A new strategy: identification of specific antibodies for neutralizing epitope on SARS-CoV-2 S protein by LC-MS/MS combined with immune repertoire.

Although the SARS-CoV-2 vaccine has been widely used worldwide, not all individuals can produce neutralization antibodies, so it is still urgent to find and prepare neutralization antibodies for COVID-19 prevention or treatment. In this study, we created a new strategy to effectively obtain neutralizing antibodies or complementary determining region 3 (CDR3) of neutralizing antibodies against SARS-CoV-2. We first predicted and synthesized several B cell epitopes on RBD and adjacent RBD of S protein, then the B cell epitopes were used to prepare affinity chromatography columns respectively and purify the binding IgG from serum samples of convalescent COVID-19 patients. After these IgGs were identified to have neutralizing activity, the peptide sequences of the antigen-binding regions (variable region) of neutralizing antibodies were analyzed by protein mass spectrometry. Subsequently, the B cells from the same individual were sorted and used to obtain their full BCR repertoire by 5' RACE combined with high-throughput of PacBio sequencing method. Then, the peptide sequence of neutralizing antibody variable region by protein mass spectrometry was mapped to the full BCR repertoire and found the full variable region sequence of neutralizing antibodies. Finally, we obtained and synthesized numerous CDR3 peptides of neutralizing antibodies to confirm the neutralizing activity for SARS-CoV-2 infection. Our results indicate that the novel scheme will be suitable for rapid screening of neutralizing antibodies, including screening neutralizing antibodies against SARS-CoV-2 and other pathogenic microorganisms.

A Preliminary Study on Change of Serum Immunoglobulin G Glycosylation in Patients With Migraine.

Background and Objective: Migraine is a common neurological disease, but its pathogenesis is still unclear. Previous studies suggested that migraine was related to immunoglobulin G (IgG). We intended to analyze the immune characteristics of migraine from the perspective of IgG glycosylation and provide theoretical assistance for exploring its pathogenesis. Methods: The differences in the serum level of IgG glycosylation and glycopeptides between patients with episodic migraine and healthy controls were analyzed by applying the poly(glycerol methacrylate)@chitosan (PGMA@CS) nanomaterial in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). We constructed a binary classification model with a feedforward neural network using PyTorch 1.6.0 in Python 3.8.3 to classify the episodic migraine and healthy control groups. Results: Twenty patients with migraine and 20 healthy controls were enrolled and the blood samples and clinical information were collected. Forty-nine IgG N-glycopeptides were detected in the serum of the subjects. The serum level of N-glycopeptide IgG1 G0-NF (p = 0.012) was increased in patients with migraine. The serum level of N-glycopeptide IgG3/4 G2FS (p = 0.041) was decreased in patients with migraine with family history of headache. It was found that the serum level of the IgG1 G1 (p = 0.004) and IgG2 G0 (p = 0.045) was increased in patients with migraine with aura, while the serum level of IgG2 G0N (p = 0.043) in patients with migraine with aura was significantly lower than that in patients with migraine without aura. In addition, a linear feedforward neural network (FFNN) was used to construct a binary classification model by detected IgG N-glycopeptides. The area under the curve (AUC) value of the binary classification model, which was constructed with 7 IgG N-glycopeptides, was 0.857, suggesting a good prediction performance. Among these IgG N-glycopeptides that were constructed the model, IgG1 G0-NF was overlapped with the differential IgG N-glycopeptide between patients with migraine and healthy controls detected with MALDI-TOF-MS. Conclusion: Our results indicated that the serum level of N-glycopeptides IgG1 G0-NF might be one of the important biomarkers for the diagnosis of migraine. To the best of our knowledge, this is the first study about the changes of IgG N-glycosylation in patients with migraine by the method of MALDI-TOF-MS. The results indicated a relationship between the migraine and immune response.

Nanoprotein Interaction Atlas Reveals the Transport Pathway of Gold Nanoparticles across Epithelium and Its Association with Wnt/ß-Catenin Signaling.

