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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 (Cd

To analyze the phenotype and genotype of two Chinese family with inherited dysfibrinogenemia and the molecular pathogenic mechanism.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.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.1115TA (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.1115TA 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.Inherited dysfibrinogenemia of the probands maybe caused by variant of FGB c.1115 TA (p.Val372Glu), and the variant was firstly reported.

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 Ti

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.

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.

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.

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.

To analyze the phenotype and genotype of a patient affected with inherited antithrombin deficiency.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).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.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.

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.

Human serum is a complex body fluid that contains various N-linked and O-linked glycoproteins. Compared with N-linked glycoproteins, the serum O-linked glycoproteins are not well-studied due to their high heterogeneity and their low abundance. Herein, we presented a novel chemoenzymatic method to analyze core-1 type of O-GalNAcylation in human serum. In this approach, the tryptic digest of serum was first subjected to PNGase F treatment to release the N-glycan and was then treated with strong acid to release sialic acid residues from mucin-type O-glycans. In this way, the internal Gal/GalNAc residues were exposed and were oxidized by the galactose oxidase to carry the aldehyde groups. The oxidized O-GalNAcylated peptides were then captured by hydrazide beads and eluted with methoxylamine for LC-MS/MS analysis. The de-N-deglycosylation decreased the abundance of N-glycopeptides, the desialylation simplified the O-glycans and the enzymatic oxidization conferred the enrichment specificity. We have demonstrated that this method was fitted to analyze O-GalNAcylated peptides with high confidence. This method was applied to analyze human serum, which resulted in the identification of 59 O-GalNAc modified peptide sequences corresponding to 38 glycoproteins from 50 μL of serum. This method is expected to have broad applications in the analysis of O-glycoproteome.

Multisite phosphorylation of a protein, generally occurring in biological processes, plays important roles in the regulation of cellular functions. However, the identification of multi-phosphopeptides especially at low abundance is a big challenge as the extreme hydrophilicity and poor ionization efficiency of the multiphosphorylated peptides restrict the deep inspection of multisite phosphorylation processes. In this study, the highly specific enrichment of multiphosphorylated peptides was achieved via the modification of the dual-metal-centered zirconium-organic framework with the diphosphorylated fructose. The diphosphorylated fructose-modified dual-metal-centered zirconium-organic framework (DZMOF-FDP) demonstrated the highly specific affinity to the multiple phosphorylated peptides, with the density functional theory calculations explaining the plausible mechanism for multi-phosphopeptides on the DZMOF-FDP. The selective capture of multi-phosphopeptides from mimic samples confirmed the superior performance of the DZMOF-FDP, with comprehensive comparisons to other modification agents, such as orthophosphate and pyrophosphate. A number of 1871 multiphosphorylated peptides captured by DZMOF-FDP from tryptic digests of HeLa cell lysate could be identified, significantly higher than that by the pristine DZMOF. The deliberately designed modification with diphosphorylated fructose for the dual-zirconium-centered metal-organic framework materials suggests an efficient strategy to develop new enrichment methods in the selective capture of target analytes by judiciously optimizing specific modifiers for adsorbents.

With the advance of mass spectrometry based proteomics techniques, thousands of phosphorylation sites can be identified from a single cell line or a single tissue sample by large scale global analysis. However, such large scale analysis is time consuming. And, due to the high complexity of the phosphoproteome sample, this approach is often unable to identify low abundance regulatory phosphorylation sites. Alternatively, the “divide and conquer” strategy by targeted analysis of a subset of phosphoproteome, i.e. subphosphoproteome, can improve the detection sensitivity of these phosphorylation sites. In this review, we summarized the analytical methods in this field. The enrichment approaches for the analysis of subphosphoproteomes generated by tyrosine kinases and basophilic kinases were introduced. The strategies for the analysis of the subphosphoproteomes composed by endogenous phosphopeptides, multiply phosphorylated peptides, etc., were also reviewed.

Selective enrichment and sensitive detection of phosphopeptides are of great significance in many bioapplications. In this work, dendritic mesoporous silica nanoparticles modified with polydopamine and chelated Ti4+ (denoted DMSNs@PDA-Ti4+) were developed to improve the enrichment selectivity of phosphopeptides. The unique central-radial pore structures endowed DMSNs@PDA-Ti4+ with a high surface area (362 m2 g–1), a large pore volume (1.37 cm3 g–1), and a high amount of chelated Ti4+ (75 μg mg–1). Compared with conventional mesoporous silica-based materials with the same functionalization (denoted mSiO2@PDA-Ti4+) and commercial TiO2, DMSNs@PDA-Ti4+ showed better selectivity and a lower detection limit (0.2 fmol/μL). Moreover, 2422 unique phosphopeptides were identified from HeLa cell extracts with a high specificity (>95%) enabled by DMSNs@PDA-Ti4+, better than those in previous reports.

