Virtual Sensing of Key Variables in the Hydrogen Production Process: A Comparative Study of Data-Driven Models.

Hydrogen is an ideal energy carrier manufactured mainly by the natural gas steam reforming hydrogen production process. The concentrations of CH4, CO, CO2, and H2 in this process are key variables related to product quality, which thus need to be controlled accurately in real-time. However, conventional measurement methods for these concentrations suffer from significant delays or huge acquisition and upkeep costs. Virtual sensors effectively compensate for these shortcomings. Unfortunately, previously developed virtual sensors have not fully considered the complex characteristics of the hydrogen production process. Therefore, a virtual sensor model, called "moving window-based dynamic variational Bayesian principal component analysis (MW-DVBPCA)" is developed for key gas concentration estimation. The MW-DVBPCA considers complicated characteristics of the hydrogen production process, involving dynamics, time variations, and transportation delays. Specifically, the dynamics are modeled by the finite impulse response paradigm, the transportation delays are automatically determined using the differential evolution algorithm, and the time variations are captured by the moving window method. Moreover, a comparative study of data-driven virtual sensors is carried out, which is sporadically discussed in the literature. Meanwhile, the performance of the developed MW-DVBPCA is verified by the real-life natural gas steam reforming hydrogen production process.

[Phenotypic and genetic analysis of a Chinese pedigree affected with Hereditary antithrombin deficiency due to a novel variant of SERPINC1 gene].

OBJECTIVE: To analyze the clinical phenotype and genetic characteristics of a Chinese pedigree affected with Hereditary antithrombin deficiency. METHODS: A pedigree diagnosed at the the Second Affiliated Hospital of Wenzhou Medical University, Yuying Children's Hospital in June, 2020 was selected as the study subject. Plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), and thrombin time (TT) of the probands and their pedigree members were determined using a STA-R automatic coagulation analyzer. Antithrombin activity (AT: A) and antithrombin antigen (AT: Ag) in plasma were determined with chromogenic substrate and immunonephelometry assays. All exons and flanking sequences of the anticoagulant protein gene SERPINC1 were amplified by PCR and subjected to Sanger sequencing. Candidate variants were verified with bioinformatic tools (PolyPhen-2, SIFT, Mutation Taster and PYMOL) to explore their effect on the function and structural conformation of the protein. RESULTS: The probands (II-2, II-10), their brother (II-5) and sons (III-1, III-8) had shown normal PT, APTT, FIB, and TT, but significantly decreased AT: A and AT: Ag, with their levels being 34%, 57%, 56%, 48%, 53% and 13.51 mg/dL, 13.44 mg/dL, 18.39 mg/dL, 17.36 mg/dL, 17.71 mg/dL, respectively. The remaining pedigree members had normal values. Sanger sequencing revealed that the probands and all affected pedigree members had harbored a heterozygous c.851T>C (p.Met284Thr) missense variant in exon 5 of the SERPINC1 gene. Bioinformatic analysis and simulation suggested that the variant has resulted in alteration of hydrogen bonds at the c.851 position, which may affect the structure of the protein. Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), the variant was classified as pathogenic (PS1+PM1+PM5+PP1+PP4). CONCLUSION: The probands and other affected members were all diagnosed with type I hereditary AT deficiency, for which the c.851T>C (p.Met284Thr) variant of the SERPINC1 gene may be accountable.

Inhaled nanoparticles for treating idiopathic pulmonary fibrosis by inhibiting honeycomb cyst and alveoli interstitium remodeling.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with high mortality. The Food and Drug Administration-approved drugs, nintedanib and pirfenidone, could delay progressive fibrosis by inhibiting the overactivation of fibroblast, however, there was no significant improvement in patient survival due to low levels of drug accumulation and remodeling of honeycomb cyst and interstitium surrounding the alveoli. Herein, we constructed a dual drug (verteporfin and pirfenidone)-loaded nanoparticle (Lip@VP) with the function of inhibiting airway epithelium fluidization and fibroblast overactivation to prevent honeycomb cyst and interstitium remodeling. Specifically, Lip@VP extensively accumulated in lung tissues via atomized inhalation. Released verteporfin inhibited the fluidization of airway epithelium and the formation of honeycomb cyst, and pirfenidone inhibited fibroblast overactivation and reduced cytokine secretion that promoted the fluidization of airway epithelium. Our results indicated that Lip@VP successfully rescued lung function through inhibiting honeycomb cyst and interstitium remodeling. This study provided a promising strategy to improve the therapeutic efficacy for IPF.