A tremendous number of proteins participate in the delivery and transport process of nanomedicines. Nanoprotein interactions not only mediate drug delivery but also determine drug safety. In the field of biomedical sciences, the epithelial barrier is a huge challenge for gastrointestinal, intratracheal, intranasal, vaginal, and intrauterine delivery of nanomedicines. However, the molecular mechanisms by which nanomedicines cross tissue or cell barriers are not well understood. Here, we explored the nanoprotein interactions during the transcytosis of nanoparticles across the epithelial barrier by focusing on the transport pathway and mechanisms. Due to the limitations of traditional methods in resolving nanoprotein interactions, we developed a backward analysis strategy. By simultaneously analyzing the protein corona on the particle surface and the cellular response after transcytosis, we integrated the information on both directly and indirectly interacting proteins, establishing a holistic nanoprotein interaction atlas. It revealed the dominant role of the EV/ER/Golgi/SV pathway in the transcytosis of nanoparticles. More importantly, based on the established atlas, we discovered the association of Wnt/ß-catenin signaling with nanoparticle transportation. The endocytosis for entering cells and exocytosis/transcytosis for leaving cells were differently regulated by the Wnt pathway. Notably, this regulatory effect was dependent on the particle size. Bigger nanoparticles departed from cells through the exocytosis pathway faster because of the specific bridging effect on the Wnt-Frizzled interaction and the feedback loop construction based on the exosomes. This mechanism gives an interpretation at the molecular level to the transcytosis dilemma of larger nanoparticles. Moreover, the size-dependent Wnt/ß-catenin signaling pathway provides a promising regulatory and screening platform for the transportation of different nanomedicines through the epithelial barrier.

A one-pot synthesis of hydrophilic poly(glycerol methacrylate) chitosan for highly selective enrichment of glycopeptides.

Poly(glycerol methacrylate) chitosan nanospheres were facilely one-pot synthesized. For the first time, poly(glycerol methacrylate) with a highly flexible density of hydrophilic molecules grafted on the surface of chitosan was applied to highly specific enrichment of glycopeptides.

Comprehensive analysis of human IgG Fc N-glycopeptides and construction of a screening model for colorectal cancer.

Currently, analyzing intact glycopeptides remains a challengeable task. Considerable progress has been achieved in the knowledge of immunoglobulin G (IgG) glycans in patients with colorectal cancer (CRC), whereas data on IgG Fc N-glycopeptides are scarce in the literature. To fill this gap in knowledge, we developed a rapid and effective method to obtain and analyze IgG Fc N-glycopeptides in the plasma from 46 CRC patients and 67 healthy individuals using chitosan@poly (glycidyl methacrylate) @iminodiacetic acid (CS@PGMA@IDA) nanomaterial in combination with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). A total of 29 N-glycopeptides were detected and analyzed. Compared with healthy individuals, CRC patients had increased levels of N-acteylglucosamine, yet decreased levels of galactosylation, fucosylation and sialylation. Further, a multivariate logistic regression model was developed using the levels of IgG Fc N-glycopeptides to distinguish CRC patients from healthy individuals, and the prediction performance was good, with an average AUC of the ROC curves of 0.893. SIGNIFICANCE: In this study, we proposed a strategy for obtaining and analyzing IgG glycopeptides using CS@PGMA@IDA nanomaterial in combination with MALDI-TOF-MS. Using this strategy, IgG Fc N-glycopeptides were analyzed in the plasma of CRC patients, and our findings indicated that glycosylation levels in the IgG Fc region were closely related to CRC. By using the IgG N-glycopeptide enrichment method and screening model designed in this study, early large-scale colorectal cancer screening can be implemented easily and fast.

Highly efficient enrichment method for human plasma glycoproteome analyses using tandem hydrophilic interaction liquid chromatography workflow.

