Preparation of core-shell microporous organic polymer-coated silica microspheres for chromatographic separation and N-glycopeptides enrichment

Through a “one-pot” strategy, a layer of microporous organic polymer was coated onto the surface of monodisperse amino-functionalized silica microsphere via amino-aldehyde condensation reaction with core-shell structure. The change in chemical structure of material before and after modification was determined by Fourier-transform infrared spectroscopy and X-ray photoelectron spectroscopy. Due to existence of a large number of amino and aldehyde groups in microporous organic polymer shell, the water contact angle decreased from 56.8° (silica microspheres) to 34.7° (microporous organic polymer-coated silica microspheres). Based on these properties, microporous organic polymer-coated silica microspheres were employed as the stationary phase for capillary liquid chromatography and successfully offered baseline separation of polar small molecules. Additionally, the material could also be served as the sorbent of hydrophilic interaction chromatography to enrich glycopeptides from human serum digest. A total of 470 unique N-glycopeptides and 342 N-glycosylation sites mapped to 112 N-glycosylated proteins were unambiguously identified from 2 μL of human serum, exhibiting a promising application prospect of microporous organic polymer-coated silica microspheres in the pretreatment of proteomics samples.     

Highly efficient and selective enrichment of phosphopeptides using porous anodic alumina membrane for MALDI-TOF MS analysis

Because of its good biocompatibility, high surface-to-volume ratio, and distinct surface electrical properties, porous anodic alumina (PAA) membrane has been used to selectively enrich phosphopeptides from a mixture of synthetic peptides and tryptic digest product of beta-casein by a direct MALDI-TOF MS analysis. As we reported previously, PAA membrane has strong incorporation ability to the phosphate anion. Herein, we describe the application of PAA membrane as a selective sampling absorbent for phosphopeptides. The PAA membrane could enrich phosphopeptides with high efficiency and selectivity; for example, the tryptic digest product of beta-casein at a concentration as low as 4 x 10(-9) M can be satisfactorily detected. Compared to that from the nonenriching peptide mixture, the MS signal of the phosphorylated peptides enriched by the PAA membrane is remarkably improved. In addition, acidic peptides have insignificant influence on the enriching process. Results show that the adsorption of phosphate anions on the PAA membrane plays a determining role in achieving highly selective enriching capacity toward phosphopeptides. The feasibility of PAA membranes as specific absorbents for phosphopeptides is also demonstrated.     

Proteomic analysis of detergent-resistant membrane rafts

A combined, detergent- and organic solvent-based proteomic method for the analysis of detergent-resistant membrane rafts (DRMR) is described. These specialized domains of the plasma membrane contain a distinctive and dynamic protein and/or lipid complement, which can be isolated from most mammalian cells. Lipid rafts are predominantly involved in signal transduction and adapted to mediate and produce different cellular responses. To facilitate a better understanding of their biology and role, DRMR were isolated from Vero cells as a Triton X-100 insoluble fraction. After detergent removal, sonication in 60% buffered methanol was used to extract, solubilize and tryptically digest the resulting protein complement. The peptide digestate was analyzed by microcapillary reversed-phase liquid chromatography-tandem mass spectrometry. Gas-phase fractionation in the mass-to-charge range was employed to broaden the selection of precursor ions and increase the number of identifications in an effort to detect less abundant proteins. A total of 380 proteins were identified including all known lipid raft markers. A total of 91 (24%) proteins were classified as integral alpha-helical membrane proteins, of which 51 (56%) were predicted to have multiple transmembrane domains.     

Comparison of surfactant-assisted shotgun methods using acid-labile surfactants and sodium dodecyl sulfate for membrane proteome analysis

