exhibited good reproducibility with within-run coefficient variations (CVs) of 1.96-2.00%, 4.06-5.75%, and 3.89-4.90% for MN, NMN, and 3-MT, respectively. The total CVs for MN, NMN, and 3-MT were 1.96-2.80%, 5.12-5.75%, and 5.44-6.27%, respectively. The relative recoveries for MN, NMN, and 3-MT varied between 93.5 and 107.4%, whereas their biases were all within 10%. The results for MN, NMN, and 3-MT extracted via MBE compared with SPE exhibited excellent correlation, with r > 0.99; the mean bias% for MN, NMN, and 3-MT were small (-2.9%, -3.2%, and -3.2%, respectively). In conclusion, the automated MBE method for measuring plasma metanephrines and 3-MT can be applied in future routine clinical diagnostics, and the MBE principle may indicate a new era for LC-MS/MS in clinical application.

Acridinium-conjugated aromatic heterocycles as highly potent FtsZ inhibitors: Design, synthesis, and biological evaluation.

The epidemic of multidrug resistance (MDR) is a serious threat to public health, and new classes of antibiotics with novel mechanisms of action are in critical need. We rationally designed and efficiently synthesized three series of new chemical entities with potential antibacterial activity targeting filamenting temperature-sensitive mutant Z (FtsZ). Evaluation of these compounds against a panel of Gram-positive bacteria including MDR and vancomycin-resistant Enterococcus strains indicated that most compounds showed enhanced antibacterial efficacy, comparable or even superior to the reference drugs. The newly synthesized compounds proved to be substrates of the Escherichia coli efflux pump AcrB, thus affecting the activity. Their structure-activity relationships were summarized in detail. The most potent compound 10f quickly eliminated bacteria in a bactericidal mode, with low susceptibility to induce bacterial resistance. Further mechanistic studies with the BsFtsZ protein revealed that 10f functioned as an effective FtsZ inhibitor through altering the dynamics of FtsZ self-polymerization via a stimulatory mechanism, which leads to inhibition of cell division and cell death. Besides, 10f not only displayed no obvious cytotoxicity to mammalian cells but also had a high efficacy in a murine model of bacteremia in vivo. Regarded as a whole, our findings highlight 10f as a promising new FtsZ-targeting bactericidal agent.

Identification of Estradiol Benzoate as an Inhibitor of HBx Using Inducible Stably Transfected HepG2 Cells Expressing HiBiT Tagged HBx.

HBx plays a significant role in the cccDNA epigenetic modification regulating the hepatitis B virus (HBV) life cycle and in hepatocyte proliferation and carcinogenesis. By using the sleeping-beauty transposon system, we constructed a tetracycline-induced HBx-expressing stable cell line, SBHX21. HBx with a HiBiT tag can be quickly detected utilizing a NanoLuc-based HiBiT detection system. By screening a drug library using SBHX21 cells, we identified estradiol benzoate as a novel anti-HBx agent. Estradiol benzoate also markedly reduced the production of HBeAg, HBsAg, HBV pgRNA, and HBV DNA in a dose-dependent manner, suggesting that estradiol benzoate could be an anti-HBV agent. Docking model results revealed that estradiol benzoate binds to HBx at TRP87 and TRP107. Collectively, our results suggest that estradiol benzoate inhibits the HBx protein and HBV transcription and replication, which may serve as a novel anti-HBV molecular compound for investigating new treatment strategies for HBV infection.

A deep learning quantified stroma-immune score to predict survival of patients with stage II-III colorectal cancer.

BACKGROUND: Profound heterogeneity in prognosis has been observed in colorectal cancer (CRC) patients with intermediate levels of disease (stage II-III), advocating the identification of valuable biomarkers that could improve the prognostic stratification. This study aims to develop a deep learning-based pipeline for fully automatic quantification of immune infiltration within the stroma region on immunohistochemical (IHC) whole-slide images (WSIs) and further analyze its prognostic value in CRC. METHODS: Patients from two independent cohorts were divided into three groups: the development group (N = 200), the internal (N = 134), and the external validation group (N = 90). We trained a convolutional neural network for tissue classification of CD3 and CD8 stained WSIs. A scoring system, named stroma-immune score, was established by quantifying the density of CD3+ and CD8+ T-cells infiltration in the stroma region. RESULTS: Patients with higher stroma-immune scores had much longer survival. In the development group, 5-year survival rates of the low and high scores were 55.7% and 80.8% (hazard ratio [HR] for high vs. low 0.39, 95% confidence interval [CI] 0.24-0.63, P < 0.001). These results were confirmed in the internal and external validation groups with 5-year survival rates of low and high scores were 57.1% and 78.8%, 63.9% and 88.9%, respectively (internal: HR for high vs. low 0.49, 95% CI 0.28-0.88, P = 0.017; external: HR for high vs. low 0.35, 95% CI 0.15-0.83, P = 0.018). The combination of stroma-immune score and tumor-node-metastasis (TNM) stage showed better discrimination ability for survival prediction than using the TNM stage alone. CONCLUSIONS: We proposed a stroma-immune score via a deep learning-based pipeline to quantify CD3+ and CD8+ T-cells densities within the stroma region on WSIs of CRC and further predict survival.

The value of the tumour-stroma ratio for predicting neoadjuvant chemoradiotherapy response in locally advanced rectal cancer: a case control study.