Selective enrichment of glycopeptides from complex sample with hydrophilic interaction liquid chromatography (HILIC) method, followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites is used extensively as a sample preparation for comprehensive glycoproteome analysis. However, the coenrichment of hydrophilic nonglycosylated peptides and the released N-glycans seriously affect the identification of deglycopeptides with nano-LC-MS/MS. Here, we developed a new method for highly efficient and specific enrichment of human plasma N-glycopeptides using HILIC-PNGaseF-HILIC workflow (HPH). The first HILIC enriches the N-glycopeptides from the complex peptide mixtures. After the enriched N-glycopeptides are deglycosylated with PNGase F, the second HILIC captures the coenrichment of hydrophilic nonglycosylated peptides and the N-glycans, and then further enriches the deglycosylated peptides. The glycopeptide enrichment efficiency can be notably improved by employing HPH, evaluated by the highly recovery (more than 93.6%) and specific capturing glycopeptides from tryptic digest of IgG and BSA up to the molar ratios of 1:200. Meanwhile, we found that the alkylated proteins with IAA can affect the enrichment efficiency for N-glycopeptides with HILIC method. Moreover, after optimism the protein digestion, this novel HPH strategy allowed for the identified 722 N-glycopeptides within 202 unique glycoproteins from 1 µL human plasma digest using PNGase F in H216O. Meanwhile, this new HPH strategy identified an average 501 N-glycopeptides within averagely 134 unique glycoproteins from 1 µL human plasma digest using PNGase F in H218O. The enhanced glycopeptide detection was promoted by a substantial depletion of nonglycosylated peptides in the second HILIC. It was found that 52.2% more N-glycosylation peptides were identified by the HPH strategy compared with the using one HILIC enrichment alone.

Transferrin Functionization Elevates Transcytosis of Nanogranules across Epithelium by Triggering Polarity-Associated Transport Flow and Positive Cellular Feedback Loop.

Overcoming the epithelial barriers to enhance drug transport is a focused topic for gastrointestinal, intratracheal, intranasal, vaginal, and intrauterine delivery. Nanomedicines with targeting functionization promote such a process owing to specific ligand-receptor interaction. However, compared to the cell uptake of targeting nanotherapies, currently few studies concentrate on their transcytosis including endocytosis for "in" and exocytosis for "out". In fact, the cellular regulatory mechanism for these pathways as well as the principle of ligand's effect on the transcytosis are almost ignored. Here, we fabricated transferrin (Tf) functionalized nanogranules (Tf-NG) as the nanomedicine model and confirmed the difference in polar distributions of Tf receptors (TfRs) between two epithelium models (bipolarity for Caco-2 and unipolarity for MDCK cells). Compared to the nonspecific reference, Tf-conjugation boosted the endocytosis by different pathways in two cell models and transformed the intracellular route of Tf-NG in both cells differently, affecting exocytosis, recycling, and degradation but not the secretion pathway. Only bipolar cells could establish a complete transport flow from "in" to "out", leading to the enhanced transcytosis of Tf-NG. Importantly, epithelia could make responses to Tf-NG transcytosis. Based on the quantitative proteomics, the intracellular trafficking of Tf-NG altered the protein expression profiles, in which the endocytosis- and transcytosis-related proteins were specifically upregulated. Particularly, only bipolar cells could positively feed back to such trafficking via accelerating the subsequent Tf-NG transcytosis. Here, all the cell transport of Tf-NG was polarity associated. In summary, Tf modification elevated the transcytosis of Tf-NG across the epithelium by triggering the polarity-associated transport flow and positive cell feedback loop. These findings provided an insight into the targeting nanodelivery for efficient transport through epithelial barriers.

Single-walled carbon-nanohorns improve biocompatibility over nanotubes by triggering less protein-initiated pyroptosis and apoptosis in macrophages.

Single-walled carbon-nanohorns (SNH) exhibit huge application prospects. Notably, spherical SNH possess different morphology from conventional carbon nanotubes (CNT). However, there is a tremendous lack of studies on the nanotoxicity and mechanism of SNH, and their comparison with nanotubes. Here, the dissimilarity between SNH and CNT is found in many aspects including necrosis, pyroptosis, apoptosis, protein expression, hydrolases leakage, lysosome stress, membrane disturbance and the interaction with membrane proteins. The improved biocompatibility of SNH over four types of established CNT is clearly demonstrated in macrophages. Importantly, a key transmembrane protein, glycoprotein nonmetastatic melanoma protein B (GPNMB) is discovered to initiate the nanotoxicity. Compared to CNT, the weaker nano-GPNMB interaction in SNH group induces lower degree of cascade actions from nano/membrane interplay to final cell hypotoxicity. In conclusion, the geometry of single-construct unit, but not that of dispersive forms or intracellular levels of nanocarbons make the most difference.