Three surfactant-assisted shotgun methods using acid labile surfactants, sodium-3-[(2-methyl-2-undecyl-1,3-dioxolan-4-yl)-methoxyl]-1-propanesulfonate (RapiGest) and 3-[3-(1,1-bisalkyloxyethyl)pyridin-1-yl]propane-1-sulfonate (PPS), and sodium dodecyl sulfate (SDS) were investigated for their applicability to membrane proteome analysis. It is shown that RapiGest is a preferred reagent for handling membrane proteomes of Escherichia coli and MCF7 cells for liquid chromatography tandem mass spectrometry (LC MS/MS) analysis of tryptic digests. The RapiGest method allowed identification of more peptides and proteins than the SDS and PPS methods and there was no apparent bias for the type of peptides and proteins identified by the RapiGest and SDS methods, while a slightly higher proportion of hydrophilic peptides and proteins were identified by the PPS method. The performance of the SDS and PPS methods is similar in terms of the numbers of peptides and proteins identified. Since the SDS method required the removal of SDS using a technique such as strong-cation exchange (SCX), we further investigated the effect of SCX on sample loss through analyzing the digest of an enriched E. coli membrane fraction as well as a standard protein, bovine serum albumin (BSA). The results showed that extensive sample loss (as much as 62%) was encountered during the SCX cleaning step. We then applied the RapiGest method in combination with two-dimensional LC MS/MS to characterize the E. coli membrane proteome. In total, 1626 unique proteins (5799 unique peptides) were identified with a peptide false discovery rate of 2.4%. About 60% of the identified proteins with known cellular locations were found to be membrane proteins. Among them, about 75% were integral membrane proteins. This work represents one of the most comprehensive profiles of E. coli membrane proteome generated by a proteomic technique.     

Isolation of bovine lactoferrin, lactoperoxidase and enzymatically prepared lactoferricin from proteolytic digestion of bovine lactoferrin using adsorptive membrane chromatography

A new downstream procedure for the isolation of bovine lactoferrin (bLf), lactoperoxidase and bovine lactoferricin (LfcinB) from sweet cheese whey was developed at the laboratory scale, based on membrane adsorber technology. The procedure was upscaled later on to an industrially relevant scale for the purificationof sweet whey concentrate with a recovery yield for lactoferrin of more than 90%. Based on these results the industrial process for 1 x 10(8) kg whey per year was projected. These high-value proteins were downstreamed by using cation-exchange membrane systems (Sartobind S, Sartorius, G?ttingen, Germany). These strongly acidic membranes trap proteins in its anionic form. The dynamic loading capacity for both proteins as well as the optimal elution profiles with sodium chloride gradients were derived from laboratory experiments using membrane modules with 15-75 cm2 membrane material. Further investigations were performed with 1 m2 modules in a continuous process mode. The enzymatic preparation of LfcinB from bLf was performed by pepsin hydrolysis and the isolation of LfcinB was directly carried out from the enzymatic digest mixture. The identification of the proteins was performed with matrix-assisted laser desorption ionisation mass spectrometry (MALDI-MS). LfcinB and bLf were both tested afterwards in biological assays in order to show not only the efficiency of the downstreaming process in regard to product quantity but also to product quality (biological activity).     

Application of solid‐phase extraction for the concentration of chromophores,fluorophores, and photosensitizers from lens protein digests

Solid‐phase extraction was applied for the separation of protein digests obtained from aged human lenses, cataractous human lenses, calf lens proteins in vitro glycated with dehydroascorbic acid and native calf lens proteins. Four fractions were collected after stepwise elution with different solvents. The first fraction contained about 80% of the digested material possessing free amino groups. At the same time, the third and the fourth fractions were enriched in chromophores, fluorophores, and photosensitizing structures that originate mainly from advanced protein glycation. The comparison between the total digest and the fourth fraction based on their UV absorption at 330 nm, intensity of fluorescence (excitation/emission 350/450 nm), and production of singlet oxygen upon UVA irradiation argues that the solid‐phase extraction was capable of concentrating the advanced glycation end‐products about a hundredfold. Thus, this technique is a useful step for separation and concentration of fluorophores, chromophores, and photosensitizers from aged and glycated lens protein digests.     