Temporal Tracking of Insulin Action on the Cell Surface of Proteins at a Resolution of Ten Seconds.

The ligand-receptor signaling occurring on the cell surface governs cell growth, proliferation, and survival via rapidly triggering a cascade of events. Here, we for the first time report an in situ perturbation-free and rapid surface proteomic profiling at a temporal resolution of ten seconds. By this innovation, about 1022 cell surface-associated proteins were reproducibly identified and quantified. It is noteworthy that, upon a model ligand insulin stimulus, a few rapid-responding proteins at 10 s to 2 min were identified, e.g., CNNM3. Moreover, temporal response patterns were established for the members of GLUT4 storage vesicles (GSVs; responsible for glucose transportation) and confirmed with five known GSV proteins. This pattern was then exploited to uncover seven new regulatory proteins (LDLR, HFE, ECE1, MRC2, CORO1C, CPD, and BST2). Collectively, we showed a powerful surface proteomic tool to decipher rapid signaling of cell-surface proteins and to uncover new subunits involved in rapidly trafficking vesicles.

Injectable rhein-assisted crosslinked hydrogel for efficient local osteosarcoma chemotherapy.

Osteosarcoma (OS) is the most common malignant tumor of the bone that affects children and adolescents, and its treatment usually involves doxorubicin hydrochloride (DOX). However, the drug resistance and side effects caused by high-dose DOX infusion greatly hinder its therapeutic effects. To achieve efficient OS treatment with low toxicity, an injectable rhein (RH)-assisted crosslinked hydrogel (PVA@RH@DOX hydrogel, PRDH) was designed, which was prepared by loading DOX and RH into a polyvinyl alcohol (PVA) solution. The cytotoxicity assay and live/dead staining results showed that the combination of RH and DOX more effectively killed OS cells, producing excellent effects at low concentrations of DOX. The wound healing and transwell test results proved that PRDH could significantly inhibit the metastasis and invasion of OS cells. PRDH showed a long-lasting antitumor effect after injection of a single dose, significantly suppressing the proliferation and metastasis of OS and achieving the strategy of a single administration for long-term treatment. Excitingly, RH facilitated hydrogel formation by assisting with PVA crosslinking. This system provides an alternative regimen and broadens the horizon for the clinical treatment of OS.

Uniform thin ice on ultraflat graphene for high-resolution cryo-EM.

Cryo-electron microscopy (cryo-EM) visualizes the atomic structure of macromolecules that are embedded in vitrified thin ice at their close-to-native state. However, the homogeneity of ice thickness, a key factor to ensure high image quality, is poorly controlled during specimen preparation and has become one of the main challenges for high-resolution cryo-EM. Here we found that the uniformity of thin ice relies on the surface flatness of the supporting film, and developed a method to use ultraflat graphene (UFG) as the support for cryo-EM specimen preparation to achieve better control of vitreous ice thickness. We show that the uniform thin ice on UFG improves the image quality of vitrified specimens. Using such a method we successfully determined the three-dimensional structures of hemoglobin (64 kDa), α-fetoprotein (67 kDa) with no symmetry, and streptavidin (52 kDa) at a resolution of 3.5 Å, 2.6 Å and 2.2 Å, respectively. Furthermore, our results demonstrate the potential of UFG for the fields of cryo-electron tomography and structure-based drug discovery.

[Analysis of two pedigrees affected with inherited dysfibrinogenemia due to a novel c.1115 T>A variant of the FGB gene].