BACKGROUND: The tumour-stroma ratio (TSR) is recognized as a practical prognostic factor in colorectal cancer. However, TSR assessment generally utilizes surgical specimens. This study aims to investigate whether the TSR evaluated from preoperative biopsy specimens by a semi-automatic quantification method can predict the response after neoadjuvant chemoradiotherapy (nCRT) of patients with locally advanced rectal cancer (LARC). METHODS: A total of 248 consecutive patients diagnosed with LARC and treated with nCRT followed by resection were included. Haematoxylin and eosin (HE)-stained sections of biopsy specimens were collected, and the TSR was evaluated by a semi-automatic quantification method and was divided into three categories, using the cut-offs determined in the whole cohort to balance the proportion of patients in each category. The response to nCRT was evaluated on the primary tumour resection specimen by an expert pathologist using the four-tier tumour regression grade (TRG) system. RESULTS: The TSR can discriminate patients that are major-responders (TRG 0-1) from patients that are non-responders (TRG 2-3). Patients were divided into stroma-low (33.5%), stroma-intermediate (33.9%), and stroma-high (32.7%) groups using 56.3 and 72.8% as the cutoffs. In the stroma-low group, 58 (69.9%) patients were major-responders, and only 39 (48.1%) patients were considered major-responders in the stroma-high group (P = 0.018). Multivariate analysis showed that the TSR was the only pre-treatment predictor of response to nCRT (adjusted odds ratio 0.40, 95% confidence interval 0.21-0.76, P = 0.002). CONCLUSION: An elevated TSR in preoperative biopsy specimens is an independent predictor of nCRT response in LARC. This semi-automatic quantified TSR could be easily translated into routine pathologic assessment due to its reproducibility and reliability.

A Dual Workflow to Improve the Proteomic Coverage in Plasma Using Data-Independent Acquisition-MS.

Plasma is one of the most important and common matrices for clinical chemistry and proteomic analyses. Data-independent acquisition (DIA) mass spectrometry has enabled the simultaneous quantitative analysis of hundreds of proteins in plasma samples in support population and disease studies. Depletion of the highest abundant proteins is a common tool to increase plasma proteome coverage, but this strategy can result in the nonspecific depletion of protein subsets with which proteins targeted for depletion interact, adversely affecting their analysis. Our work using an antibody-based depletion column revealed significant complementarity not only in the identification of the proteins derived from depleted and undepleted plasma, but importantly also in the extent to which different proteins can be reproducibly quantified in each fraction. We systematically defined four major quantitative parameters of increasing stringency in both the depleted plasma fraction and in undepleted plasma for 757 observed plasma proteins: Linearity cutoff r2 > 0.8; lower limit of quantification (LLOQ); measurement range; limit of detection (LOD). We applied the results of our study to build a web-based tool, PlasmaPilot, that can serve as a protocol decision tree to determine whether the analysis of a specific protein warrants IgY14 mediated depletion.

Reagents for Isobaric Labeling Peptides in Quantitative Proteomics.

Currently, the commercial reagents for isobaric peptides labeling (TMT and iTRAQ) have some drawbacks, such as high cost in experiments, especially in quantitation for the modified peptides, and inconvenient handling for variable sizes of samples. Herein, we developed a set of 10-plex isobaric tags (IBT) with high stability and low cost. The labeled peptides were sensitively detected on Orbitrap Q Exactive MS with an MS2 resolution of 35 000 at 30% NCE, while the peptides were efficiently labeled over 97% by IBT at a ratio of 10:1 of reagent/peptide (w/w) in 200 mM TEAB buffer for 2 h. The IBT labeling was demonstrated with a wide dynamic range of 50-fold without obvious matrix effects on quantification. Importantly, there was little quantification bias found among the individual IBT tags, indicating that the peptides labeled by different tags were quantitatively comparable. The IBT 10-plex reagents were applied for dynamically monitoring the quantitative responses of phosphoproteome stimulated by EGF treatment in HeLa cells. In total, 5 361 unique phosphopeptides were identified, which reached a similar conclusion as others reported. The IBT reagents were therefore experimentally proven as a new type of reagents for isobaric peptides labeling and useful in a large quantity peptides of quantitative proteomics.

Profiling B-Type Natriuretic Peptide Cleavage Peptidoforms in Human Plasma by Capillary Electrophoresis with Electrospray Ionization Mass Spectrometry.

B-type Natriuretic Peptide (BNP) is a biologically active circulating hormone. Plasma concentrations of BNP are routinely used in the diagnosis of heart failure, and the intravenous infusion of recombinant BNP can be used for heart failure treatment. Like many bioactive polypeptides, multiple plasma enzymes are known to cleave circulating BNP, and as part of the CVD-B/D-HPP mandate, we sought to develop a technique capable of profiling these catabolic processes in plasma. We used a neutral-coated capillary electrophoresis-electrospray ionization (CESI) separation system coupled with high-resolution mass spectrometry to profile the proteolysis of exogenous recombinant BNP1-32 in plasma. Our method utilizes electrokinetic injection of minimally processed plasma samples to simultaneously monitor the dynamic generation and breakdown of at least five BNP peptidoforms in plasma. By integrating multisegment injection, our method can produce a multipoint BNP proteolytic profile for one sample within an hour. We envision applying this method to assess the potential relation between plasma-based BNP proteolysis and heart failure as well as a means of monitoring BNP bioavailability after therapeutic infusion.