Methods for quantification of glycopeptides by liquid separation and mass spectrometry.

Recent advances in analytical techniques provide the opportunity to quantify even low-abundance glycopeptides derived from complex biological mixtures, allowing for the identification of glycosylation differences between healthy samples and those derived from disease states. Herein, we discuss the sample preparation procedures and the mass spectrometry (MS) strategies that have facilitated glycopeptide quantification, as well as the standards used for glycopeptide quantification. For sample preparation, various glycopeptide enrichment methods are summarized including the columns used for glycopeptide separation in liquid chromatography separation. For MS analysis strategies, MS1 level-based quantification and MS2 level-based quantification are described, either with or without labeling, where we have covered isotope labeling, TMT/iTRAQ labeling, data dependent acquisition, data independent acquisition, multiple reaction monitoring, and parallel reaction monitoring. The strengths and weaknesses of these methods are compared, particularly those associated with the figures of merit that are important for clinical biomarker studies and the pathological and functional studies of glycoproteins in various diseases. Possible future developments for glycopeptide quantification are discussed.

The Method of Lipemia Clearance Should be Based on the Characteristics and the Method of Testing in the Emergency Laboratory.

BACKGROUND: This study aimed to compare the lipemia removal efficiency of highspeed centrifugation, lipid scavengers, and dilution for biochemical analytes. METHODS: We collected 30 cases of lipemic plasma in an emergency laboratory and divided them into 4 aliquots. Lipemia was removed by highspeed centrifugation, lipid scavenger, dilution, and ultracentrifugation, then analytes were measured by an AU5800 analyzer. Taking ultracentrifugation as reference, the efficiencies of the other three methods were evaluated based on the deviation. RESULTS: When highspeed centrifugation was used for lipemia removal, DBIL (18.62%), and Magnesium (6.09%) could not satisfy the criterion. When lipid scavengers were applied to remove lipemia, CRP (-86.70%), TP (-8.29%), CKMB (-44.85%), DBIL (37.96%), Glu (4.20%) and phosphate (14.32%) were not suggested as lipid scavengers. For dilution, nearly half of the analytes could satisfy the criterion, including AMY (2.41%), CRP (5.54%), ALT (2.85%), GGTL (-1.73%), ALP (-0.04%), Glu (-0.84%), LDH (0.06%), CK (0.68%), BUN (3.80%), CREA (-1.54%), UA (5.42%), and magnesium (0.43%). CONCLUSIONS: Neither of the methods for lipid removal could satisfy all emergency department tests for lipid removal. This finding suggests that removing lipemia in the clinical laboratory should be based on the characteristics and the method of testing.

Outcomes of Aortomitral Continuity Reconstruction During Concomitant Aortic and Mitral Valve Replacement.

OBJECTIVES: To describe outcomes of reconstruction of the aortomitral continuity (AMC) during concomitant aortic and mitral valve replacement (ie, the "Commando" procedure). DESIGN: A retrospective study of consecutive cardiac surgeries from 2010 to 2022. SETTING: At a single institution. PARTICIPANTS: All patients undergoing double aortic and mitral valve replacement. INTERVENTIONS: Patients were dichotomized by the performance (or not) of AMC reconstruction. MEASUREMENTS AND MAIN RESULTS: A total of 331 patients underwent double-valve replacement, of whom 21 patients (6.3%) had a Commando procedure. The Commando group was more likely to have had a previous aortic valve replacement (AVR) or mitral valve replacement (MVR) (66.7% v 27.4%, p < 0.001), redo cardiac surgery (71.4% v 31.3%, p < 0.001), and emergent/salvage surgery (14.3% v 1.61%, p = 0.001), whereas surgery was more often performed for endocarditis in the Commando group (52.4% v 22.9%, p = 0.003). The Commando group had higher operative mortality (28.6% v 10.7%, p = 0.014), more prolonged ventilation (61.9% v 31.9%, p = 0.005), longer cardiopulmonary bypass time (312 ± 118 v 218 ± 85 minutes, p < 0.001), and longer ischemic time (252 ± 90 v 176 ± 66 minutes, p < 0.001). Despite increased short-term morbidity in the Commando group, Kaplan-Meier survival estimation showed no difference in long-term survival between each group (p = 0.386, log-rank). On multivariate Cox analysis, the Commando procedure was not associated with an increased hazard of death, compared to MVR + AVR (hazard ratio 1.29, 95% CI: 0.65-2.59, p = 0.496). CONCLUSIONS: Although short-term postoperative morbidity and mortality were found to be higher for patients undergoing the Commando procedure, AMC reconstruction may be equally durable in the long term.