OBJECTIVE: To analyze the phenotype and genotype of two Chinese family with inherited dysfibrinogenemia and the molecular pathogenic mechanism. METHODS: In the probands and their family members, coagulation routine, fibrinogen activity (Fg: A) and fibrinogen antigen (Fg: Ag) were detected. To find the mutation and exclude single nucleotide polymorphisms, all the exons and exons-intron boundaries of fibrinogen genes (FGA, FGB and FGG) were amplified by Ploymerase Chain Reaction (PCR), then sequenced. Bioinformatics prediction softwares were used to predict and score the change of function caused by the variant. PyMol were used to analyze the structure of protein caused by the variant. Clustal X software was used to analyze the conservation of the mutant amino acids. RESULTS: The thrombin time (TT) of the two was slightly prolonged and could not be corrected by protamine sulfate, and the fibrinogen activity was significantly reduced (1.25 g/L and 1.17 g/L), but the fibrinogen antigen content was normal, respectively (3.50 g /L and 3.81 g/L). Genetic analysis showed that both probands were heterozygous missense variants (FGB exon 7 c.1115T>A (p.Val372Glu)), both of which originated from the paternal line. The prediction results of the four bioinformatics softwares indicate that this variant could be disease causing. Clustal X software showed that Val372 is highly conserved among homologous species. Based on the guidelines of the American College of Medical Genetics and Genomics, c.1115T>A was predicted to be likely pathgenic (PM2+PP1+PP2+PP3+PP4). PyMol showed that the secondary structure and three-dimensional structure of fibrinogen protein were changed by p.Val372Glu variant. CONCLUSION: Inherited dysfibrinogenemia of the probands maybe caused by variant of FGB c.1115 T>A (p.Val372Glu), and the variant was firstly reported.

A Tyrosine Phosphoproteome Analysis Approach Enabled by Selective Dephosphorylation with Protein Tyrosine Phosphatase.

Protein tyrosine phosphorylation (pTyr) plays a prominent role in signal transduction and regulation in all eukaryotic cells. In conventional immunoaffinity purification (IP) methods, phosphotyrosine peptides are isolated from the digest of cellular protein extracts with a phosphotyrosine-specific antibody and are identified by tandem mass spectrometry. However, low sensitivity, poor reproducibility, and high cost are universal concerns for IP approaches. In this study, we presented an antibody-free approach to identify phosphotyrosine peptides by using protein tyrosine phosphatase (PTP). It was found that most of the PTPs including PTP1B, TCPTP, and SHP1 can efficiently and selectively dephosphorylate phosphotyrosine peptides. We then designed a workflow by combining two Ti4+-IMAC-based phosphopeptide enrichment steps with PTP-catalyzed dephosphorylation for tyrosine phosphoproteomics analysis. This workflow was first validated by selective detection of phosphotyrosine peptides from semicomplex samples and then applied to analyze the tyrosine phosphoproteome of Jurkat T cells. Around 1000 putative former phosphotyrosine peptides were identified from less than 500 µg of cell lysate. The tyrosine phosphosites on the majority of these peptides could be unambiguously determined for over 70% of them possessing only one tyrosine residue. It was also found that the tyrosine sites identified by this method were highly complementary to those identified by the SH2 superbinder-based method. Therefore, the combination of Ti4+-IMAC enrichment with PTP dephosphorylation provides an alternative and cost-effective approach for tyrosine phosphoproteomics analysis.

Highly efficient enrichment of intact phosphoproteins by a cadmium ion-based co-precipitation strategy.

Selective separation and enrichment of phosphoproteins are essential for understanding their important functions in almost all cellular processes. Here, taking advantage of the feature that cadmium ion (Cd2+ ) has an overwhelming preference for phosphates, we developed a robust and simple Cd2+ co-precipitation strategy for the selective isolation of intact phosphoproteins. After evaluating the feasibility of Cd2+ in phosphoprotein precipitation, we compared the washing protocols for the removal of non-specific binding proteins and then used the best-performing protocol for the isolation of phosphoproteins from different complex samples. It was found that phosphoproteins can be specifically enriched from artificial protein mixtures containing α-casein, ß-casein, and bovine serum albumin or plasma, in which bovine serum albumin or plasma were served as interferences with very high molar ratios. Applying this method to enrich phosphoproteins from complex cell lysates, a high specificity was confirmed by western blotting analysis with a phosphoprotein-specific kit. Finally, we successfully applied this method to the purification of caseins from drinking milk, highlighting its potential application in the studies where purified phosphoproteins were required. In a word, this Cd2+ co-precipitation method enables universal and effective capture, enrichment, and detection of intact phosphoproteins, making it a powerful tool for the comprehensive analysis of the phosphoproteome.

Dynamic changes of platelets before and after surgery predict the prognosis of patients with operable non-small cell lung cancer.