A facile approach was developed to prepare hybrid monoliths with different functions via two-step photo-initiated reactions. Firstly, acrylopropyl polyhedral oligomertic silsesquioxane (acryl-POSS) and propargyl acrylate (PA) were used as precursors to synthesize alkynyl-functionalized hybrid monoliths via photo-initiated free radical polymerization. Secondly, the hybrid monoliths were modified with 1-octadecanethiol (ODT) and sodium 3-mercapto-1-propanesulfonate (SMPS) via photo-initiated thiol-yne click reaction to prepare reversed-phase (RP) and strong cation-exchange (SCX) hybrid monoliths, respectively. The results of chromatographic characterization indicated that the column efficiencies for alkylbenzenes on ODT-modified hybrid monolith reached 84,000-87,700 plates per meter at the velocity of 0.58mm/s, and also revealed a retention-independent efficient performance of small molecules in isocratic elution. The SMPS-modified hybrid monolith exhibited both hydrophobicity and ion-exchange mechanisms, and the dynamic binding capacity was calculated to be 1.4×10-4μmol/cm. Human Hela cells tryptic digest was well separated on ODT-modified hybrid monolith in one-dimensional RPLC-MS/MS, and 2786 unique peptides and 685 proteins were identified. Furthermore, the SMPS-modified monolith coupled with ODT-modified monolith was used for two-dimensional separation of human Hela cells tryptic digest in SCX-RPLC-MS/MS, and the results showed that 9744 unique peptides and 2749 proteins were identified. Compared to those identified in one-dimensional RP system, the total numbers of unique peptides and proteins identified in SCX-RP system increased by 249.7% and 301.3%, respectively.

An open tubular (OT) column (25 μm i.d.) was prepared by in situ ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxanes (POSS-epoxy) with 4-aminophenyl disulfide (APDS) in a binary porogenic system of ethanol/H2O. It was found that porogenic composition played an important role in the formation of OT stationary phases. The ratio of ethanol/H2O at 6/1 (v/v) would lead to the fabrication of hybrid monoliths, while the ratio of ethanol/H2O at 13/1 (v/v) would result in the synthesis of OT phases. In addition, the effects of precursor content and reaction duration on the thickness of OT stationary phases were investigated. Either lower precursor content or shorter reaction duration would produce thinner layer of OT column. The repeatability of OT columns was evaluated through relative standard deviation (RSD%) with benzene as the analyte. The run-to-run, column-to-column and batch-to-batch repeatabilities were 1.7%, 4.8% and 5.6%, respectively, exhibiting satisfactory repeatability of the OT column. Then tryptic digest of mouse liver proteins was used to evaluate the performance of the resulting OT columns (25 μm i.d. × 2.5 m in length) by cLC-MS/MS analysis, demonstrating their potential in proteome analysis.

In this study, we developed a centrifugation assisted solid phase extraction (SPE) method for the selective enrichment of phosphopeptides using a new Ti4+-IMAC material synthesized in-house. This new material has the feature of big size and large specific surface area which makes it more suitable to enrich phosphopeptides in a SPE way. The spin tips loaded with the Ti4+-IMAC material were applied to enrich phosphopeptides from the complex protein digests. It was found that phosphopeptides can be specifically enriched from tryptic digest of bovine serum albumin and β-casein at a molar ratio up to 1000:1. And about 4700 unique phosphorylated peptides can be identified with the specificity as high as 99% from the tryptic digest of HeLa cell proteins. This tip was demonstrated to have good column-to-column reproducibility. Furthermore, it is fitted to analyze minute amount of sample. Compared with the conventional solution method, the SPE method facilitated the rapid and complete separation of the material with solution, which making it a time-saving and convenient method for phosphopeptide enrichment. Compared with the commercial TiO2 material, this new materials yielded much more phosphopeptide identifications and much higher enrichment specificity.

Hierarchically porous materials have become a key feature of biological materials and have been widely applied for adsorption or catalysis. Herein, we presented a new approach to directly prepare a phosphate-functionalized hierarchically porous hybrid monolith (HPHM), which simultaneously contained mesopores and macropores. The design was based on the copolymerization of polyhedral oligomeric vinylsilsesquioxanes (vinylPOSS) and vinylphosphonic acid (VPA) by adding degradable polycaprolactone (PCL) additive. The phosphate groups could be directly introduced into the hybrid monoliths. This approach was simple and time-saving, and overcame the defect of a rigorous, complex process for preparing traditional Ti4+-immobilized metal ion affinity chromatography (IMAC) materials. The specific surface area of an optimal hybrid monolith could reach 502 m2/g obtained by nitrogen adsorption/desorption measurements, which originated from the degradation of PCL. Meanwhile, the characterization of scanning electron micr...