Comprehensive analysis of host cell impurities in monoclonal antibodies with improved sensitivity by capillary zone electrophoresis mass spectrometry

Four methods were compared for analysis of host‐cell protein (HCP) impurities in a recombinant mAb. First, CZE‐MS/MS was used to analyze the digest of an HCP sample following extraction of the mAb with proteins A and L affinity columns; 220 protein groups and 976 peptides were identified from the depleted HCP digest. Second, a nanoACQUITY UltraPerformance LCH system was also used to analyze the depleted HCP digest; 34 protein groups and 53 peptides from 50 ng of the depleted HCP digest and 290 protein groups and 1011 peptides were identified from 1 μg of the depleted HCP digest. Third, 185 protein groups and 709 peptides were identified by CZE‐MS/MS from the HCP digest without depletion. Fourth, a strong cation exchange SPE was coupled to CZE‐ESI‐MS/MS using online pH gradient elution for analysis of the HCP digest without depletion. A series of five pH bumps were applied to elute peptides from the strong cation exchange monolith followed by analysis using CZE coupled to a Q Exactive HF mass spectrometer; 230 protein groups and 796 peptides were identified from the HCP digest without depletion.     

Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties

Recent efforts at the proteomic level were employed to describe the protein equipment of the plasma membrane of the model plant Arabidopsis thaliana. These studies had revealed that the plasma membrane is rich in extrinsic proteins but came up against two major problems: (i) few hydrophobic proteins were recovered in two-dimensional electrophoresis gels, and (ii) many plasma membrane proteins had no known function or were unknown in the database despite extensive sequencing of the Arabidopsis genome. In this paper, several methods expected to enrich a membrane sample in hydrophobic proteins were compared. The optimization of solubilization procedures revealed that the detergent to be used depends on the lipid content of the sample. The corresponding proteomes were compared with the statistical model AMMI (additive main effects with multiplicative interaction) that aimed at regrouping proteins according to their solubility and electrophoretic properties. Distinct groups emerged from this analysis and the identification of proteins in each group allowed us to assign specific features to several of them. For instance, two of these groups regrouped very hydrophobic proteins, one group contained V-ATPase subunits, another group contained proteins with one transmembrane domain as well as proteins known to interact with membrane proteins. This study provides methodological tools to study particular classes of plasma membrane proteins and should be applicable to other cellular membranes.     

Enrichment and desalting of tryptic protein digests and the protein depletion using boron nitride

Sample preparation still remains a great challenge in modern bioanalysis and the interest in new efficient solid phase extraction (SPE) materials still remains high. In this work, hexagonal boron nitride (h-BN) is introduced as a new SPE material for the isolation and enrichment of peptides. The h-BN is isoelectronic and structurally similar to graphite. It has remarkable properties including good thermal conductivity, excellent thermal and chemical stability and a better oxidation resistance than graphite. BN attracts increasing interest because of its wide range of applicability. In the present work, the great potential of h-BN, as a new SPE-material, on the enrichment, preconcentration and desalting of tryptic digest of model proteins is demonstrated. A special attention was dedicated to the efficient enrichment of hydrophilic phosphopeptides. Two elution protocols were developed for the enrichment of peptides compatible for subsequent MALDI-MS and ESI-MS analysis. In addition, the recoveries of 5 peptides and 3 phosphopeptides with wide range of pI values utilizing h-BN materials with different surface areas were investigated. 84–106% recovery rate could be achieved using h-BN materials. The results were compared with those obtained using graphite and silica C18 under the same elution conditions, and lower recoveries were obtained. In addition, h-BN was found to have a capability of protein depletion, which is requisite for the peptide profiling.     

Synthesis of an adsorbed reversed-phase packing material for the separation of proteins and peptides

We have described the preparation and chromatographic evaluation of an adsorbed hydrophobic stationary phase suitable for reversed-phase chromatography of proteins and peptides. The synthetic procedure involves three steps: the adsorption of a polyamine to the silica surface; crosslinking of the adsorbed polyamine layer with a bis-phenyl difunctional epoxide; and the benzoylation of the remaining accessible amino groups. Performance of this chromatographic material compared favorably with SynChropak RP-8 silica (SynChrom, Linden, IN, U.S.A.) and was stable to 40% formic acid. Good separations were obtained between the components of sample mixtures containing proteins or the cyanogen bromide fragments of sperm whale myoglobin. However, in both cases, the adsorbed hydrophobic stationary phase was less retentive. Furthermore, this medium exhibited slightly different selectivity. Whereas the heme which was present in the cyanogen bromide digest of myoglobin desorbed as the second peak from the RP-8 column, it eluted last from the adsorbed stationary phase. Comparable performance, acid stability and alternate selectivity suggest that this material is an interesting alternative to organosilane reversed-phase coatings.     

