Hongqiang Qin

Polyhedral Oligomeric Silsesquioxane as a Cross-linker for Preparation of Inorganic−Organic Hybrid Monolithic Columns

An inorganic-organic hybrid monolithic capillary column was synthesized via thermal free radical copolymerization within the confines of a capillary using a polyhedral oligomeric silsesquioxane (POSS) reagent as the inorganic-organic hybrid cross-linker and a synthesized long carbon chain quaternary ammonium methacrylate of N-(2-(methacryloyloxy)ethyl)-dimethyloctadecylammonium bromide (MDOAB) as the organic monomer. The preparation process was as simple as pure organic polymer-based monolithic columns instead of using POSS as the nanosized inorganic-organic hybrid blocks (cross-linker) of the monolithic matrix. The pore properties and permeability could be tuned by the composition of the polymerization mixture. The characterization and evaluation results indicated that the synthesized MDOA-POSS hybrid monolith possessed the merits of organic polymer-based monoliths and silica-based monoliths with good mechanical and pH (pH 1-11) stabilities, which may be attributed to the incorporation of the rigid nanosized silica core of POSS. Column efficiencies of 223 000 and 50 000 N/m were observed in capillary electro-driven chromatography (CEC) and mu-HPLC, respectively. Peptides and standard proteins were baseline separated by this hybrid column in CEC and mu-HPLC, respectively, as well. The separation of bovine serum albumin (BSA) tryptic digest was also attempted to show its potential application in proteome analysis.

Highly efficient extraction of serum peptides by ordered mesoporous carbon.

Human serum is of great importance for organ function and the potential diagnosis of diseases. Serum biomarkers have been widely applied in clinical diagnosis and disease therapies. Endogenous serum peptides are an important class of potential biomarkers for the elucidation of biological and pathological variations in the serum. However, owing to the complexity and the highly dynamic range of protein concentrations in serum, the discovery of serum peptides, especially those at low abundance levels from minuscule blood samples, is still a challenge. With regard to obtaining serum peptides, organic solvent precipitation is a simple method for the removal of highly abundant proteins. However, the difficulty in discriminating between peptides and proteins together with the inevitable loss of peptides during the removal of proteins by solvent precipitation makes this method inefficient for the extraction of peptides. Centrifugal ultrafiltration (UF) with an accurate molecular weight cutoff is considered to be a very useful technology for the separation of proteins with low and high masses. By using UF coupled with a nanospray ionization hybrid ion trap/Fourier transform mass spectrometer, 300 unique peptides were identified from a 60 mL of serum sample. However, the coconcentration of small molecules and salts result in inefficient peptide extraction and severe interference to the MS detection. Therefore, additional peptide enrichment as well as salt removal by solid phase extraction (SPE), particularly using hydrophobic C18 adsorbents, has to be adopted before MS analysis. To simplify the extraction of peptides from serum, ordered mesoporous silica materials have been applied to enable the selective extraction of serum peptides rather than large proteins by the size-exclusion effect of the mesopores. Unfortunately, owing to the inherently insufficient hydrophobicity of silica some peptides are not extracted, thus resulting in a low number of unique peptides being identified. Surface modifications to mesoporous silica and titanium oxide materials have been developed for the enrichment of some specific posttranslational peptides, such as phosphopeptides and glycopeptides in serum. So far, a highly efficient general method for the extraction of a broad spectrum of serum peptides rather than for specific peptides is still absent; such a method would be a crucial technology for the discovery of serum biomarkers from very small blood samples. Herein, we describe the synthesis of an ordered mesoporous carbon material (OMC) and its use for the enrichment of a broad spectrum of endogenous peptides from serum. The expected high efficiency of this method for peptide enrichment is due to the distinct hydrophobicity of carbon as well as the size exclusion of the mesopores against serum proteins. The procedure for the extraction of a broad spectrum of endogenous serum peptides using OMC is illustrated in Scheme 1.

Synthesis of branched PEG brushes hybrid hydrophilic magnetic nanoparticles for the selective enrichment of N-linked glycopeptides

Hybrid Fe(3)O(4)@SiO(2)@PEG-Maltose MNPs were synthesized by SI-ATRP of branched PEG brushes on the surface and subsequent functionalization with hydrophilic maltose group, and the multifunctional materials were utilized for selective enrichment of N-linked glycopeptides from biological samples with high specifity, high sensitivity, and large binding capacity.

Recent advances of mesoporous materials in sample preparation.