Single-Step Enrichment of N-Glycopeptides and Phosphopeptides with Novel Multifunctional Ti4+-Immobilized Dendritic Polyglycerol Coated Chitosan Nanomaterials.

Protein glycosylation and phosphorylation, two of the most important post-translational modifications (PTMs) in the proteome, play a vital role in regulating a number of complex biological processes and involvement in a variety of diseases. Comprehensive characterization of the phosphoproteome and glycoproteome requires highly specific and sensitive enrichment methods of purification of phosphopeptides and glycopeptides because many glycoproteins and phosphoproteins naturally occur at low abundances and substoichiometry. Here, we reported a facile route to fabricate a novel multifunctional Ti4+-mmobilized dentritic polyglycerol CS@PGMA@IDA (CS, chitosan; PGMA, poly(glycidyl methacrylate); IDA, iminodiacetic acid) nanomaterials. The polymer surface endows the nanomaterials with biocompatibility, excellent hydrophilic property, and a large amount of Ti 4+ which have the property of immobilized metal ion affinity chromatography (IMAC)- and hydrophilic interaction liquid chromatography (HILIC)-based functional materials. The CS@PGMA@IDA-Ti4+ nanomaterials demonstrate an outstanding ability for N-glycopeptides and phosphopeptides enrichment simultaneously, evaluated by the extremely high binding capacity (150 mg g-1), sensitivity (above 0.1 fmol), and high enrichment recovery (above 75.4%). Its outstanding specificity and efficiency for purification of phosphopeptides is reflected in quantities as low as 1:5000 molar ratios of phosphopeptides which can be detected. Furthermore, we used CS@PGMA@IDA-Ti4+ to enrich for N-glycopeptides and phosphopeptides followed by PNGase F treatment, fractionated and separated N-glycopeptides and phosphopeptides with different eluents, and then analyzed by MS, a total of 423 (84.4 ID/µg, 3.525 ID/min) N-glycopeptides in 235 different glycoproteins and 422 (84.4 ID/µg, 3.517 ID/min) phosphopeptides in 256 different phosphoproteins which were finally identified in two independent LC-MS/MS runs (with a total time of 120 min) from 50 µg of mouse liver. The results demonstrated that the method based on CS@PGMA@IDA-Ti4+ to single-step enrichment of N-glycopeptides and phosphopeptides is simple, efficient, specific, and compatible to MS. It can be expected that CS@PGMA@IDA-Ti4+ would hold great applicability of modification-based proteomics to the precious and low amounts of clinical samples.

Quantitative proteomics study of the neuroprotective effects of B12 on hydrogen peroxide-induced apoptosis in SH-SY5Y cells.

B12 belongs to the coumarin class of compounds that have been shown to have various physiological and pharmacological activities including anti-inflammatory, antibacterial, and antioxidant. In the present study, we characterised the neuroprotective effects of B12 against H2O2-induced neuronal cell damage in SH-SY5Y cells. Protein expression profiling in combination with pathway analysis was deployed to investigate the molecular events associated with the neuroprotective effects in human neuronal cells using a label-free quantitative proteomics approach. A total of 22 proteins were significantly differentially expressed in H2O2-damaged cells with or without B12 treatment. Bioinformatics analysis using the Cytoscape platform indicated that poly pyrimidine tract binding protein 1 (PTBP1) was highly associated with the protective effect, and western blotting verified that PTBP1 was up-regulated in H2O2 + B12 treatment group, compared with the H2O2 treated group. PTBP RNAi experiments knocked down PTBP expression, which cancelled out the protective effect of B12 on cell viability. Thus, we infer that B12 neuroprotective activity involves up-regulation of PTBP1 and its associated signalling networks following H2O2-induced apoptosis in SH-SY5Y cells. B12 or related compounds may prove to be useful therapeutic agents for the treatment of neurodegenerative diseases such as Alzheimer's and Parkinson's.

A facile and cheap synthesis of zwitterion coatings of the CS@PGMA@IDA nanomaterial for highly specific enrichment of glycopeptides.