Purpose: To determine the prognostic significance of postoperative platelet/preoperative platelet ratio (PPR) in patients with operable non-small cell lung cancer (NSCLC), and assess its prognostic benefit compared to models relying solely preoperative platelet counts (PLT). Materials and Methods: A retrospective analysis of 403 patients who underwent radical resection of NSCLC in our institution from 2013 to 2018 was conducted to assess the prognostic significance of PLT and PPR. Progression-free survival (PFS) and overall survival (OS) were performed by the Kaplan-Meier method. Single-factor and multi-factor COX regression models were used to determine factors that affect long-term outcomes. Time-dependent ROC was used to evaluate the value of PPR in predicting the prognosis. Results: A significant association between high PLT and PPR and poor long-term patient survival outcomes was observed. The median PFS and OS of NSCLC patients with high PLT were 25 months and 29 months, which was significantly shorter than that of patients with low PLT (30 months and 33 months) (both P = 0.002). In addition, the median PFS and OS of NSCLC patients with high PPR were 18 months and 26.5 months, which was significantly shorter than that of patients with low PPR (33 months and 35 months) (both P<0.001). Univariate and Multivariate analysis using Cox regression model showed that PLT and PPR were independent factors affecting PFS and OS. Time-dependent ROC showed that the predictive capability of PLT and PPR preserved well when they were compared over time following surgery. The AUCs of PLT and PPR to predict 1-year PFS and OS, 3-year PFS and OS, 5-year PFS and OS stabilized between 0.528-0.607. PPR showed significantly higher accuracy than PLT in the prediction of 1-year and 3-year PFS and OS. Conclusions: Elevated PPR is significantly related to the adverse outcomes of patients with NSCLC. PPR can stably predict the long-term prognosis of patients, and can be used as a reliable indicator for evaluating the prognosis of patients with operable NSCLC.

Discovery of a New CDK4/6 and PI3K/AKT Multiple Kinase Inhibitor Aminoquinol for the Treatment of Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is a lethal malignancy lacking effective treatment. The Cyclin-dependent kinases 4/6 (CDK4/6) and PI3K/AKT signal pathways play pivotal roles in carcinogenesis and are promising therapeutic targets for HCC. Here we identified a new CDK4/6 and PI3K/AKT multi-kinase inhibitor for the treatment of HCC. Methods: Using a repurposing and ensemble docking methodology, we screened a library of worldwide approved drugs to identify candidate CDK4/6 inhibitors. By MTT, apoptosis, and flow cytometry analysis, we investigated the effects of candidate drug in reducing cell-viability,inducing apoptosis, and causing cell-cycle arrest. The drug combination and thermal proteomic profiling (TPP) method were used to investigate whether the candidate drug produced antagonistic effect. The in vivo anti-cancer effect was performed in BALB/C nude mice subcutaneously xenografted with Huh7 cells. Results: We demonstrated for the first time that the anti-plasmodium drug aminoquinol is a new CDK4/6 and PI3K/AKT inhibitor. Aminoquinol significantly decreased cell viability, induced apoptosis, increased the percentage of cells in G1 phase. Drug combination screening indicated that aminoquinol could produce antagonistic effect with the PI3K inhibitor LY294002. TPP analysis confirmed that aminoquinol significantly stabilized CDK4, CDK6, PI3K and AKT proteins. Finally, in vivo study in Huh7 cells xenografted nude mice demonstrated that aminoquinol exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil with the combination treatment showed the highest therapeutic effect. Conclusion: The present study indicates for the first time the discovery of a new CDK4/6 and PI3K/AKT multi-kinase inhibitor aminoquinol. It could be used alone or as a combination therapeutic strategy for the treatment of HCC.

Rapid Enzyme-Mediated Biotinylation for Cell Surface Proteome Profiling.