Sample preparation has been playing an important role in the analysis of complex samples. Mesoporous materials as the promising adsorbents have gained increasing research interest in sample preparation due to their desirable characteristics of high surface area, large pore volume, tunable mesoporous channels with well defined pore-size distribution, controllable wall composition, as well as modifiable surface properties. The aim of this paper is to review the recent advances of mesoporous materials in sample preparation with emphases on extraction of metal ions, adsorption of organic compounds, size selective enrichment of peptides/proteins, specific capture of post-translational peptides/proteins and enzymatic reactor for protein digestion.

Coupling Strong Anion-Exchange Monolithic Capillary with MALDI-TOF MS for Sensitive Detection of Phosphopeptides in Protein Digest

Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful tool for the analysis of protein phosphorylation by detection of phosphopeptides in phosphoprotein digest. Enrichment of phosphopeptides by immobilized metal ion affinity chromatography (IMAC) or metal oxide affinity chromatography (MOAC) followed with MALDI analysis is the common approach. However, the pH for loading and elution of phosphopeptides is incompatible with protein digestion as well as the preparation of the MALDI matrix solution. Therefore, some pretreatment steps, such as pH adjustment and desalting, are required, which make the approach tedious and insensitive. In this study, a strong anion-exchange (SAX) capillary monolith was prepared to enrich phosphopeptides from protein digest for MALDI-TOF MS analysis. It was found that phosphopeptides could be specifically retained on the SAX column at high pH around 8 and could be eluted by 5% formic acid. Thus, the protein digests without any pretreatment could be loaded onto the SAX column under basic pH condition; after removing nonphosphopeptides by washing, the bound phosphopeptides could be eluted directly onto a MALDI target and analyzed by MALDI-TOF MS. This approach significantly simplified the analytical procedures and reduced the sample loss. Because of the excellent MALDI MS compatible procedure and the microscale SAX column, a detection limit as low as 50 amol for the analysis of phosphopeptides from beta-casein digest was achieved. To circumvent the inconvenience of the sample loading, a new simple sample introducing method based on capillary action was proposed, which further reduced the detection limit to 10 amol.

A poly(ethylene glycol)-brush decorated magnetic polymer for highly specific enrichment of phosphopeptides

Immobilized metal affinity chromatography (IMAC) is a powerful method in phosphopeptide enrichment. However, the achievement of highly specific enrichment and sensitive detection of phosphopeptide by IMAC is still a big challenge because of the lack of high specificity and large binding capacity of conventional IMAC materials. Here, we report a novel IMAC nanoparticle to dramatically improve the enrichment specificity for the phosphopeptide by introducing a titanium phosphate moiety on a poly(ethylene glycol) methacrylate (PEG) brush decorated Fe3O4@SiO2 core–shell nanoparticle (denoted as Fe3O4@SiO2@PEG–Ti4+ IMAC nanoparticle). The thicker grafting layer of the PEG brushes has a higher chelating capacity of titanium ions. Due to the combination of the superior nonfouling property and the enhanced binding capacity of the grafted PEG brushes, the Fe3O4@SiO2@PEG–Ti4+ IMAC nanoparticle demonstrated a high phosphopeptide recovery (over 70%) and low limit of detection (0.5 fmol), along with an exceptional great specificity to capture phosphopeptides from a tryptic digest of the mixture of a nonphosphorylated protein BSA and a phosphorylated protein α-casein with molar ratios of BSA/α-casein up to 2000 : 1. In the analysis of a real complex biological sample, the tryptic digests of Arabidopsis, 2447 unique phosphopeptides have been identified, showing a superior performance of the Fe3O4@SiO2@PEG–Ti4+ IMAC nanoparticle than that of Fe3O4@SiO2–Ti4+ (1186) and commercial TiO2 microspheres (961). We believe that the PEG decoration for IMAC materials will be a convenient approach to significantly improve the specificity and the binding capacity of phosphopeptide enrichment.

Size-Selective Enrichment of N-Linked Glycans Using Highly Ordered Mesoporous Carbon Material and Detection by MALDI-TOF MS

Many diseases are characterized by the changes of either glycan structure or glycosylation site of glycoproteins. The glycan profiling can provide the overview of glycosylation in despite of the absence of the glycosylation sites, which in turn simplifies the complexity of disease diagnosis. Herein, we describe a simple method to profile the N-linked glycans by MALDI-TOF MS with the enrichment using oxidized ordered mesoporous carbon, taking advantages of the size-exclusive effect of mesopore against proteins as well as the interaction between glycans and carbon. Twenty four N-linked glycans derived from ovalbumin could be efficiently detected with high signal-to-noise (S/N) ratios and sufficient peak intensities. In the analysis of complex serum samples, 32 N-linked glycans could be profiled, and 5 (4 core-fucosylated glycans) of them were distinguished from liver cancer and healthy samples.