GlycoSLASH: Concurrent Glycopeptide Identification from Multiple Related LC-MS/MS Data Sets by Using Spectral Clustering and Library Searching.

Liquid chromatography coupled with tandem mass spectrometry is commonly adopted in large-scale glycoproteomic studies involving hundreds of disease and control samples. The software for glycopeptide identification in such data (e.g., the commercial software Byonic) analyzes the individual data set and does not exploit the redundant spectra of glycopeptides presented in the related data sets. Herein, we present a novel concurrent approach for glycopeptide identification in multiple related glycoproteomic data sets by using spectral clustering and spectral library searching. The evaluation on two large-scale glycoproteomic data sets showed that the concurrent approach can identify 105%-224% more spectra as glycopeptides compared to the glycopeptide identification on individual data sets using Byonic alone. The improvement of glycopeptide identification also enabled the discovery of several potential biomarkers of protein glycosylations in hepatocellular carcinoma patients.

Oscillator Strengths and Cross Sections of the Valence-Shell Excitations of HBr Studied by Fast Electron Impact.

The oscillator strengths and cross sections of the valence-shell excitations of HBr were determined by fast electron scattering with an incident electron energy of 1500 eV and an energy resolution of 80 meV. The momentum transfer dependence behaviors of the generalized oscillator strengths have been used to elucidate the transition characteristics. The present results show that the strong spin-orbital interaction results in the observation of some triplet states in the (Λ, S) coupling and the constant generalized oscillator strength ratios for the pair states with the same electronic configuration and quantum number Ω, and the quantitative spin-orbit coupling coefficients of b3Π1(v = 0) and C1Π(v = 0) are determined. The optical oscillator strengths of the valence-shell excitations were obtained by extrapolating the generalized oscillator strengths to the limit of zero squared momentum transfer. The present optical oscillator strengths give an independent cross-check of the previous experimental and theoretical results, and the comparison shows that the line-saturation effect is more severe for the high Rydberg states with large intensities and narrow natural widths. The integral cross sections of the valence-shell excitations of HBr were obtained from the excitation threshold to 5000 eV by the BE-scaling method. The present oscillator strengths and cross sections supplement the fundamental molecular database of HBr and can be used for modeling in the semiconductor industry, astrophysics, and atmospheric chemistry.

H-type hypertension is a risk factor for chronic total coronary artery occlusion: a cross-sectional study from southwest China.

BACKGROUND: Chronic total coronary occlusion (CTO) is serious and the "last bastion" of percutaneous coronary intervention. Hypertension and hyperhomocysteinemia (HHCY) are synergistic and significantly increase cardiovascular event risk. The relationship between H-type hypertension and CTO remains unclear; thus, this cross-sectional study investigated this potential association. METHODS: Between January 2018 and June 2022, 1446 individuals from southwest China were recruited to participate in this study. CTO was defined as complete coronary artery occlusion persisting for over three months. H-type hypertension was defined as hypertension with plasma homocysteine levels ≥ 15 µmol/L. Multivariate logistic regression models were applied to assess the association between H-type hypertension and CTO. Receiver operating characteristic (ROC) curves were generated to determine the accuracy of H-type hypertension in predicting CTO. RESULTS: Of the 1446 individuals, 397 had CTO, and 545 had H-type hypertension. After multivariate adjustment, the odds ratio (OR) for CTO in individuals with H-type hypertension was 2.3-fold higher (95% CI 1.01-5.26) than that in healthy controls. The risk of CTO is higher in individuals with H-type hypertension than in those with isolated HHCY and hypertension. The area under the ROC curve for CTO was 0.685 (95% CI, 0.653-0.717) for H-type hypertension. CONCLUSIONS: In southwest China, H-type hypertension is significantly related to the occurrence of CTO. TRIAL REGISTRATION: This retrospective study was registered with the Chinese Clinical Trials Registry ( http://www.chictr.org.cn , ChiCTR2100050519.2.2).

