Yejing Weng

Gold nanoparticles immobilized hydrophilic monoliths with variable functional modification for highly selective enrichment and on-line deglycosylation of glycopeptides

The poly (glycidyl methacrylate-co-poly (ethylene glycol) diacrylate) monoliths modified with gold nanoparticles, with advantages of enhanced reactive sites, good hydrophilicity and facile modification, were prepared as the matrix, followed by variable functionalization with cysteine and PNGase F for glycopeptide enrichment and on-line deglycosylation respectively. By the cysteine functionalized monolithic column, glycopeptides could be efficiently and selectively enriched with good reproducibility based on hydrophilic interaction chromatography (HILIC). Furthermore, the enrichment was specially achieved in weak alkaline environment, with 10 mM NH4HCO3 as the elution buffer, compatible with deglycosylation conditions. Therefore, the glycopeptides could be on-line deglycosylated with high efficiency and throughput by directly coupling the PNGase F functionalized monolithic column with the enrichment column during elution without the requirement of buffer exchange and pH adjustment. By such a method, within only 70-min pretreatment, 196 N-linked glycopeptides, corresponding to 122 glycoproteins, could be identified from 5 μg of human plasma with 14 high-abundant proteins removed, and the N-linked glycopeptides occupied 81% of all identified peptides, achieving to the best of our knowledge, the highest selectivity of HILIC-based methods. All the results demonstrated the high efficiency, selectivity and throughput of our proposed strategy for the large scale glycoproteome analysis.

An integrated sample pretreatment platform for quantitative N-glycoproteome analysis with combination of on-line glycopeptide enrichment, deglycosylation and dimethyl labeling

Relative quantification of N-glycoproteomes shows great promise for the discovery of candidate biomarkers and therapeutic targets. The traditional protocol for quantitative analysis of glycoproteomes is usually off-line performed, and suffers from long sample preparation time, and the risk of sample loss or contamination due to manual manipulation. In this study, a novel integrated sample preparation platform for quantitative N-glycoproteome analysis was established, with combination of online N-glycopeptide capture by a HILIC column, sample buffer exchange by a N2-assisted HILIC-RPLC interface, deglycosylation by a hydrophilic PNGase F immobilized enzymatic reactor (hIMER) and solid dimethyl labeling on a C18 precolumn. To evaluate the performance of such a platform, two equal aliquots of immunoglobulin G (IgG) digests were sequentially pretreated, followed by MALDI-TOF MS analysis. The signal intensity ratio of heavy/light (H/L) labeled deglycosylated peptides with the equal aliquots was 1.00 (RSD=6.2%, n=3), much better than those obtained by the offline protocol, with H/L ratio as 0.76 (RSD=11.6%, n=3). Additionally, the total on-line sample preparation time was greatly shortened to 160 min, much faster than that of offline approach (24h). Furthermore, such an integrated pretreatment platform was successfully applied to analyze the two kinds of hepatocarcinoma ascites syngeneic cell lines with high (Hca-F) and low (Hca-P) lymph node metastasis rates. For H/L labeled Hca-P lysates with the equal aliquots, 99.6% of log2 ratios (H/L) of quantified glycopeptides ranged from -1 to 1, demonstrating high accuracy of the developed sample preparation strategy. By triplicated analysis of glycopeptides and non-glycopeptides of Hca-F and Hca-P lysates, 43 up-regulated and 30 down-regulated (Hca-F/P) N-glycosylation sites, and 11 significantly changed N-glycoproteins were successfully quantified, and most of them were related to tumorigenesis and tumor metastasis. All these results demonstrate the developed integrated N-glycoprotein pretreatment platform is of great power for the accurate, precise and high-throughput analysis of N-glycoproteomes.

Releasing N-glycan from Peptide N-terminus by N-terminal Succinylation Assisted Enzymatic Deglycosylation

Due to the important roles of N-glycoproteins in various biological processes, the global N-glycoproteome analysis has been paid much attention. However, by current strategies for N-glycoproteome profiling, peptides with glycosylated Asn at N-terminus (PGANs), generated by protease digestion, could hardly be identified, due to the poor deglycosylation capacity by enzymes. However, theoretically, PGANs occupy 10% of N-glycopeptides in the typical tryptic digests. Therefore, in this study, we developed a novel strategy to identify PGANs by releasing N-glycans through the N-terminal site-selective succinylation assisted enzymatic deglycosylation. The obtained PGANs information is beneficial to not only achieve the deep coverage analysis of glycoproteomes, but also discover the new biological functions of such modification.