Synthesis of zwitterionic polymer brushes hybrid silica nanoparticles via controlled polymerization for highly efficient enrichment of glycopeptides

Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) materials have been increasingly attractive in glycopeptide enrichment. However, the traditional ZIC-HILIC materials are modified with monolayer zwitterionic molecules on the surface, therefore, the hydrophilicity, detection sensitivity and loading capacity are limited. In this work, we synthesized novel silica nanoparticles with uniform poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sul-fopropyl)ammonium hydroxide (PMSA) brushes grafted onto the surface via reversible addition-fragmentation chain transfer (RAFT) polymerization (denoted as SiO2-RAFT@PMSA). The resulting SiO2-RAFT@PMSA nanoparticles demonstrated low detection limit (10 fmol) and high recovery yield (over 88%) for glycopeptide enrichment from tryptic digest of human IgG. The SiO2-RAFT@PMSA nanoparticles were further applied for the analysis of mouse liver glycoproteome, a total number of 303 unique N-glycosylation sites corresponding to 185 glycoproteins was reliably profiled in three replicate nano-LC-MS/MS runs. Significantly, more glycopeptides were identified than those of nanoparticles, monolayer MSA molecules modified SiO2@single-MSA and nonuniform multi-layer PMSA brushes coated SiO2@PMSA, as well as commercial ZIC@HILIC beads and Click Maltose beads. The excellent performance of SiO2-RAFT@PMSA nanoparticles results from the non-fouling property, a large quantity of functional molecules and suitable link arms provided by uniform PMSA brushes, as well as efficient interaction between glycopeptides and uniform PMSA brushes. It is concluded that the synthesized SiO2-RAFT@PMSA nanoparticles exhibit great potential in glycoproteome analysis. Moreover, this strategy to modify nanopaticles with uniform polymer brushes via RAFT polymerization can also be explored to design other types of materials for bioseparation application.

Interference in autophagosome fusion by rare earth nanoparticles disrupts autophagic flux and regulation of an interleukin-1β producing inflammasome.

Engineered nanomaterials (ENMs) including multiwall carbon nanotubes (MWCNTs) and rare earth oxide (REO) nanoparticles, which are capable of activating the NLRP3 inflammasome and inducing IL-1β production, have the potential to cause chronic lung toxicity. Although it is known that lysosome damage is an upstream trigger in initiating this pro-inflammatory response, the same organelle is also an important homeostatic regulator of activated NLRP3 inflammasome complexes, which are engulfed by autophagosomes and then destroyed in lysosomes after fusion. Although a number of ENMs have been shown to induce autophagy, no definitive research has been done on the homeostatic regulation of the NLRP3 inflammasome during autophagic flux. We used a myeloid cell line (THP-1) and bone marrow derived macrophages (BMDM) to compare the role of autophagy in regulating inflammasome activation and IL-1β production by MWCNTs and REO nanoparticles. THP-1 cells express a constitutively active autophagy pathway and are also known to mimic NLRP3 activation in pulmonary macrophages. We demonstrate that, while activated NLRP3 complexes could be effectively removed by autophagosome fusion in cells exposed to MWCNTs, REO nanoparticles interfered in autophagosome fusion with lysosomes. This leads to the accumulation of the REO-activated inflammasomes, resulting in robust and sustained IL-1β production. The mechanism of REO nanoparticle interference in autophagic flux was clarified by showing that they disrupt lysosomal phosphoprotein function and interfere in the acidification that is necessary for lysosome fusion with autophagosomes. Binding of LaPO4 to the REO nanoparticle surfaces leads to urchin-shaped nanoparticles collecting in the lysosomes. All considered, these data demonstrate that in contradistinction to autophagy induction by some ENMs, specific materials such as REOs interfere in autophagic flux, thereby disrupting homeostatic regulation of activated NLRP3 complexes.

Size-selective proteolysis on mesoporous silica-based trypsin nanoreactor for low-MW proteome analysis

In this study, the concept of size-selective proteolysis was first described by using the mesoporous silica-based trypsin nanoreactor. For analysis of a complex protein sample, low-MW proteins were preferentially digested for identification while high-MW proteins were excluded from digestion.