CS@PGMA@IDA nanomaterials were facilely synthesized, the zwitterion polymer surface PGMA@IDA endows the nanomaterial with biocompatibility, excellent hydrophilic properties and a large amount of functional groups on the polymer chains that can selectively bind to glycopeptides based on hydrophilic interaction.

Enhanced production of secretory glycoprotein VSTM1-v2 with mouse IgGκ signal peptide in optimized HEK293F transient transfection.

VSTM1-v2 is a secretory glycoprotein identified by our laboratory. Our previous study revealed that VSTM1-v2 could promote differentiation and activation of Th17 cells. To explore the role of VSTM1-v2 in the immune system further, a source of abundant high-quality recombinant protein is warranted. However, high-level expression of bioactive VSTM1-v2 is difficult due to its weak secretion capacity. To obtain sufficient recombinant VSTM1-v2, we developed an improved expression and purification system by replacing the native signal peptide with a mouse IgGκ signal peptide that did not alter the protein cleavage site. We also optimized parameters for a transient gene expression system in HEK293F cells suspended in serum-free media with polyethyleneimine. Finally, 3.6 mg/L recombinant VSTM1-v2 protein with N-glycosylation and no less than 95% purity was obtained through one-step purification with Ni affinity chromatography. The final yield after purification was increased by more than 7-fold compared to the yield from our previously reported HEK293T system (from 0.5 mg/L to 3.6 mg/L). More importantly, VSTM1-v2 protein exhibited excellent bioactivity. In conclusion, the improved system is not only a dependable source of abundant bioactive VSTM1-v2 for functional studies but also demonstrates a highly efficient approach for enhancing the production of proteins in a short time period, especially for secretory proteins with poor yields.

Proteomics and bioinformatics analysis of mouse hypothalamic neurogenesis with or without EPHX2 gene deletion.

The aim of this study was to identify differently expressed proteins in the presence and absence of EPHX2 gene in mouse hypothalamus using proteomics profiling and bioinformatics analysis. This study was performed on 3 wild type (WT) and 3 EPHX2 gene global knockout (KO) mice (EPHX2(-/-)). Using the nano- electrospray ionization (ESI)-LC-MS/MS detector, we identified 31 over-expressed proteins in WT mouse hypothalamus compared to the KO counterparts. Gene Ontology (GO) annotation in terms of the protein-protein interaction network indicated that cellular metabolic process, protein metabolic process, signaling transduction and protein post-translation biological processes involved in EPHX2(-/-) regulatory network. In addition, signaling pathway enrichment analysis also highlighted chronic neurodegenerative diseases and some other signaling pathways, such as TGF-beta signaling pathway, T cell receptor signaling pathway, ErbB signaling pathway, Neurotrophin signaling pathway and MAPK signaling pathway, were strongly coupled with EPHX2 gene knockout. Further studies into the molecular functions of EPHX2 gene in hypothalamus will help to provide new perspective in neurogenesis.

PTENα, a PTEN isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism.

PTEN is one of the most frequently mutated genes in human cancer. It is known that PTEN has a wide range of biological functions beyond tumor suppression. Here, we report that PTENα, an N-terminally extended form of PTEN, functions in mitochondrial metabolism. Translation of PTENα is initiated from a CUG codon upstream of and in-frame with the coding region of canonical PTEN. Eukaryotic translation initiation factor 2A (eIF2A) controls PTENα translation, which requires a CUG-centered palindromic motif. We show that PTENα induces cytochrome c oxidase activity and ATP production in mitochondria. TALEN-mediated somatic deletion of PTENα impairs mitochondrial respiratory chain function. PTENα interacts with canonical PTEN to increase PINK1 protein levels and promote energy production. Our studies demonstrate the importance of eIF2A-mediated alternative translation for generation of protein diversity in eukaryotic systems and provide insights into the mechanism by which the PTEN family is involved in multiple cellular processes.

Synthesis and characterization of a novel boronic acid-functionalized chitosan polymeric nanosphere for highly specific enrichment of glycopeptides.