Quantitative secretomic analysis of pancreatic cancer cells in serum-containing conditioned medium

Pancreatic cancer is a highly metastatic and chemo-resistant disease. Secreted proteins involved in cell-cell interactions play an important role in changing the tumor microenvironment. Previous studies generally focus on the secretome of cancer cell line from serum-free media, due to the serious interference of fetal bovine serum (FBS). However, serum-starvation may alter expression patterns of secreted proteins. Hence, efforts to decrease the interference of serum in proteomic analysis of serum-containing media have been hampered to quantitatively measure the tumor secretion levels. Recently, the metabolic labeling, protein equalization, protein fractionation and filter-aided sample preparation (FASP) strategy (MLEFF) has been successfully used to avoid the disturbance of serum on secretome analysis. Here, this efficient method was applied for comparative secretome analysis of two hamster pancreatic cancer cells with differentially metastatic potentials, enabling the observation of 161 differentially expressed proteins, including 106 proteins that had been previously reported and detected in plasma. By integrated analysis of our data and publicly available bioinformatics resources, we found that a combination panel consisting of CDH3, PLAU, and LFNG might improve the prognosis of overall pancreatic cancer survival. These secreted proteins may serve as a potential therapeutic targets for pancreatic cancer metastasis.

Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres To Achieve Automated Proteome Quantification

Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility, and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultraperformance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and 18O labeling, but also the online integration with nano-high-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoHPLC-ESI-MS/MS). Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and 18O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility, and throughput for proteome quantification.

3-Carboxybenzoboroxole Functionalized Polyethylenimine Modified Magnetic Graphene Oxide Nanocomposites for Human Plasma Glycoproteins Enrichment under Physiological Conditions

Boronate affinity materials have been successfully used for the selective recognition of glycoproteins. However, by such materials, the large-scale glycoproteins enrichment from human plasma under physiological conditions is rarely reported. In this work, 3-carboxybenzoboroxole (CBX) functionalized polyethylenimine (PEI) modified magnetic graphene oxide nanocomposites were synthesized. Benefitting from the low pKa value of CBX (∼6.9) and PEI dendrimer-assisted multivalent binding, the Freundlich constant (KF) for the adsorption of horseradish peroxidase (HRP) was 3.0-7.3 times higher than that obtained by previous work, displaying the high enrichment capacity. Moreover, PEI could improve the hydrophilicity of nanocomposites and reduce nonglycoprotein adsorption. Therefore, such nanocomposites were successfully applied to the analysis of human plasma glycoproteome under physiological conditions, and the identified glycoproteins number and recognition selectivity was increased when compared to the results obtained by previous boronic acid-functionalized particles (Sil@Poly(APBA-co-MBAAm)) under common alkaline condition (137 vs 78 and 67.8% vs 57.8%, respectively). In addition, thrombin (F2), an important plasma glycoprotein, labile under alkaline conditions, was specifically identified by our method, demonstrating the great promise of such nanocomposites in the deep-coverage glycoproteome analysis.

Ionic liquid-based method for direct proteome characterization of velvet antler cartilage

The cartilage zone of the velvet antler is richly vascularized, this being a major difference to the classical cartilage, in which there are no blood vessels. Angiogenesis and rapid growth of vasculature in velvet antler cartilage (VAC) make it an ideal model for discovering the novel angiogenic regulatory factors. However, the proteomic analysis of VAC is challenging due to the serious interference of proteoglycans (PGs) and collagens. To achieve a comprehensive proteome characterization of VAC, herein, we developed an ionic liquid-based method using 1-dodecyl-3-methylimidazolium chloride ([C12-mim]Cl) for selective extraction of cellular proteins from VAC. Compared with the previous cetylpyridinium chloride (CPC)-based method, the developed [C12-mim]Cl-based method takes much less processing time, shows facile preparation procedure and good compatibility towards downstream proteomic analysis, leading to the identification of more protein groups (1543 vs 753), membrane proteins (663 vs 279) and transmembrane proteins (217 vs 58).

Fast MS/MS acquisition without dynamic exclusion enables precise and accurate quantification of proteome by MS/MS fragment intensity.