The Long-Term Impact of Diastolic Dysfunction After Routine Cardiac Surgery.

OBJECTIVE: To determine the impact of diastolic dysfunction (DD) on survival after routine cardiac surgery. DESIGN: This was an observational study of consecutive cardiac surgeries from 2010 to 2021. SETTING: At a single institution. PARTICIPANTS: Patients undergoing isolated coronary, isolated valvular, and concomitant coronary and valvular surgery were included. Patients with a transthoracic echocardiogram (TTE) longer than 6 months prior to their index surgery were excluded from the analysis. INTERVENTIONS: Patients were categorized via preoperative TTE as having no DD, grade I DD, grade II DD, or grade III DD. MEASUREMENTS AND MAIN RESULTS: A total of 8,682 patients undergoing a coronary and/or valvular surgery were identified, of whom 4,375 (50.4%) had no DD, 3,034 (34.9%) had grade I DD, 1,066 (12.3%) had grade II DD, and 207 (2.4%) had grade III DD. The median (IQR) time of the TTE prior to the index surgery was 6 (2-29) days. Operative mortality was 5.8% in the grade III DD group v 2.4% for grade II DD, 1.9% for grade I DD, and 2.1% for no DD (p = 0.001). Atrial fibrillation, prolonged mechanical ventilation (>24 hours), acute kidney injury, any packed red blood cell transfusion, reexploration for bleeding, and length of stay were higher in the grade III DD group compared to the rest of the cohort. The median follow-up was 4.0 (IQR: 1.7-6.5) years. Kaplan-Meier survival estimates were lower in the grade III DD group than in the rest of the cohort. CONCLUSIONS: These findings suggested that DD may be associated with poor short-term and long-term outcomes.

A sonogram radiomics model for differentiating granulomatous lobular mastitis from invasive breast cancer: a multicenter study.

PURPOSE: To construct a nomogram based on sonogram features and radiomics features to differentiate granulomatous lobular mastitis (GLM) from invasive breast cancer (IBC). MATERIALS AND METHODS: A retrospective collection of 213 GLMs and 472 IBCs from three centers was divided into a training set, an internal validation set, and an external validation set. A radiomics model was built based on radiomics features, and the RAD score of the lesion was calculated. The sonogram radiomics model was constructed using ultrasound features and RAD scores. Finally, the diagnostic efficacy of the three sonographers with different levels of experience before and after combining the RAD score was assessed in the external validation set. RESULTS: The RAD score, lesion diameter, orientation, echogenicity, and tubular extension showed significant differences in GLM and IBC (p < 0.05). The sonogram radiomics model based on these factors achieved optimal performance, and its area under the curve (AUC) was 0.907, 0.872, and 0.888 in the training, internal, and external validation sets, respectively. The AUCs before and after combining the RAD scores were 0.714, 0.750, and 0.830 and 0.834, 0.853, and 0.878, respectively, for sonographers with different levels of experience. The diagnostic efficacy was comparable for all sonographers when combined with the RAD score (p > 0.05). CONCLUSION: Radiomics features effectively enhance the ability of sonographers to discriminate between GLM and IBC and reduce interobserver variation. The nomogram combining ultrasound features and radiomics features show promising diagnostic efficacy and can be used to identify GLM and IBC in a noninvasive approach.

