Solid-phase capture and release of arginine peptides by selective tagging and boronate affinity chromatography

A method for the selection of arginine-containing peptides from a mixture by a solid phase capture and release technique is presented. The method is based on the covalent modification of the guanidine group of arginine with 2,3-butanedione and phenylboronic acid under alkaline conditions. Using polymeric materials with immobilised phenylboronic acid the arginine-peptides can be captured on a solid support while arginine-free peptides are not covalently bound and can be washed away. Finally, the arginine-peptides can be cleaved again from the boronic acid beads due to the reversibility of the reaction. The recovered peptides are then analysed by liquid chromatography-tandem mass spectrometry. The method was optimised with model peptides with regard to the non-specific binding of arginine-free peptides and quantitative cleavage of the label after the selection step. Using an adequate protocol, the applicability towards more complex samples was successfully tested with a tryptic digest of a mixture of three standard proteins.     

Zirconium arsenate-modified magnetic nanoparticles: preparation, characterization and application to the enrichment of phosphopeptides

Phosphorylation, one of the most important post-translational modifications of protein, plays a crucial role in a large number of biological processes. Large-scale identification of protein phosphorylation by mass spectrometry is still a challenging task because of the low abundance of phosphopeptides and sub-stoichiometry of phosphorylation. In this work, a novel strategy based on the specific affinity of zirconium arsenate to the phosphate group has been developed for the effective enrichment of phosphopeptides. Zirconium arsenate-modified magnetic nanoparticles (ZrAs-Fe(3)O(4)@SiO(2)) were prepared by covalent immobilization of zirconium arsenate on Fe(3)O(4)@SiO(2) magnetic nanoparticles under mild conditions, and characterized by transmission electron microscope (TEM), Fourier transform infrared (FT-IR) spectroscopy, energy dispersive X-ray spectroscopy (EDX) and vibrating sample magnetometer (VSM). The prepared ZrAs-Fe(3)O(4)@SiO(2) was applied for the selective enrichment of phosphopeptides from the digestion mixture of phosphoproteins and bovine serum albumin (BSA). Our results demonstrated that the ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles possess higher selectivity for phosphopeptides and better capture capability towards multiply-phosphorylated peptides than commercial zirconium dioxide (ZrO(2)), which has been widely employed for the enrichment of phosphopeptides. In addition, endogenous phosphopeptides from human serum can be effectively captured by ZrAs-Fe(3)O(4)@SiO(2) magnetic nanoparticles. It is the first report, to the best of our knowledge, in which the zirconium arsenate-modified magnetic nanoparticles were successfully applied to the enrichment of phosphopeptides, which offers the potential application of this new material in phosphoproteomics study.     

Improved method for fatty acid analysis in herbage based on direct transesterification followed by solid-phase extraction

Direct transesterification (DT) and solvent extraction with acid or basic derivatization procedure for fatty acid (FA) analysis in herbage were compared. The highest total FA, alpha-linolenic and linoleic acid contents were obtained with DT. However, DT also produced the highest amount of interfering compounds, identified as phytadienes and sugar derivative products, which may co-elute with FA. An additional step based on solid-phase extraction to produce clean samples was proposed. This procedure is simple and gives good recoveries for the FA fortified samples. Additionally, structural characterization of 16:1 trans-3 was conducted by covalent adduct chemical ionization tandem mass spectrometry.     

Dynamic identification of phosphopeptides using immobilized metal ion affinity chromatography enrichment, subsequent partial beta-elimination/chemical tagging and matrix-assisted laser desorption/ionization mass spectrometric analysis

The enrichment of phosphopeptides using immobilized metal ion affinity chromatography (IMAC) and subsequent mass spectrometric analysis is a powerful protocol for detecting phosphopeptides and analyzing their phosphorylation state. However, nonspecific binding peptides, such as acidic, nonphosphorylated peptides, often coelute and make analyses of mass spectra difficult. This study used a partial chemical tagging reaction of a phosphopeptide mixture, enriched by IMAC and contaminated with nonspecific binding peptides, following a modified beta-elimination/Michael addition method, and dynamic mass analysis of the resulting peptide pool. Mercaptoethanol was used as a chemical tag and nitrilotriacetic acid (NTA) immobilized on Sepharose beads was used for IMAC enrichment. The time-dependent dynamic mass analysis of the partially tagged reaction mixture detected intact phosphopeptides and their mercaptoethanol-tagged derivatives simultaneously by their mass difference (-20 Da for each phosphorylation site). The number of new peaks appearing with the mass shift gave the number of multiply phosphorylated sites in a phosphopeptide. Therefore, this partial chemical tagging/dynamic mass analysis method can be a powerful tool for rapid and efficient phosphopeptide identification and analysis of the phosphorylation state concurrently using only MS analysis data.     