聚酰基氨基脲类多孔有机聚合物的制备及其在糖肽富集中的应用

通过均苯三甲酰肼(BTZ)和对苯二异氰酸酯(PDI)的聚合反应制备了一种新型的聚酰基氨基脲类多孔有机聚合物(POP)。采用傅里叶变换红外光谱(FT-IR)、交叉极化魔角自旋碳13核磁共振(CP-MAS13C NMR)、氮气物理吸附/脱附实验和水接触角等手段对POP-1的物理性质进行表征。其中比表面积为166 m~2/g,水接触角为46.4°,表明这种多孔材料具有良好的亲水性。因此,尝试将POP-1应用于标准蛋白质和复杂生物样品中糖肽的亲水色谱富集。通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/MS)分析,从500 fmol人血清免疫球蛋白G(IgG)酶解液中共鉴定到19条典型的N-连接糖肽;通过毛细管液相色谱-串联质谱(c LC-MS/MS)分析,从100μg鼠肝蛋白酶解液中共鉴定到属于605个糖蛋白的1 919个糖肽的1 350个N-糖基化位点。这些结果表明POP-1在糖蛋白质组学研究中具有较强的应用潜力。     

超疏水聚苯胺/聚四氟乙烯复合膜的制备及油-水乳液分离性能

以苯胺为原料, 采用原位聚合法在聚四氟乙烯(PTFE)基体上合成聚苯胺/聚四氟乙烯(PANI/PTFE)复合膜. 利用光学显微镜、 扫描电子显微镜(SEM)、 傅里叶变换红外光谱(FTIR)、 紫外-可见吸收光谱(UV-Vis)和静态水接触角测试对PANI/PTFE复合膜的形貌、 结构和浸润性进行分析, 并对其油包水乳液分离性能、 通量和循环使用性能进行了测试. 研究结果表明, PANI/PTFE复合膜仅在重力条件就能有效分离油包水乳液; 而且重复数十次过滤后, PANI/PTFE复合膜仍具有良好的抗污能力和分离性能.     

原位复合法制备层状结构的壳聚糖/羟基磷灰石纳米材料

用原位复合法制备了高性能的壳聚糖/羟基磷灰石(CS/HA)纳米复合材料.用预先沉积的壳聚糖膜将含有羟基磷灰石前驱体的壳聚糖溶液与凝固液隔离,同时控制壳聚糖沉积与羟基磷灰石前驱体转化为羟基磷灰石的过程,使其缓慢且有序地进行.当pH值改变时,质子化的壳聚糖分子链在负电层诱导下有序沉积并形成层状结构与羟基磷灰石原位生成CS/HA,并实现二者分子级复合.XRD和TEM测试证实原位生成的磷酸盐是羟基磷灰石,且其颗粒长约为100nm,宽30～50nm.SEM结果表明,用原位复合法制备的材料具有层状结构,CS/HA(质量比100/5)纳米复合材料弯曲强度高达86MPa,比松质骨的高3～4倍,相当于密质骨的1/2,有望用于可承重部位的组织修复材料.     

Integration of solid-phase extraction membranes for sample multiplexing: application to rapid protein identification from gel-isolated protein extracts

The present report describes the design and application of a dual sprayer system for high-throughput proteome analysis. This system comprises parallel solid-phase extraction cartridges used for preconcentration and desalting of proteolytic digests prior to nanoelectrospray mass spectrometry analyses. Tryptic peptides from in-gel digest of protein bands/spots are first adsorbed on styrene divinyl benzene membrane and subsequently eluted with a short plug of organic buffer prior to infusion to the mass spectrometer at a flow rate of typically 500 nL/min. Tryptic peptide eluting from the membrane are analyzed by the mass spectrometer by moving in turn each sprayer in front of the sampling orifice. Sequential injection, preconcentration and analyses of tryptic digests are typically achieved with a throughput of up to 3.5 min/sample and a detection limit of approximately 8-80 fmol per injection. Replicate injections of peptide mixtures indicated that reproducibility of peak areas ranged from relative standard deviations (RSD) of 1.1% to 4.5%. The application of this device is demonstrated for digests of gel-isolated proteins obtained from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation of rat liver plasma membrane and from two-dimensional gel electrophoresis of total cell lysate extracts from human prostatic cancer cell.     