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.

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.

Selective enrichment and sensitive detection of phosphopeptides are of great significance in many bioapplications. In this work, dendritic mesoporous silica nanoparticles modified with polydopamine and chelated Ti

In this study, we presented an enrichment-free approach for the sensitive analysis of protein phosphorylation in minute amounts of samples, such as purified protein complexes. This method takes advantage of the high sensitivity of parallel reaction monitoring (PRM). Specifically, low confident phosphopeptides identified from the data-dependent acquisition (DDA) data set were used to build a pseudotargeted list for PRM analysis to allow the identification of additional phosphopeptides with high confidence. The development of this targeted approach is very easy as the same sample and the same LC-system were used for the discovery and the targeted analysis phases. No sample fractionation or enrichment was required for the discovery phase which allowed this method to analyze minute amount of sample. We applied this pseudotargeted MS method to quantitatively examine phosphopeptides in affinity purified endogenous Shc1 protein complexes at four temporal stages of EGF signaling and identified 82 phospho-sites. To our knowledge, this is the highest number of phospho-sites identified from the protein complexes. This pseudotargeted MS method is highly sensitive in the identification of low abundance phosphopeptides and could be a powerful tool to study phosphorylation-regulated assembly of protein complex.

Selective enrichment of glycopeptides from complex sample followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites has become the popular protocol for comprehensive glycoproteome analysis. On account of the high enrichment specificity, hydrazide chemistry based solid-phase extraction of N-linked glycopeptides technique has sparked numerous interests. However, the enzymatic release of glycopeptides captured by hydrazide beads through direct incubation of the beads with PNGase F is not efficient due to the inherent steric hindrance effect. In this study, we developed a hydroxylamine assisted PNGase F deglycosylation (HAPD) method using the hydroxylamine to release glycopeptides captured on the hydrazide beads through the cleavage of hydrazone bonds by transamination followed with the PNGase F deglycosylation of the released glycopeptides in the free solution. Because of the homogeneous condition for the deglycosylation, the recovery of deglycosylated peptides (deglycopeptides) was improved significantly. It was found that 27% more N-glycosylation sites were identified by the HAPD strategy compared with the conventional method. Moreover, the ratio of identified N-terminal glycosylated peptides was improved over 5-fold.

In this study, we present a method to specifically capture phosphotyrosine (pTyr) peptides from minute amount of sample for the sensitive analysis of protein tyrosine phosphorylation. We immobilized SH2 superbinder on a monolithic capillary column to construct a microreactor to enrich pTyr peptides. It was found that the synthetic pTyr peptide could be specifically enriched by the microreactor from the peptide mixture prepared by spiking of the synthetic pTyr peptide into the tryptic digests of α-casein and β-casein with molar ratios of 1:1000:1000. The microreactor was further applied to enrich pTyr peptides from pervanadate-treated HeLa cell digests for phosphoproteomics analysis, which resulted in the identification of 796 unique pTyr sites. In contrast, the conventional SH2 superbinder-based method identified 41 pTyr sites for the same sample, only 5.2% of the number achieved by the microreactor. Finally, this microreactor was also applied to analyze the pTyr in Shc1 complex, an immunopurified protein complex, which resulted in the identification of 15 pTyr sites. Together, this technique is best fitted to analyze the pTyr in minute amount of sample and will have broad application in fields where only a limited amount of sample is available.

Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered as a hallmark in cellular signal transduction. However, its analysis at the proteome level remains challenging. The conventional immunopurification (IP) approach using antibodies specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. Our recent study indicated that SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we presented an efficient SH2 superbinder based workflow for the sensitive analysis of tyrosine phosphoproteome. This new method can identify 41% more pTyr peptides than the previous method. Its excellent performance was demonstrated by the analysis of a variety of different samples. For the highly tyrosine phosphorylated sample, for example, pervanadate-treated Jurkat T cells, it identified over 1800 high confident pTyr sites from only 2 mg of proteins. For the unstimulated Jurkat cells, where the pTyr events rarely occurred, it identified 343 high confident pTyr sites from 5 mg of proteins, which was 31% more than that obtained by the antibody-based method. For the heterogeneous sample of tissue, it identified 197 high confident pTyr sites from 5 mg protein digest of mouse skeletal muscle. In general, it is a sensitive, robust and cost-effective approach and would have wide applications in the study of the regulatory role of tyrosine phosphorylation in diverse physiological and pathological processes.