Development of a monolithic silica extraction tip for the analysis of proteins

In proteomics, pre-treatment of sample is the most important procedure to remove the matrix for interfacing with mass spectrometry (MS). Additionally, for the samples with low concentration, the process of pre-concentration is required before MS analysis. We have newly developed solid-phase extraction (SPE) tool with pipette-tip shape for purification of bio-samples of various characteristics, utilizing monolithic silica gel as medium. The monolithic silica surface was modified with a C18 phase or coated with titania phase. A C18-bonded tip and a non-modified tip were used for sample concentration, desaltination and removal of detergents from sample. A titania-coated tip was also applied for purification and concentration of phosphorylated peptides. This novel pre-treatment method using monolithic silica extraction tip is much effective and suitable for protein analysis.     

Autonomous protein sample processing on-chip using solid-phase microextraction, capillary force pumping, and microdispensing

A capillary force filling microsystem consisting of a chip-integrated solid-phase microextraction (SMEC) array and a microdispenser for sample purification and trace enrichment of peptides is described. The microextraction array was loaded with solid-phase media (50 microm Poros R2 beads) for purification and enrichment of proteomic samples. Samples bound to the SMEC were eluted in a volume of 200 nL. A piezo-electric microdispenser was docked to the array and the samples bound to the SMEC were eluted in a volume of 200 nL using capillary forces. The purified and enriched samples were dispensed onto the matrix-assisted laser desorption/ionization (MALDI) target, providing quality data from samples in the picomolar range. The nanoproteomic platform was compared to corresponding commercial preparation protocols, showing higher mass spectrometry (MS) signal intensities for peptides generated from an alpha-casein digest. The platform was also evaluated with regards to two-dimensional (2-D) gel-derived protein digests from both fibroblast and epithelial target cells.     

Identification of food-derived bioactive peptides in blood and other biological samples

Recent studies have demonstrated the presence of food-derived peptides in human blood after ingestion of enzymatic hydrolysates of food proteins, while most peptides in food are degraded into amino acids during digestion and absorption. To capture and clarify the food-derived peptides in blood, solid-phase extraction (SPE) using a mini-spin column packed with a strong cation exchanger was developed. This technique allows the use of a nonvolatile acid such as trichloroacetic acid, a strong protein denaturant, for the deproteinizing procedure. To improve resolution of hydrophilic peptide and increase specificity and sensitivity in the detection of peptide by reversed-phase high-performance liquid chromatography (RP-HPLC) after subfractionation by size-exclusion chromatography (SEC), peptides are derivatized with phenyl isothiocyanate. The resultant phenyl thiocarbamyl (PTC)-peptides can be resolved with high resolution and sensitivity by RP-HPLC. By comparing chromatograms of PTC derivatives from blood before and after ingestion of a peptide sample, food-derived peptide can be detected. The isolated PTC-peptide can be applied to a peptide sequencer based on the Edman degradation reaction.     

Enhancement of ionization efficiency and selective enrichment of phosphorylated peptides from complex protein mixtures using a reversible poly-histidine tag

To improve the detection of phosphorylated peptides/proteins, a combination of optimized MS-based strategies were used involving chemical derivatization with a polyhistidine-tag (His-tag) and affinity enrichment of the resulting His-tag peptides on a nanoscale Ni(2+)-IMAC column. The phosphoserine and phosphothreonine peptides were derivatized using a one-pot beta-elimination/Michael addition reaction with a reversible His-tag possessing a thiol-containing Cys residue. The His-tag peptides were enriched selectively by Ni(2+)-IMAC and released using either imidazole or cleavage with Factor Xa. This novel capture and enzyme-mediated release provided an additional element of selectivity and yielded phosphopeptide-specific modifications with enhanced MS ionization characteristics. The eluted peptides were mapped using MALDI-TOF MS and QTRAP ESI-MS/MS techniques. The results obtained for a model peptide and two tryptic protein digests show that the method is highly specific and allows selective enrichment of phosphorylated peptides at low concentrations of femtomoles per microliter.     