Cell surface is the primary site for sensing extracellular stimuli. The knowledge of the transient changes on the surfaceome upon a perturbation is very important as the initial changed proteins could be driving molecules for some phenotype. In this study, we report a fast cell surface labeling strategy based on peroxidase-mediated oxidative tyrosine coupling strategy, enabling efficient and selective cell surface labeling within seconds. With a labeling time of 1 min, 2684 proteins, including 1370 (51%) cell surface-annotated proteins (cell surface/plasma membrane/extracellular), 732 transmembrane proteins, and 81 cluster of differentiation antigens, were identified from HeLa cells. By comparison with the negative control experiment using quantitative proteomics, 500 (68%) out of the 731 significantly enriched proteins (p-value < 0.05, ≥2-fold) in positive experimental samples were cell surface-annotated proteins. Finally, this technology was applied to track the dynamic changes of the surfaceome upon insulin stimulation at two time points (5 min and 2 h) in HepG2 cells. Thirty-two proteins, including INSR, CTNNB1, TFRC, IGF2R, and SORT1, were found to be significantly regulated (p-value < 0.01, ≥1.5-fold) after insulin exposure by different mechanisms. We envision that this technique could be a powerful tool to analyze the transient changes of the surfaceome with a good time resolution and to delineate the temporal and spatial regulation of cellular signaling.

Economical and easily detectable markers of digestive tumors: platelet parameters.

Aim: This study aimed to evaluate the clinical values of platelet parameters in patients with digestive tumors. Patients & methods: A total of 974 people were classified into three groups: malignant group, patients with digestive malignant tumors; benign group, patients with benign tumors; and normal group: healthy individuals. Results: Compared with the benign and normal groups, the malignant group showed significantly increased platelet count (PLT) and plateletcrit (PCT) and significantly reduced mean platelet volume (MPV) and platelet-large cell rate (P-LCR, p < 0.001). Elevated PLT and PCT and reduced MPV and P-LCR indicated poor overall survival in patients with digestive tumors. Conclusion: PLT, PCT, MPV and P-LCR were proven to be predictive biomarkers for patients with digestive malignant tumors. Elevated PLT and PCT or decreased MPV and P-LCR indicated poor overall survival.

Facile preparation of bifunctional adsorbents for efficiently enriching N-glycopeptides and phosphopeptides.

In bottom-up strategy, specific enrichment of glycopeptides and phosphopeptides from complicated biological samples is a prerequisite for efficient identifying glycosylation and phosphorylation by mass spectrometry. Although there were a plethora of materials used as either hydrophilic interaction liquid chromatography (HILIC) or immobilized metal affinity chromatography (IMAC) adsorbents, even several bifunctional materials for simultaneous enrichment of glycopeptides and phosphopeptides, most of them are not easily commercialized as many other well-performing adsorbents due to the complicated preparation process. In our case, a one-step modification strategy was developed to prepare bifunctional adsorbents for HILIC and IMAC, employing O-phospho-l-serine as the modifier and poly(GMA-co-EDMA) microspheres, a kind of macroporous adsorption resin (MAR) with epoxy groups, as the matrix. The MARs were directly modified with O-phospho-l-serine under facile condition for HILIC strategy and further chelated with Ti4+ for IMAC strategy. A total of 522 unique N-glycopeptides and 442 unique N-glycosylation sites mapped to 275 N-glycoproteins was identified from HeLa cell proteins, showing excellent enrichment efficiency in HILIC. Additionally, 3141 unique phosphopeptides were unambiguously identified from 200 µg of digest of HeLa cell proteins, demonstrating great enrichment efficiency in IMAC. Moreover, these materials have been successfully applied in the analysis of multiple biological samples including human serum and milk, demonstrating their feasibility for real sample applications and potential business value.

[Clinical and genetic analysis of a pedigree affected with type I hereditary antithrombin deficiency due to a g.2736dupT variant of the AT gene].

OBJECTIVE: To analyze the phenotype and genotype of a patient affected with inherited antithrombin deficiency. METHODS: All exons and exon-intron boundaries of the AT genes were subjected to PCR amplification and Sanger sequencing. The influence of variants on the disease was predicted using bioinformatic software (MutationTaster). RESULTS: The results of all coagulation tests were normal, though the antithrombin activity and antigen content of the proband and his father have decreased significantly (34%, 48% and 12.97 mg/dL, 15.60 mg/dL, respectively). His mother was normal. Genetic analysis revealed that the proband and his father both carried a heterozygous g.2736dupT variant of the AT gene. Bioinformatic analysis suggested that the variant may be pathogenic. CONCLUSION: The proband and his father both had type I hereditary antithrombin deficiency caused by a g.2736dupT variant of the AT gene. The variant was unreported previously.

Targeting the EphB4 receptor tyrosine kinase sensitizes HER2-positive breast cancer cells to Lapatinib.