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 PLCβ3 and PLCβ3 pS1105 in Bel/5-Fu cells were increased as compared to Bel cells. Furthermore, the protein levels of SRC and PKCδ, which could phosphorylate PLCβ3 at ser1105, were higher in Bel/5-Fu cells than in Bel cells. The knockdown of SRC, PKCδ and PLCβ3 increased the susceptibility of Bel/5-Fu cells to 5-Fu. Besides, the increased transcription levels of PLCβ3, 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 PLCβ3 pS1105 and the protein levels of PLCβ3, 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 PLCβ3, 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.

A simple approach was developed for rapid preparation of polymeric monolithic columns in UV-transparent fused-silica capillaries via photoinitiated thiol-acrylate polymerization of pentaerythritol diacrylate monostearate (PEDAS) and trimethylolpropane tris(3-mercaptopropionate) (TPTM) within 10min, in which the acrylate homopolymerized and copolymerized with the thiol simultaneously. The morphology, permeability and chromatographic performance of the resulting poly(PEDAS-co-TPTM) monoliths were studied. It could be observed from SEM that the morphology of poly(PEDAS-co-TPTM) monolith was rather different from that of poly(PEDAS) monolith, which was fabricated via photo-induced free radical polymerization using PEDAS as the sole monomer. Compared with poly(PEDAS) monolith, poly(PEDAS-co-TPTM) monolith possessed better permeability when they were fabricated under the same preparation conditions. By adjusting the composition of porogenic solvents, poly(PEDAS-co-TPTM) monolith exhibited lower plate heights (15.7-17.7μm) than poly(PEDAS) monolith (19.1-37.9μm) in μLC. In addition, 66 unique peptides were positively identified on poly(PEDAS-co-TPTM) monolith when tryptic digest of four proteins was separated by μLC-MS/MS, demonstrating its potential in proteome analysis.

Enrichment of glycopeptides by hydrazide chemistry (HC) is a popular method for glycoproteomics analysis. However, possible side reactions of peptide backbones during the glycan oxidation in this method have not been comprehensively studied. Here, we developed a proteomics approach to locate such side reactions and found several types of the side reactions that could seriously compromise the performance of glycoproteomics analysis. Particularly, the HC method failed to identify N-terminal Ser/Thr glycopeptides because the oxidation of vicinal amino alcohol on these peptides generates aldehyde groups and after they are covalently coupled to HC beads, these peptides cannot be released by PNGase F for identification. To overcome this drawback, we apply a peptide N-terminal protection strategy in which primary amine groups on peptides are chemically blocked via dimethyl labeling, thus the vicinal amino alcohols on peptide N-termini are eliminated. Our results showed that this strategy successfully prevented the oxidation of peptide N-termini and significantly improved the coverage of glycoproteome.

A method for the determination of thiocyanate in dairy products by headspace gas chromatography was established. At first, the thiocyanate in dairy products was extracted by water. Then, the zinc acetate solution was added to the crude product for protein precipitation. The extract obtained above was centrifuged and the supernatant was added with chloramine T, which derivatized the thiocyanate ions to cyanogen chloride. The head-space vapor of the final extract was injected into a BP10 (14% cyanopropyl phenyl polysiloxane) gas chromatographic column, and detected by an electron capture detector (ECD). The target compound was quantified by external standard. The results showed that there was a good linearity between 0.005 mg/L and 0.1 mg/L with the correlation coefficient (r) of 0.997, and the limit of detection (signal-to-noise ratio (S/N)or = 10) was 0.1 mg/kg. The recoveries were 90.0%-110.0% with the relative standard deviations (RSDs) (n = 10) of 4.98%-7.89% at the three spiked levels of 1.0, 2.0, 10.0 mg/kg. In conclusion, this method is simple, rapid and accurate. It can be applied in the determination of thiocyanate in dairy products, and meets the requirements of the daily testing. The method has been successfully used to test 18 kinds of commercially available dairy products and it was found that all the dairy products tested contained thiocyanate about 0.5-10 mg/kg

In the past decade, with the introduction of multi-layer low-temperature co-fired ceramic (LTCC) technology, terahertz(THz) filters embedded in multi-layer LTCC substrates, are shown to be promising in many applications of the microwave communication domain. This paper proposes two sub-millimeter wave filters and one THz band-pass filter designs based on waveguides and elements. They are rectangular bandpass filters featuring coupling cavity, metal posts and the combination of both metal posts and coupling cavity respectively. The simulation results of the three bandpass filters show that, these filters which innovatively make uses of rectangular waveguide structures embedded in multi-layer LTCC substrate, metal posts and coupling cavities, may meet the designing target and may function well in sub-millimeter wave or THz bands.