共价有机骨架功能材料及其在糖肽选择性富集中的应用

蛋白质糖基化是生物体中最重要的翻译后修饰手段之一,糖蛋白/糖肽的有效分离和富集成为目前糖蛋白组学研究的首要问题。对于复杂的生物样本,糖蛋白的数量较少,酶解后大量高丰度非糖基化修饰肽的存在,使得低丰度糖肽的检测更加困难。因此,需要一些手段来有效地富集糖肽以提高其检测丰度,发展高选择性的糖肽富集材料及方法就成为在分子水平上有效地监测糖蛋白或糖肽的重要途径。相对于传统的糖肽富集材料,共价有机骨架材料具有比表面积大和可修饰位点丰富的优点,在糖肽富集领域具有很大的应用潜力。该文制备了一种新型的共价有机骨架材料(O-T-D-COFs),利用1,3,5-三(4-氨苯基)苯和2,5-二甲氧基苯-1,4-二甲醛作为反应单体通过共聚缩合反应生成的席夫碱构成了材料的框架,对合成后的中间体材料进行氧化处理,从而提高材料的富集性能。利用扫描电镜、透射电镜、红外光谱和固体核磁等表征技术对材料的结构进行了表征,并将其应用于糖肽的选择性富集。分别对富集过程的上样条件、淋洗条件、洗脱条件进行了优化,结合质谱检测技术,从人血清免疫球蛋白G酶解液中观察到32个明显的糖肽信号峰。通过模拟复杂样本体系验证材料富集选择性,在人血清免疫球蛋白G和牛血清白蛋白的酶解液混合物摩尔比达到1∶50时,该材料仍然保持了良好的选择性。此外,还考察了材料的检测限、富集容量、回收率等富集性能,及在实际样品中的应用潜力。以人血清免疫球蛋白G为评价对象,O-T-D-COFs具有较低的检测限(2.5 fmol/μL)、较高的富集容量(120 mg/g),及较好的富集回收率(103.5%±6.6%、101.5%±10.4%)。在血清样品中富集到来自53个N-糖蛋白中的86个N-糖肽序列,并鉴定到了94个N-糖基化位点。这些结果都表明,该材料在糖肽富集领域有较好的应用前景。     

Identification of carbonylation sites in apomyoglobin after exposure to 4‐hydroxy‐2‐nonenal by solid‐phase enrichment and liquid chromatography–electrospray ionization tandem mass spectrometry

Identification of protein carbonylation because of covalent attachment of a lipid peroxidation end‐product was performed by combining proteolytic digestion followed by solid‐phase hydrazide enrichment and liquid chromatography (LC)–electrospray ionization (ESI) tandem mass spectrometry (MS/MS) using both collision‐induced dissociation (CID) and electron capture dissociation (ECD). To evaluate this approach, we selected apomyoglobin and 4‐hydroxy‐2‐nonenal (4‐HNE) as a model protein and a representative end‐product of lipid peroxidation, respectively. Although the characteristic elimination of 4‐HNE (156 Da) in CID was found to serve as a signature tag for the modified peptides, generation of nearly complete fragment ion series because of efficient peptide backbone cleavage (in most cases over 75%) and the capability to retain the labile 4‐HNE moiety of the tryptic peptides significantly aided the elucidation of primary structural information and assignment of exact carbonylation sites in the protein, when ECD was employed. We have concluded that solid‐phase enrichment with both CID‐ and ECD‐MS/MS are advantageous during an in‐depth interrogation and unequivocal localization of 4‐HNE‐induced carbonylation of apomyoglobin that occurs via Michael addition to its histidine residues. Copyright © 2010 John Wiley & Sons, Ltd.     