Unlocking the Interfacial Adsorption-Intercalation Pseudocapacitive Storage Limit to Enabling All-Climate, High Energy/Power Density and Durable Zn-Ion Batteries.

Sluggish storage kinetics and insufficient performance are the major challenges that restrict the transition metal dichalcogenides (TMDs) applied for zinc ion storage, especially at the extreme temperature conditions. Herein, a multiscale interface structure-integrated modulation concept was presented, to unlock the omnidirectional storage kinetics-enhanced porous VSe2-x ⋅n H2 O host. Theory research indicated that the co-modulation of H2 O intercalation and selenium vacancy enables enhancing the interfacial zinc ion capture ability and decreasing the zinc ion diffusion barrier. Moreover, an interfacial adsorption-intercalation pseudocapacitive storage mechanism was uncovered. Such cathode displayed remarkable storage performance at the wide temperature range (-40-60 °C) in aqueous and solid electrolytes. In particular, it can retain a high specific capacity of 173 mAh g-1 after 5000 cycles at 10 A g-1 , as well as a high energy density of 290 Wh kg-1 and a power density of 15.8 kW kg-1 at room temperature. Unexpectedly, a remarkably energy density of 465 Wh kg-1 and power density of 21.26 kW kg-1 at 60 °C also can be achieved, as well as 258 Wh kg-1 and 10.8 kW kg-1 at -20 °C. This work realizes a conceptual breakthrough for extending the interfacial storage limit of layered TMDs to construct all-climate high-performance Zn-ion batteries.

Phosphorus-Based Materials for High-Performance Alkaline Metal Ion Batteries: Progress and Prospect.

Alkaline metal-ion batteries (AIBs) such as lithium-ion batteries (LIBs), sodium-ion batteries (NIBs), and potassium-ion batteries (KIBs) are potential energy storage systems. Currently, although LIBs are widely used in consumer electronics and electric vehicles, the electrochemical performance, safety, and cost of current AIBs are still unable to meet the needs for many future applications, such as large-scale energy storage, due to the low theoretical capacity of cathode/anode materials, flammability of electrolytes and limited Li resources. It is thus imperative to develop new materials to improve the properties of AIBs. Several promising cathodes, anodes, and electrolytes have been developed and among the new battery materials, phosphorus-based (P-based) materials have shown great promise. For example, P and metal phosphide anodes have high theoretical capacity, resource abundance, and environmental friendliness boding well for future high-energy-density AIBs. Besides, phosphate cathode materials have the advantages of low cost, high safety, high voltage, and robust stability, and P-based materials like LiPF6 and lithium phosphorus oxynitride are widely used electrolytes. In this paper, the latest development of P-based materials in AIBs, challenges, effective solutions, and new directions are discussed.

Sweet taste perception in mice is blunted by PTBP1-regulated skipping of Tas1r2 exon 4.

Taste perception, initiated by activation of taste receptors in taste bud cells, is crucial for regulating nutrient intake. Genetic polymorphisms in taste receptor genes cannot fully explain the wide individual variations of taste sensitivity. Alternative splicing (AS) is a ubiquitous posttranscriptional mode of gene regulation that enriches the functional diversity of proteins. Here, we report the identification of a novel splicing variant of sweet taste receptor gene Tas1r2 (Tas1r2_∆e4) in mouse taste buds and the mechanism by which it diminishes sweet taste responses in vitro and in vivo. Skipping of Tas1r2 exon 4 in Tas1r2_∆e4 led to loss of amino acids in the extracellular Venus flytrap domain, and the truncated isoform reduced the response of sweet taste receptors (STRs) to all sweet compounds tested by generating nonfunctional T1R2/T1R3 STR heterodimers. The splicing factor PTBP1 (polypyrimidine tract-binding protein 1) promoted Tas1r2_∆e4 generation through binding to a polypyrimidine-rich splicing silencer in Tas1r2 exon 4, thus decreasing STR function and sweet taste perception in mice. Taken together, these data reveal the existence of a regulated AS event in Tas1r2 expression and its effect on sweet taste perception, providing a novel mechanism for modulating taste sensitivity at the posttranscriptional level.