In this study we describe a method for highly specific enrichment of glycopeptides with boronic acid-functionalized chitosan polymeric nanospheres and matrix assisted laser desorption-ionization mass spectrometry (MALDI-MS). This is the first time chitosan has been used to create nanosphere support material for selective enrichment of glycopeptides by modification with glycidyl methacrylate (GMA) and derivatization with 3-aminophenylboronic acid (APB). Due to their multifunctional chemical moieties, these 20-100 nm chitosan-GMA-APB nanospheres have unique properties, such as good dispersibility, good biocompatibility and chemical stability, as well as augmented specificity with glycopeptides. Enrichment conditions were optimized by using trypsin digested glycoprotein horseradish peroxidase. The high specificity of chitosan-GMA-APB nanospheres was demonstrated by effectively enriching glycopeptides from a digest mixture of horseradish peroxidase and nonglycoproteins (bovine serum albumin (BSA)).

Highly specific capture and direct MALDI-MS analysis of phosphorylated peptides using novel multifunctional chitosan-GMA-IDA-Fe (III) nanosphere.

In this study, we describe a method for highly specific enrichment of phosphopeptides with multifunctional chitosan-glycidyl methacrylate (GMA)-iminodiacetic acid (IDA)-Fe (III) nanospheres for direct analysis by matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS). This is the first time that chitosan has been used to create nanospheres support material for selective enrichment of phosphopeptides by modification with GMA, derivatization with IDA, and loading with Fe (III) ions. Chitosan-GMA-IDA-Fe (III) nanospheres with a diameter of 20 to 100 nm have multifunctional chemical moieties which confer unique properties, good dispersibility in highly acidic binding buffers, as well as good biocompatibility and chemical stability which improves their specific interaction with phosphopeptides using various types of acid binding buffers. The process of enrichment is very simple, quick, efficient, and specific. Its high specificity and efficiency for purification of phosphopeptides is reflected in the very low and substoichiometric amounts of phosphopeptides which can be detected, in quantities as low as 1:3,000 M ratios. Compared with other state-of the-art technologies such as the use of conventional Fe(3+)-IMAC and TiO(2), these chitosan nanosphere techniques show superior specificity and sensitivity. Moreover, the resultant chitosan-GMA-IDA-Fe(3+) nanosphere-absorbed phosphopeptides can be either directly analyzed by MALDI-TOF MS analysis or eluted and further analyzed by nano-LC-MS/MS.

Novel multifunctional chitosan-GMA-IDA-Cu(II) nanospheres for high dynamic range characterization of the human plasma proteome.

In this study, we describe characterization of the human plasma proteome based on analysis with multifunctional chitosan-GMA-IDA-Cu(II) nanospheres. Chitosan-GMA-IDA-Cu(II) nanospheres with diameters of 20 to 100 nm have unique properties due to multifunctional chemical moieties, high surface area, high capacity, good dispersibility in buffer solution as well as good biocompatibility and chemical stability which improves their specific interaction with peptides and proteins of the human plasma using different binding buffers. Combining these chitosan-GMA-IDA-Cu(II) nanospheres with MS spectrometry results in a novel strategy which should make it possible to characterize the plasma proteome in a single test. Peptides and proteins adsorbed on the nanosphere can be directly detected by MALDI-TOF-MS. The eluted lower molecular weight peptides and proteins are identified by nano-LC-ESI-MS/MS. A total of 842 unique LMW peptides and 1,682 human unredundant proteins IDs were identified in two different binding buffers, which included relatively low-level proteins (e.g., pg/mL of IL3 Interleukin-3) co-distributed with high-abundance proteins (e.g., 35-55 mg/mL level serum albumin). As such, this nanosphere technique selectively enabled the identification of proteins over a dynamic range of greater than 8 orders of magnitude. Considering this capacity for selective enrichment of peptides and proteins in human plasma, and the large number of LMW peptides and proteins which can be identified, this method promises to accelerate discovery of biomarkers for clinical application.

[Two types of MWNTs with different surface modifications induce differential expression of proteins in RAW264.7 cells].