Clinical data analysis reveals that the expression of the EphB4 receptor tyrosine kinase is significantly elevated in HER2-positive breast cancer and high levels of EphB4 strongly correlate with poor disease prognosis. However, the impact of EphB4 activation on HER2-positive breast cancer cells and the potential of EphB4 as a therapeutic target remain to be explored. Here, we show that EphB4 overexpression confers gain-of-function activities to HER2-positive breast cancer cells, rendering resistance to a HER2/EGFR inhibitor Lapatinib. Furthermore, using integrated transcriptomic and tyrosine phosphoproteomic analyses, followed by biochemical confirmation, we establish that EphB4 activation engages the SHP2/GAB1-MEK signaling cascade and downstream c-MYC activation, and thereby limits the overall drug responses to Lapatinib. Finally, we demonstrate that, in HER2-positive breast tumors, inhibition of EphB4 combined with Lapatinib is more effective than either alone. These findings provide new insights into the signaling networks dictating therapeutic response to Lapatinib as well as a rationale for co-targeting EphB4 in HER2-positive breast cancer.

Quantitative proteomic and phosphoproteomic studies reveal novel 5-fluorouracil resistant targets in hepatocellular carcinoma.

The development of chemoresistance remains the major obstacles to successful chemotherapy of hepatocellular carcinoma. The molecular mechanisms of drug resistance are complex. Identifying the key markers is crucial for development of therapeutic strategies to overcome resistance. In this study, we employed a cell-line model consisting of the 5-fluorouracil resistant Bel/5-Fu cell line and its parental Bel cell line. Using stable isotope dimethyl labeling combined with high-resolution mass spectrometry, in total, 8272 unique proteins and 22,095 phosphorylation sites with high localization confidence were identified. Our data indicated that the GnRH signaling pathway was involved in acquiring drug resistance, which has not been well elucidated. The western blotting results confirmed that the expression levels of PLCß3 and PLCß3 pS1105 in Bel/5-Fu cells were increased as compared to Bel cells. Furthermore, the protein levels of SRC and PKCδ, which could phosphorylate PLCß3 at ser1105, were higher in Bel/5-Fu cells than in Bel cells. The knockdown of SRC, PKCδ and PLCß3 increased the susceptibility of Bel/5-Fu cells to 5-Fu. Besides, the increased transcription levels of PLCß3, PKCδ and SRC were significantly associated with decreased overall survival. Together, our deep proteomic and phosphoproteomic data reveal novel therapeutic targets for attenuating 5-Fu resistance in anti-cancer therapy. SIGNIFICANCE: It was reported that many hepatocellular carcinoma patients are resistance to 5-Fu. Although some studies related to drug resistance have been reported, the underlying mechanisms were not well elucidated. Unlike many single molecular studies, we focused on the global proteome and phosphoproteome analysis of Bel and Bel5-/Fu cell line using stable isotope dimethyl labeling to identify the previously unrecognized signaling pathway for causing 5-Fu resistance. Our results showed that the phosphorylation levels of PLCß3 pS1105 and the protein levels of PLCß3, PKCδ and SRC, which are major components of GnRH signaling pathway were higher in Bel/5-Fu cells than in Bel cells. Furthermore, knockdown of PLCß3, PKCδ and SRC increased the susceptibility of Bel/5-Fu cells to 5-Fu. Overall, this is the first comprehensive proteomic and phosphoproteomic studies on 5-Fu resistant cell line Bel/5-Fu to identify the potential targets of attenuating chemoresistance in hepatocellular carcinoma.

One-Step SH2 Superbinder-Based Approach for Sensitive Analysis of Tyrosine Phosphoproteome.