Blood cadmium, lead, manganese, mercury, and selenium levels in American Indian populations: The Strong Heart Study.

BACKGROUND: Many American Indian (AI) communities are in areas affected by environmental contamination, such as toxic metals. However, studies assessing exposures in AI communities are limited. We measured blood metals in AI communities to assess historical exposure and identify participant characteristics associated with these levels in the Strong Heart Study (SHS) cohort. METHOD: Archived blood specimens collected from participants (n = 2014, all participants were 50 years of age and older) in Arizona, Oklahoma, and North and South Dakota during SHS Phase-III (1998-1999) were analyzed for cadmium, lead, manganese, mercury, and selenium using inductively coupled plasma triple quadrupole mass spectrometry. We conducted descriptive analyses for the entire cohort and stratified by selected subgroups, including selected demographics, health behaviors, income, waist circumference, and body mass index. Bivariate associations were conducted to examine associations between blood metal levels and selected socio-demographic and behavioral covariates. Finally, multivariate regression models were used to assess the best model fit that predicted blood metal levels. FINDINGS: All elements were detected in 100% of study participants, with the exception of mercury (detected in 73% of participants). The SHS population had higher levels of blood cadmium and manganese than the general U.S. population 50 years and older. The median blood mercury in the SHS cohort was at about 30% of the U.S. reference population, potentially due to low fish consumption. Participants in North Dakota and South Dakota had the highest blood cadmium, lead, manganese, and selenium, and the lowest total mercury levels, even after adjusting for covariates. In addition, each of the blood metals was associated with selected demographic, behavioral, income, and/or weight-related factors in multivariate models. These findings will help guide the tribes to develop education, outreach, and strategies to reduce harmful exposures and increase beneficial nutrient intake in these AI communities.

The Stability of Intact Parathyroid Hormone (PTH) in Different Types of Blood Collection Tubes.

BACKGROUND: Over past decades, the instability of parathyroid hormone (PTH) causes great interference for the clinical laboratory. Contradictory results were reported in many reports about storage conditions and suitable blood collection tubes to ensure PTH stability in the pretreatment phase. METHODS: This study recruited 30 participants including 10 healthy persons, 10 hemodialysis, and 10 hyperparathyroidism patients. Five types of blood collection tubes (EDTA-K3 tube, coagulant tube, heparin anticoagulant tube, gel separating tube, and plain tube) were included to determine whether they were suitable as blood-collecting vessels. The time points and conditions for testing samples included less than 2 hours, 4 hours, and 8 hours at room temperature, and, in parallel, 24 hours, 48 hours, and 72 hours in refrigeration. Two different judgement criteria were used to compare the stability of PTH in different blood vessels. RESULTS: Purely statistical analysis showed that 4 types of blood collection tubes could not perform the same storage ability as EDTA-K3 tube at "T0" time point. Plain tube had the largest drop among all types of blood collection tubes. Compared by pairwise t-test, EDTA-K3 tube could maintain intact PTH for 8 hours (p = 0.998) at room temperature and 24 hours (p = 0.053) in refrigeration. When comparing the total change limit (TCL = 18.8%), at room temperature, EDTA-K3 tube (7.0%), heparin tube (12.7%), coagulant tube (16.2%), and plain tube (17.6%) could maintain intact PTH for 8 hours, and GST can preserve PTH for 4 hours (18.2%). In refrigeration, EDTA-K3 tube could maintain PTH for 72 hours (7.5%) and heparin tube could maintain 24 hours (18.4%). The other three blood collection tubes could not preserve PTH in refrigeration (GST = 22.1%, coagulant tube = 20.3%, plain tube = 20.8%). CONCLUSIONS: PTH seems more stable in the EDTA-K3 tube than any other blood collection tubes and is followed next by the heparin anticoagulant tube. Plain tube and GST have faster degradation than other tubes and are not suggested to preserve intact PTH.