糖蛋白质组学中基于化学反应的富集方法研究进展

蛋白质糖基化是一种广泛存在的重要的蛋白质翻译后修饰,糖基化肽在总酶解肽中占的比例不超过5%,这使得糖肽的分离富集成为糖蛋白质组学研究发展的关键技术之一。在诸多糖蛋白质组富集技术中,化学方法是富集技术的主导,本文从化学反应的角度介绍糖基化蛋白质富集的技术进展。富集过程按照连接和释放分别讨论,在连接过程中,重点介绍硼酸化学法、肼化学法、胺化学法和肟点击法;在释放过程中,以N-糖蛋白的酶释放法和O-糖蛋白的β-消除法为主导,一并介绍了最新的氧化断裂释放的化学法。最后,讨论总体富集策略的发展现状。该文以糖蛋白富集的共价反应为核心,分析不同方法的优缺点以及各技术在糖蛋白质组学研究中的应用和贡献。     

Cysteine‐reactive covalent capture tags for enrichment of cysteine‐containing peptides

Considering the tremendous complexity and the wide dynamic range of protein samples from biological origin and their proteolytic peptide mixtures, proteomics largely requires simplification strategies. One common approach to reduce sample complexity is to target a particular amino acid in proteins or peptides, such as cysteine (Cys), with chemical tags in order to reduce the analysis to a subset of the whole proteome. The present work describes the synthesis and the use of two new cysteinyl tags, so‐called cysteine‐reactive covalent capture tags (C³T), for the isolation of Cys‐containing peptides. These bifunctional molecules were specifically designed to react with cysteines through iodoacetyl and acryloyl moieties and permit efficient selection of the tagged peptides. To do so, a thioproline was chosen as the isolating group to form, after a deprotection/activation step, a thiazolidine with an aldehyde resin by the covalent capture (CC) method. The applicability of the enrichment strategy was demonstrated on small synthetic peptides as well as on peptides derived from digested proteins. Mass spectrometric (MS) analysis and tandem mass spectrometric (MS/MS) sequencing confirmed the efficient and straightforward selection of the cysteine‐containing peptides. The combination of C³T and CC methods provides an effective alternative to reduce sample complexity and access low abundance proteins. Copyright © 2009 John Wiley & Sons, Ltd.     

Solid-phase extraction for multiresidue determination of sulfonamides, tetracyclines, and pyrimethamine in Bovine's milk

This paper describes a systematic approach to the development of a solid-phase extraction method for simultaneous extraction of 10 antibiotic residues in bovine milk, belonging to groups of sulfonamides, tetracyclines, and pyrimethamine. The sample preparation steps include acidic deproteinization of milk proteins followed by sample enrichment and cleanup using a polymer-based Oasis HLB solid-phase extraction cartridge. The analyses were carried out by using a method based on liquid chromatography-electrospray ionisation-mass spectrometry with positive ion mode. The parameters affecting the extraction efficiency such as sample loading pH, SPE wash solvent composition, and eluting solution pH were carefully investigated and optimized. The developed solid-phase extraction procedure coupled to multiresidue liquid chromatography-electrospray ionization-mass spectrometry method was applied for the analysis of 10 antibiotic residues in milk samples, and it proved to be simple, sensitive, and selective providing a recovery ranging from 70 to 106%.     

Isotope-coded N-terminal sulfonation of peptides allows quantitative proteomic analysis with increased de novo peptide sequencing capability

Recently various methods for the N-terminal sulfonation of peptides have been developed for the mass spectrometric analyses of proteomic samples to facilitate de novo sequencing of the peptides produced. This paper describes the isotope-coded N-terminal sulfonation (ICenS) of peptides; this procedure allows both de novo peptide sequencing and quantitative proteomics to be studied simultaneously. As N-terminal sulfonation reagents, 13C-labeled 4-sulfophenyl[13C6]isothiocyanate (13C-SPITC) and unlabeled 4-sulfophenyl isothiocyanate (12C-SPITC) were synthesized. The experimental and reference peptide mixtures were derivatized independently using 13C-SPITC and 12C-SPITC and then combined to generate an isotopically labeled peptide mixture in which each isotopic pair differs in mass by 6 Da. Capillary reverse-phase liquid chromatography/tandem mass spectrometry experiments on the resulting peptide mixtures revealed several immediate advantages of ICenS in addition to the de novo sequencing capability of N-terminal sulfonation, namely, differentiation between N-terminal sulfonated peptides and unmodified peptides in mass spectra, differentiation between N- and C-terminal fragments in tandem mass spectra of multiply protonated peptides by comparing fragmentations of the isotopic pairs, and relative peptide quantification between proteome samples. We demonstrate that the combination of N-terminal sulfonation and isotope coding in the mass spectrometric analysis of proteomic samples is a viable method that overcomes many problems associated with current N-terminal sulfonation methods.     