OBJECTIVE: To compare the different expression of protein in RAW264.7 macrophage cells induced by two types of MWNTs (multi-walled carbon nanotubes) with different surface modifications (acid-treated MWNTs and tau-MWNTs modified by taurine). METHODS: Treating cells with both types of MWNTs in 20 mg/L and 24 h, with a blank-control group set. Cells are lysed by using urea and by ultrasonicating in ice bath, then total proteins of cells are extracted. Using two-dimensional gel electrophoresis to separate total proteins of cells, searching for the differential expressed protein spots on the images of the gels with silver staining. Identifying the differentially expressed proteins via mass spectrometry, and studying the mechanism of effects on cells imposed by two types of MWNTs at protein level. RESULTS: There are 13 spots of protein with notably differential expression among three treated groups (including blank controls). Their functions involve apoptosis-related, calcium-binding, cell-cycle related, DNA synthesis, folding of proteins, and energy metabolism, etc. The results are consistent with our previous studies about the cytotoxicity of Both types of MWNTs including induction of apoptosis and mitochodira damage. CONCLUSION: Both two types of MWNTs could induce alteration of protein expression in RAW264.7 cells. With different surface modifications, they imposed different effects. High throughout proteomics could be applied in toxicity assessment and mechanism investigation about carbon nanotubes.

Ultra-fast mass fingerprinting by high-affinity capture of peptides and proteins on derivatized poly(glycidyl methacrylate/divinylbenzene) for the analysis of serum and cell lysates.

The development of support materials in mass fingerprinting is an important task required for diagnostic markers in conjunction with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The material-based approach, which we introduced as material-enhanced laser desorption/ionization (MELDI), focuses not only on different functionalities, but also emphasizes the morphology, i.e. porosity or particle size of the carrier material. As a result, it provides a quick and sensitive platform for effective binding of peptides and proteins out of different biofluids, e.g. serum, spinal fluid, urine or cell lysates, and to subsequently analyze them with MALDI-TOF MS. This approach includes a built-in desalting step for serum protein profiling and is sensitive enough to detect proteins and peptides down to 100 fmol/microL. Here we co-polymerized glycidyl methacrylate (GMA) with divinylbenzene (DVB) using thermal polymerization to yield a GMA/DVB polymer for further modifications. Different affinities have been created, such as immobilized metal ion affinity (IDA-Cu2+), reversed-phase (RP) and anion-exchanger (AX) chromatography. The diverse derivatizations and the dispersity of the particles created by different chemical synthetic approaches were confirmed by characteristic infrared (IR) peaks. The polymerization carried out by non-stirring yielded an average pore radius of 6.1 microm (macro-pores) that enhanced the binding capacity enormously by offering enlarged surface areas. Moreover, atomic absorption spectrometry (AAS) provided the metal content loaded on iminodiacetic acid (IDA) in the case of poly(GMA/DVB)-IDA-Cu2+. To summarize, the optimized MELDI approach is sensitive in its performance, extremely fast and can be adapted to robotic systems for routine analysis, allowing sample preparation in less than 5 min in contrast to the conventional surface-enhanced laser desorption/ionization (SELDI) methods.

Probing the subtle conformational state of N138ND2-Q106O hydrogen bonding deletion mutant (Asn138Asp) of staphylococcal nuclease using time of flight mass spectrometry with limited proteolysis.

Recent studies indicate that the N138ND2-Q106O hydrogen bonding deletion in staphylococcal nuclease significantly alters the conformational integrity and stability of the nuclease. To find out the structural basis of the changes, mass spectrometry and limited proteolysis methods were combined to probe the subtle conformational changes in the SNaseN138D mutant and SNaseN138D-Ca2+-pdTp complex. The results reveal that the N138ND2-Q106O hydrogen bonding deletion makes the C-terminal part of alpha-helix 1 and alpha-helix 2 in the C-terminal subdomain of SNaseN138D unfold to some extent, but does not have much effect on the N-terminal part of alpha-helix 1, alpha-helix 3, and the N-terminal beta-barrel subdomain of SNaseN138D. Binding of ligands makes the alpha-helices 1 and 2 more resistant to protease Glu-C attack and converts the partially unfolded state to a native-like state. This study also demonstrates how mass spectrometry can be combined with limited proteolysis to observe conformational changes induced by ligand binding.