Sex-based outcomes after surgery for acute type A aortic dissection.

BACKGROUND: While prior data have suggested worse outcomes in women after acute type A aortic dissection (ATAAD) repair when compared to men, results have been inconsistent across studies over time. This study sought to evaluate the impact of sex on short- and long-term outcomes after ATAAD repair. METHODS: This was a retrospective study utilizing an institutional database of ATAAD repairs from 2007 to 2021. Patients were stratified according to sex. Kaplan-Meier survival estimation and multivariable Cox regression were performed. Supplementary analysis using propensity score matching was also performed. RESULTS: Of the 601 patients who underwent ATAAD repair, 361 were males (60.1%) and 240 (39.9%) were females. Females were significantly older, more likely to have hypertension, and more likely to have chronic lung disease. Females were also significantly more likely than males to undergo hemiarch replacement, while males were significantly more likely than females to undergo total arch replacement and frozen elephant trunk. Operative mortality was 9.4% among males and 13.8% among females, though this was not a statistically significant difference (p = .098). Postoperative complications were comparable between groups. Kaplan-Meier survival estimates were similar for men and women, and, on multivariable Cox regression, sex was not significantly associated with long-term survival (hazard ratio: 1.00, 95% confidence interval: 0.73, 1.37, p = .986). Outcomes remained comparable after supplementary propensity score matched analysis. CONCLUSION: ATAAD repair can be performed with comparable short-term and long-term outcomes in both men and women.

The impact of prior cardiac surgery on patients undergoing surgical repair for acute type A aortic dissection.

OBJECTIVE: To determine the impact of reoperative versus first-time sternotomy for emergent open repair of acute Type A aortic dissection (ATAAD). METHODS: This was an observational study of consecutive aortic surgeries from 2007 to 2021. Kaplan-Meier survival estimation and multivariable Cox regression analysis were performed to assess the impact of reoperative versus first-time sternotomy upon survival after ATAAD repair. RESULTS: A total of 601 patients with ATAAD were identified, of which 72 (12%) underwent reoperative sternotomy. The reoperative group had a higher prevalence of baseline comorbidities, including hypertension, diabetes, peripheral vascular disease, atrial fibrillation, and coronary artery disease. Central cannulation of the aorta was achieved at a similar rate across each group (81.9% vs. 81.5%, p = .923), and cardiopulmonary bypass (CPB) time was similar across each group (204 ± 84.8 vs. 203 ± 72.4 min, p = .923). Postoperative outcomes were similar across both groups, including in-hospital mortality, stroke, pulmonary complications, renal failure, and reexploration for excessive bleeding. Five-year survival was 74.5% (70.5, 78.3) for the first-time group and was 71.6% (60.0, 81.9) for the reoperative group. After multivariable Cox regression, reoperative sternotomy was not significantly associated with an increased hazard of death compared to first-time sternotomy (hazards ratio: 0.90, 95% confidence interval: 0.56, 1.43, p = .642). CONCLUSIONS: These findings suggest that re-sternotomy can be safely performed with similar outcomes as first-time sternotomy. Central initiation of CPB after sternal reentry limits CPB time and may therefore represent a protective strategy that enhances outcomes for patients presenting with ATAAD and prior cardiac surgery.

The long-term impact of acute renal failure after aortic arch replacement for acute type A aortic dissection.