Profiling and comprehensive expression analysis of ABC transporter solute-binding proteins of Bacillus subtilis membrane based on a proteomic approach

We analyzed ABC transporter solute-binding proteins (SBPs) of the Bacillus subtilis membrane using a proteomic approach. We prepared a washed cell membrane fraction that was insoluble in 134 mM nondetergent sulfobetaine and then extracted proteins using mixtures of detergents in a stepwise manner. The membrane proteins were resolved by three two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) or two one-dimensional (1-D) PAGE procedures, electroblotted, and digested in the presence of 5% or 80% acetonitrile. Thereafter, matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS) identified 637 proteins corresponding to 15.9% of the total cellular proteins. We predicted that among these, 256 were membrane proteins, 101 were lipoproteins or secretory proteins and 280 were soluble proteins containing peripheral proteins that function in both the cytoplasm and the cell membrane such as SecA and FtsY. Among the 637 proteins, we identified 30 SBPs among 38 importers predicted by a bioinformatic search of the genome. We confirmed expression of the genes for the 30 SBPs using DNA microarray analysis. We compared the 2-D gel separation profiles of submembrane fractions solubilized by 1% n-dodecyl-beta-D-maltoside from cells cultured on Luria Bertani (LB), S7, and S7 medium without glutamate as well as DNA microarray data on LB and S7. The results suggested that YcdH, YtmK and YurO are binding proteins for Mn(++), glutamate and glucose, respectively, and that YqiX and YxeM are binding proteins for amino acids (tryptophan in S7 medium).     

Proteomics based on selecting and quantifying cysteine containing peptides by covalent chromatography

This paper describes a procedure in which cysteine containing peptides from tryptic digests of complex protein mixtures were selected by covalent chromatography based on thiol-disulfide exchange. identified by mass spectrometry, and quantified by differential isotope labeling. Following disruption of disulfide bridges with 2,2'-dipyridyl disulfide, all proteins were digested with trypsin and acylated with succinic anhydride. Cysteine containing peptides were then selected from the acylated digest by disulfide interchange with sulfhydryl groups on a thiopropyl Sepharose gel. Captured cysteine containing peptides were released from the gel with 25 mM dithiothreitol (pH 7.5) containing 1 mM (ethylenedinitrilo)tetraacetic acid disodium salt and alkylated with iodoacetic acid subsequent to fractionation by reversed-phase liquid chromatography (RPLC). Fractions collected from the RPLC column were analyzed by matrix-assisted laser desorption ionization mass spectrometry. Based on isotope ratios of peptides from experimental and control samples labeled with succinic and deuterated succinic anhydride, respectively, it was possible to determine the relative concentration of each peptide species between the two samples. Peptides obtained from proteins that were up-regulated in the experimental sample were easily identified by an increase of the relative amount of the deuterated peptide. The results of these studies indicate that by selecting cysteine containing peptides, the complexity of protein digest could be reduced and database searches greatly simplified. When coupled with the isotope labeling strategy for quantification it was possible to determine proteins that were up-regulated in plasmid bearing Escherichia coli when expression of plasmid proteins was induced. Up-regulation of several proteins of E. coli origin was also noted.     

PREPARATION AND CHARACTERIZATION OF ION EXCHANGE MEMBRANES BASED ON POLYVINYLIDENE FLUORIDE

A new ion exchange membrane based on polyvinylidene fluoride (PVDF) and sulfonated poly(styrenedivinylbenzene) was prepared by in-situ polymerization. The incorporation of sulfonic groups into the polyvinylidene fluoride composite membrane was confirmed by infrared spectroscopy (IR), ion exchange capacity (IEC) and energy dispersive X-ray analysis (EDAX). Area resistance, IEC and water uptake of the treated membrane were evaluated. When area resistance in NaCl aqueous solution at 25℃, IEC is as high as 2.43 millimoles per gram of the wet membrane. The hydrophilicity of PVDF membrane is also significantly improved after treatment. When 60% of crosslinked membrane was sulfonated at 80℃ for 6 h, water uptake of the treated membrane can attain 64.7%.     