Most currently proteomic studies use data-dependent acquisition with dynamic exclusion to identify and quantify the peptides generated by the digestion of biological sample. Although dynamic exclusion permits more identifications and higher possibility to find low abundant proteins, stochastic and irreproducible precursor ion selection caused by dynamic exclusion limit the quantification capabilities, especially for MS/MS based quantification. This is because a peptide is usually triggered for fragmentation only once due to dynamic exclusion. Therefore the fragment ions used for quantification only reflect the peptide abundances at that given time point. Here, we propose a strategy of fast MS/MS acquisition without dynamic exclusion to enable precise and accurate quantification of proteome by MS/MS fragment intensity. The results showed comparable proteome identification efficiency compared to the traditional data-dependent acquisition with dynamic exclusion, better quantitative accuracy and reproducibility regardless of label-free based quantification or isobaric labeling based quantification. It provides us with new insights to fully explore the potential of modern mass spectrometers. This strategy was applied to the relative quantification of two human disease cell lines, showing great promises for quantitative proteomic applications.

Identification of PGAM1 as a putative therapeutic target for pancreatic ductal adenocarcinoma metastasis using quantitative proteomics

Background Pancreatic ductal adenocarcinoma (PDAC) is an aggressive gastrointestinal cancer characterized by an extremely low survival rate because of early metastasis. Identifying satisfactory therapeutic targets associated with metastasis is crucial to improve the treatment effect of PDAC. Materials and methods In this research, we used stable isotope labeling by amino acids in cell culture, 1-dodecyl-3-methylimidazolium chloride-assisted sample preparation method preparing protein sample and nano-reversed-phase liquid chromatography-mass spectrometry/mass spectrometry analysis to perform the comparative proteomics of two homologous hamster pancreatic cancer cell lines that are different in metastatic ability: PC-1.0 (highly metastatic) and PC-1 (weakly metastatic). Verifications are through immunohistochemistry on clinical human PDAC pathologic tissues as well as by Western blot of human pancreatic cancer cell lines. siRNA silencing methods were used to study the effect of molecules on invasion and metastasis of pancreatic cancer cell lines. Results Bioinformatic analysis indicated that a total of 141 differentially expressed proteins (82 upregulated and 59 downregulated in PC-1.0 cells) were identified showing obviously differential expression (>1.5-fold change). These differentially expressed proteins were involved in a number of different biologic functions, metabolic pathways, and pathophysiologic processes. Phosphoglycerate mutase 1 (PGAM1) and HSPE1 are the top two upregulated proteins, and PDIA3 and CALR are the top two downregulated proteins in PC-1.0 cells compared to PC-1 cells. PGAM1 and HSPE1 showed higher expressions in PDAC tissue with clinical metastasis and highly metastatic pancreatic cancer cell lines PC-1.0 and Aspc-1. PDIA3 and CALR showed higher expressions in weakly metastatic pancreatic cancer cell lines PC-1 and Capan-2. The Western blot results were consistent with the MS quantification data. Silencing PGAM1 was found to decrease the migration and invasion of pancreatic cancer cell lines with statistical significance, especially in highly metastatic PC-1.0 and Aspc-1 cell lines. Conclusion These data indicated that PGAM1 may be a potential therapeutic target for PDAC metastasis.

Depletion of internal peptides by site-selective blocking, phosphate labeling, and TiO2 adsorption for in-depth analysis of C-terminome

AbstractThe analysis of protein C-termini is of great importance, because it not only provides valuable information about protein function, but also facilitates the elucidation of proteolytic processing. However, even with the recent methods for the global profiling of protein C-termini, the identification of C-termini is still far behind that of N-termini due to the lack of basic residue and low reactive carboxyl group. Therefore, an unbiased and complementary method for C-termini profiling is imperative. In this work, we developed a negative enrichment strategy to achieve the in-depth analysis of C-terminome. Proteins were firstly amidated to block carboxyl groups, followed by lysyl endoproteinase (LysC) digestion to generate C-terminal peptides with α-amines and internal peptides bearing both α- and ε-amines. After the α-amines were blocked by site-selective dimethylation or succinylation, the remaining ε-amines on internal peptides were labeled with phosphate groups. Finally, internal peptides were depleted by TiO2, leaving exclusively the fraction of C-terminal peptides for LC-MS/MS analysis. With Escherichia coli (E. coli) digests as the sample, the efficiency of amidation, dimethylation/succinylation, phosphate labeling and TiO2 depletion was proved high. With the combination of dimethyl and succinic blocking strategy, our method enabled the identification of 477 unique C-terminal peptides in E. coli. In comparison with the C-terminal amine-based isotope labeling of substrates (C-TAILS) method, 83 C-termini were identified by both methods, whereas 369 C-termini were unique to C-TAILS and 394 to our dataset. The method proposed is therefore efficient and possibly promotes the comprehensive profiling of C-termini. Graphical AbstractNegative isolation of C-terminal peptides with combination of site-selective blocking, phosphate labeling, and TiO2 adsorption