OBJECTIVE: To determine the long-term impact of developing acute renal failure (ARF) on survival after open aortic arch reconstruction for acute type A aortic dissection (ATAAD). METHODS: This was an observational study of consecutive aortic surgeries from 2007 to 2021. Patients with ATAAD were identified via a prospectively maintained institutional database and were stratified by the presence or absence of postoperative ARF (by RIFLE criteria). Kaplan-Meier survival estimation and multivariable Cox regression analysis were performed. RESULTS: A total of 601 patients undergoing open surgery for ATAAD were identified, of which 516 (85.9%) did not develop postoperative ARF, while 85 (14.1%) developed ARF, with a median follow-up time of 4.6 years (1.6, 7.9). Baseline characteristics were similar across each group, except for higher rates of branch vessel malperfusion and lower preoperative ejection fraction in the ARF group. Patients with ARF underwent more total arch replacement and elephant trunk procedures, with longer cardiopulmonary bypass and circulatory arrest times than patients without ARF. ARF was associated with worse short-term outcomes, including increased in-hospital mortality, prolonged mechanical ventilation, higher rates of sepsis, more blood transfusions, and longer length of hospital stay. Unadjusted Kaplan-Meier survival estimates were significantly lower in the ARF group, compared to the group without ARF (p < .001, log-rank test). After multivariable adjustment, the development of postoperative ARF was significantly associated with an increased hazard of death over the study's follow-up time-period (hazard ratio: 2.74, 95% confidence interval: 1.95, 3.86, p < .001). CONCLUSIONS: ARF is a highly morbid postoperative event that may adversely impact long-term survival after aortic surgery.

GlycoHybridSeq: Automated Identification of N-Linked Glycopeptides Using Electron Transfer/High-Energy Collision Dissociation (EThcD).

Glycosylation is one of the most common post-translational modifications (PTM) occurring in a large variety of proteins with important biological functions in human and other higher organisms. Liquid chromatography tandem mass spectrometry (LC-MS/MS) has been routinely used to characterize site-specific protein glycosylation at high throughput in complex glycoproteomic samples. Recently, electron transfer/high-energy collision dissociation (EThcD) was introduced for glycopeptide identification, which offers rich structural information on glycopepides with the fragment ions from the cleavages of both the glycan and the peptide backbone. Herein, we present the software GlycoHybridSeq for automated interpretation of EThcD-MS/MS spectra from glycoproteomic data using a customized scoring function, which enables the functionalities of identifying glycopeptides, characterizing glycosylation sites, and distinguishing some isomeric glycans. We evaluate GlycoHybridSeq on glycoproteomic data collected for cancer biomarker discovery. The results showed that it achieved comparable or better performance than that of Byonic and MSFragger. GlycoHybridSeq is released as an open source software and is ready to be used in large-scale glycoproteomic data analyses.

Column-based Technology for CD9-HPLC Immunoaffinity Isolation of Serum Extracellular Vesicles.

Serum-derived extracellular vesicles (EVs) are a promising source of biomarkers; however, major challenges in EV separation and proteomic profiling remain for isolating EVs from a small amount, that is, on the microliter scale, of human serum while minimizing the contamination of blood proteins and lipoprotein particles coeluting in EV preparations. Herein we have developed a column-based CD9-antibody-immobilized high-performance liquid chromatography immunoaffinity chromatography(CD9-HPLC-IAC) technology for EV isolation from a microliter scale of serum for downstream proteomic analysis. The CD9-HPLC-IAC method achieved EV isolation from 40 µL of serum in 30 min with a yield of 8.0 × 109 EVs, where EVs were further processed with a postcolumn cleaning step using the 50 kDa molecular weight cut-off filter for the buffer exchange, concentration, and reduction of potentially coeluting serum proteins. In total, 482 proteins were identified in EVs by using liquid chromatography tandem mass spectrometry, including the common exosomal markers such as CD63, CD81, CD82, Alix, and TSG101. The statistical analysis of EV protein content showed that the top 10 serum proteins in EVs were significantly decreased by using the CD9-HPLC-IAC method compared with the use of ultracentrifugation (p = 0.001) and size exclusion chromatography (p = 0.009), and apolipoproteins were significantly reduced 4.8-fold compared with the SEC method (p < 0.001). The result demonstrates the potential of the CD9-HPLC-IAC method for the efficient isolation and proteomic characterization of EVs from a microscale volume of serum.