Growth of two-layer copolymer as the stationary phase with very high separation efficiency for separating peptides in capillary electrochromatography

Open tubular CEC (OT-CEC) column with a very high separation efficiency was prepared for peptides separation. A pretreated silica-fused capillary was reacted with 3-(methacryloxy) propyltrimethoxysilane followed by vinylbenzyl chloride and divinylbenzene to produce first thin monolithic monolayer. The second copolymer layer was formed on thin monolithic monolayer of the capillary by reversible addition-fragmentation transfer polymerization of N-phenylacrylamide and styrene. The key parameters including buffer pH value and organic modifier were systematically evaluated to provide the optimal chromatographic condition. The resultant OT-CEC columns were validated by separating a synthetic mixture of peptides and cytochrome C tryptic digest in capillary electrochromatography. The number of theoretical plates as high as 2.4 million per column was achieved for synthetic mixture peptides. In addition, the fabricated OT-CEC column also resolved more than 18 high-efficiency digestion peptides from a mixture containing tryptic digest of cytochrome C. The column to column and inter- to intraday repeatabilities of OT-CEC column through RSD% were found better than 3.0%, exhibiting satisfactory stability and repeatability of the two-layer deposited OT-CEC column. The results reveal that the open tubular capillary column modified with two-layer copolymer shows the great prospect for the separation of proteins in capillary electrochromatography.     

聚丙烯腈和二氧化硅复合纤维膜富集-高分辨质谱法测定烟气中的有害化合物

以聚丙烯腈(PAN)和二氧化硅(Si02)纳米粒子的混合溶液为纺丝溶液,采用静电纺丝技术制备具有高吸附性能的新型PAN-SiO2疏水性复合纤维膜,对香烟燃烧产生的烟气进行富集,用甲醇作为洗脱溶剂,对待测组分进行洗脱,并使用高分辨串联质谱技术进行测定.实验结果表明,通过调控PAN和SiO2纳米粒子的浓度,优化静电纺丝参数,得到对小分子有机化合物吸附性较强的纤维膜.对所收集到的烟气进行质谱测定,在正离子模式下,检测到的主要成分有尼古丁、丙酮、苯乙烯、丙烯醛、异戊二烯、丙烯腈等21种小分子有机化合物;在负离子模式下,检测到的主要成分有水杨酸、苹果酸和乳酸等6种有机酸.同时,对烟气中的主要有害成分尼古丁进行定量分析,方法的检出限为0.071 ng/L,定量限为0.236 ng/L.     

Highly selective enrichment of phosphopeptides using poly(dibenzo‐18‐crown‐6) as a solid‐phase extraction material

A poly(dibenzo‐18‐crown‐6) was used as a new solid‐phase extraction material for the selective enrichment of phosphopeptides. Isolation of phosphopeptides was achieved based on specific ionic interactions between poly(dibenzo‐18‐crown‐6) and the phosphate group of phosphopeptides. Thus, a method was developed and optimized, including loading, washing and elution steps, for the selective enrichment of phosphopeptides. To assess this potential, tryptic digest of three proteins (α‐ casein, β‐casein and ovalbumin) was applied on poly(dibenzo‐18‐crown‐6). The nonspecific products were removed by centrifugation and washing. The spectrometric analysis was performed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Highly selective enrichment of both mono‐ and multiphosphorylated peptides was achieved using poly(dibenzo‐18‐crown‐6) as solid‐phase extraction material with minimum interference from nonspecific compounds. Furthermore, evaluation of the efficiency of the poly(dibenzo‐18‐crown‐6) was performed by applying the digest of egg white. Finally, quantum mechanical calculations were performed to calculate the binding energies to predict the affinity between poly(dibenzo‐18‐crown‐6) and various ligands. The newly identified solid‐phase extraction material was found to be a highly efficient tool for phosphopeptide recovery from tryptic digest of proteins.