Tyrosine phosphorylation plays a major role in regulating cell signaling pathways governing diverse biological functions such as proliferation and differentiation. Systemically mapping phosphotyrosine (pTyr) sites is the key to understanding molecular mechanisms underlining pTyr-dependent signaling. Although mass spectrometry-based technologies have been widely used for pTyr site profiling and quantification, their applications are often hindered by the poor efficiency in current multistep enrichment procedures for inherently low abundance pTyr peptides, especially under physiological conditions. Taking advantage of the sequence-independent high affinity of SH2 superbinder toward pTyr residues, we have developed a simplified one-step pTyr peptide enrichment method that uses immobilized SH2 superbinder for unbiased and robust enrichment of endogenous pTyr peptides from biological samples. By eliminating the prerequisite global phosphopeptide enrichment step in our previously developed two-step method, we minimized sample loss and improved peptide capture efficiency. Applying this method to Jurkat cells at resting state, where the tyrosine phosphorylation level is low, both the number of identified pTyr peptides and sites are increased by three folds compared to the two-step method. Specifically, we were able to identify 511 nonredundant pTyr peptides, corresponding to 403 high confidence pTyr sites, from Jurkat cells with high level technical reproducibility (Pearson's correlation coefficient as high as 0.94). Further applying this method to two human breast cancer cell lines, BT474 and HCC1954, before and after EGF stimulation, we demonstrated that this approach could be a powerful tool for illustrating pTyr-dependent signaling network controlling cellular behaviors such as drug resistance.

Sensitive profiling of cell surface proteome by using an optimized biotinylation method.

Cell surface proteins are responsible for many critical functions. Systematical profiling of these proteins would provide a unique molecular fingerprint to classify cells and provide important information to guide immunotherapy. Cell surface biotinylation method is one of the effective methods for cell surface proteome profiling. However, classical workflows suffer the disadvantage of poor sensitivity. In this work, we presented an optimized protocol which enabled identification of more cell surface proteins from a smaller number of cells. When this protocol was combined with a tip based fractionation scheme, 4510 proteins, including 2055 annotated cell surface-associated proteins, were identified with only 20 microgram protein digest, showing the superior sensitivity of the approach. To enable process 10 times fewer cells, a pipet tip based protocol was developed, which led to the identification of about 600 cell surface-associated proteins. Finally, the new protocol was applied to compare the cell surface proteomes of two breast cancer cell lines, BT474 and MCF7. It was found that many cell surface-associated proteins were differentially expressed. The new protocols were demonstrated to be easy to perform, time-saving, and yielding good selectivity and high sensitivity. We expect this protocol would have broad applications in the future. SIGNIFICANCE: Cell surface proteins confer specific cellular functions and are easily accessible. They are often used as drug targets and potential biomarkers for prognostic or diagnostic purposes. Thus, efficient methods for profiling cell surface proteins are highly demanded. Cell surface biotinylation method is one of the effective methods for cell surface proteome profiling. However, classical workflows suffer the disadvantage of poor sensitivity. In this work, we presented an optimized protocol which enabled identification of more cell surface proteins from a smaller number of starting cells. The new protocol is easier to perform, time-saving and has less protein loss. By using a special pipet tip, sensitive and in-depth cell surface proteome analysis could be achieved. In combination with label-free quantitative MS, the new protocol can be applied to the differential analysis of the cell surface proteomes between different cell lines to find genetically- or drug-induced changes. We expect this protocol would have broad application in cell surface protein studies, including the discovery of diagnostic marker proteins and potential therapeutic targets.

Sol-gel preparation of titanium (IV)-immobilized hierarchically porous organosilica hybrid monoliths.

Hierarchically porous monoliths as a key feature of biological materials have been applied in enrichment and separation. In this work, a metal immobilized hierarchically porous organosilica hybrid monolith was synthesized by hydrolysis and condensation of tetraethoxysilane (TEOS) and diethoxyphosphorylethyl-triethoxysilane (DPTS) under alkaline environment. Phosphonate ester groups were firstly introduced by the employment of DPTS as functional monomer, and then acidified to phosphonic acids. The surface area of optimal monolith could reach to 1170 m2/g, which simultaneously contained micropores and mesopores (4 nm) obtained from nitrogen sorption measurement. Meanwhile, mercury intrusion porosimetry (MIP) further demonstrated that macropores (1-3 µm) existed in monoliths. Followed by chelating with titanium ion (Ti4+), the hierarchically porous organosilica hybrid monoliths could be applied as IMAC materials. This synthesized process was easy-operating and time-saving, and avoided the tedious and complex process of traditional Ti4+-IMAC materials. Furthermore, the Ti4+-IMAC monoliths exhibited high adsorption capacity for pyridoxal 5'-phosphate (82.6 mg/g). The 3282 unique phosphopeptides could be identified from 100 µg of HeLa digests after enrichment with the monolith, exhibiting excellent enrichment performance of low-abundance phosphopeptides.