A Panel of Glycopeptides as Candidate Biomarkers for Early Diagnosis of NASH Hepatocellular Carcinoma Using a Stepped HCD Method and PRM Evaluation.

Changes in N-glycosylation on specific peptide sites of serum proteins have been investigated as potential markers for diagnosis of nonalcoholic steatohepatitis (NASH)-related HCC. To accomplish this work, a novel workflow involving broad-scale marker discovery in serum followed by targeted marker evaluation of these glycopeptides were combined. The workflow involved an LC-Stepped HCD-DDA-MS/MS method coupled with offline peptide fractionation for large-scale identification of N-glycopeptides directly from pooled serum samples (each n = 10) as well as differential determination of N-glycosylation changes between disease states. We then evaluated several potentially diagnostic N-glycopeptides among 78 individual patient samples (40 cirrhosis, 28 early stage NASH HCC, and 10 late-stage NASH HCC) by LC-Stepped HCD-PRM-MS/MS to quantitatively analyze 65 targeted glycopeptides from 7 glycoproteins. Of these targets, we found site-specific N-glycopeptides n169GSLFAFR_HexNAc(4)Hex(5)NeuAc(2) and n242ISDGFDGIPDNVDAALALPAHSYSGR_HexNAc(5)Hex(6)Fuc(1)NeuAc(3) from VTNC were significantly increased comparing samples from patients with NASH cirrhosis and NASH HCC (p < 0.05). When combining results of these 2 glycopeptides with AFP, the ROC curve analysis demonstrated the AUC value increased to 0.834 (95% CI, 0.748-0.921) and 0.847 (95% CI, 0.766-0.932), respectively, as compared to that of AFP alone (AUC = 0.791, 95% CI, 0.690-0.892). These 2 glycopeptides may serve as potential biomarkers for early HCC diagnosis in patients with NASH related cirrhosis.

Chronic treatment with the complex I inhibitor MPP+ depletes endogenous PTEN-induced kinase 1 (PINK1) via up-regulation of Bcl-2-associated athanogene 6 (BAG6).

Mitochondrial dysfunction is implicated in sporadic and familial Parkinson's disease (PD). However, the mechanisms that impair homeostatic responses to mitochondrial dysfunction remain unclear. Previously, we found that chronic, low-dose administration of the mitochondrial complex I inhibitor 1-methyl-4-phenylpyridinium (MPP+) dysregulates mitochondrial fission-fusion, mitophagy, and mitochondrial biogenesis. Given that PTEN-induced kinase 1 (PINK1) regulates mitochondrial function, dynamics, and turnover, we hypothesized that alterations in endogenous PINK1 levels contribute to depletion of mitochondria during chronic complex I injury. Here we found that chronic MPP+ treatment of differentiated SH-SY5Y neuronal cells significantly decreases PINK1 expression prior to reductions in other mitochondrial components. Furthermore, Bcl2-associated athanogene 6 (BAG6, BAT3, or Scythe), a protein involved in protein quality control and degradation, was highly up-regulated during the chronic MPP+ treatment. BAG6 interacted with PINK1, and BAG6 overexpression decreased the half-life of PINK1. Conversely, siRNA-mediated BAG6 knockdown prevented chronic MPP+ stress-induced loss of PINK1, reversed MPP+-provoked mitochondrial changes, increased cell viability, and prevented MPP+-induced dendrite shrinkage in primary neurons. These results indicate that BAG6 up-regulation during chronic complex I inhibition contributes to mitochondrial pathology by decreasing the levels of endogenous PINK1. Given that recessive mutations in PINK1 cause familial PD, the finding of accelerated PINK1 degradation in the chronic MPP+ model suggests that PINK1 loss of function represents a point of convergence between the neurotoxic and genetic causes of PD.