基于共价色谱分离的含半胱氨酸肽段富集策略

多维色谱分离、串联质谱分析技术已在蛋白质组研究中得到广泛应用。然而生物样品的蛋白质以及全酶切肽段具有高度的复杂性,这严重干扰了蛋白质高通量、规模化的分析。通过标签肽段富集进行样品预分离可以降低体系的复杂程度。本文建立了一种基于共价色谱技术选择性分离富集含半胱氨酸肽的方法,从而降低了样品体系的复杂程度。首先以牛血清白蛋白(BSA)的酶切肽段为模型,对富集条件进行了优化和考察,并在此基础上通过5种蛋白质酶切肽段混合物的富集对该方法进行了验证。结果证明此方法的重现性好,富集效率高,富集特异性好,能有效地富集鉴定含半胱氨酸肽段。所建立的方法在复杂体系的蛋白质组研究中具有广泛的应用前景,为复杂样品的蛋白质高通量、自动化、规模化鉴定和定量研究提供了实用技术。     

Polymeric integrated selective enrichment target (ISET) for solid-phase-based sample preparation in MALDI-TOF MS

A polymer microfabricated proteomic sample preparation and MALDI MS sample presentation device, the integrated selective enrichment target (ISET), comprising an array of perforated nanovials is reported. Each perforated nanovial can be filled with selective extraction media (microbeads) for purification and concentration of protein/peptides prior to matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). The main areas covered are the influence of the molding-process-induced surface roughness and how to address the lack of inherent conductivity in the polyetheretherketone (PEEK) material for optimal MALDI MS readout. Application of the disposable polymeric ISET devices for solid-phase extraction and phosphopeptide capture is also demonstrated.     

水热碳材料在分离分析中的应用研究进展

水热碳材料是一种由糖类或含碳有机物经过水热反应制备的新型功能材料,具有来源丰富、绿色环保、亲水性、易修饰等优点,已广泛应用于催化剂载体、能源电极材料、环境吸附剂等方面。其中,水热碳在吸附领域的应用显示出其与特定分子的相互作用,近年来,水热碳材料作为分离富集固相基质,在色谱固定相以及生物样品处理领域逐渐得到应用。该文主要综述了水热碳材料在离子化合物、极性化合物、磷酸化肽段和糖基化肽段分离分析等方面的最新应用,讨论了水热碳材料在实际分离分析应用中的优点和局限性,并就水热碳材料在该领域的应用前景进行了展望。     

A novel class of chemically modified iodo-containing resins: design, synthesis and application to mass spectrometry-based proteome analysis

A novel class of chemically modified iodo-containing resins with isotope-labeled tagging for mass spectrometry-based proteome analysis is described. This iodo-containing resin contains a thiol-reactive group that is used to capture the cysteine (Cys)-containing peptides from peptide mixtures, one 'tag' amino acid, and an aminomethyl polystyrene resin with Rink Amide Linker. The 'tag' amino acid is synthesized in both heavy and light isotope-coded forms and therefore permits the direct relative quantification of peptides/proteins through mass spectrometric analysis. In the iodo-containing resin strategy, the Cys-containing peptides of two samples covalently captured by either light or heavy iodo-containing resin were mixed and washed extensively under stringent conditions. Then the Cys-containing peptides were retrieved by acid-catalyzed elution. Finally, the eluted peptides were directly analyzed by micro liquid chromatography/mass spectrometry for identification and relative quantification. The iodo-containing resins were synthesized by a simple but effective method. Their abilities to identify and quantify the Cys-containing part in two samples were proved by the analysis of mixtures of amino acids, peptides and proteins.     

Determination of Chloroacetic Acids in Drinking Water by Reaction Gas Chromatography

A procedure was developed for determining chloroacetic acids in drinking water. The procedure is based on solid-phase extraction followed by the conversion of chloroacetic acids to trifluoroacetic esters and their separation and determination by gas–liquid chromatography with an electron capture detector. Esterification conditions were optimized. The procedure was used for analyzing samples of drinking water.