介孔氧化铁材料快速、高效富集分离磷酸化肽段和蛋白的新方法研究

以0.1μmol/Lβ-casein磷酸化蛋白酶解液为对象,利用在酸性条件下对PO43-能够特异性吸附的氧化铁材料为新载体,对介孔氧化铁材料富集分离磷酸化肽段的孵育液酸含量、孵育液有机溶剂含量和洗脱液选择的条件进行了优化,结果表明:室温条件下,在含有0.1%乙酸和30%乙腈的孵育液中孵育5min后,经1mol/LNH3.H2O溶液的洗脱,介孔氧化铁可有效地将磷酸化肽段从蛋白酶解液中富集分离。本方法也可以选择性地提取α-casein磷酸化蛋白,实现了简单、快速、高效的磷酸化肽段和蛋白的富集分离。同时,通过MALDI-TOF串级质谱分析,成功地完成了在优化条件下分离出的磷酸化肽段磷酸位点的鉴定。     

基于Fmoc策略的O-磷酸化多肽化学合成研究

以Fmoc-策略固相合成方法为基础,以亚磷酰胺为磷酸化试剂,分别以总体磷酸化法和单体磷酸化法合成了多种磷肽、修饰磷肽及其对应的非磷酸化多肽,并以乙腈/水/0.06%三氟乙酸为洗脱体系,用HPLC对磷肽和多肽进行分离.肽链的长度增加,总体法的磷酸化效率降低;这种基于Fmoc-策略的单体磷酸化法目前只适用于含酪氨酸磷肽的合成.     

基于特征肽段的液相色谱-质谱技术鉴定胶原蛋白的物种来源

建立了一种基于特征肽段的液相色谱-质谱技术鉴定胶原蛋白物种来源的方法。样品经蛋白提取,还原,烷基化,胰蛋白酶消化后,采用Eksigent C18色谱柱(75μm×150 mm,3μm)分离,用流动相0. 1%甲酸水-乙腈溶液(98∶2)和0. 1%甲酸乙腈-水溶液(98∶2)梯度洗脱,在正离子模式下,通过纳升电喷雾四极杆飞行时间质谱进行检测,数据经Protein PilotTM软件及blast分析,筛选出潜在的特征肽段。消化后的样品再采用Eclipse Plus C18色谱柱(2. 1 mm×100 mm,1. 8μm)分离,用流动相乙腈和1%甲酸水溶液梯度洗脱,在正离子模式下,通过电喷雾四极杆/线性离子阱串联质谱的多反应监测触发增强子离子扫描模式进行检测,进一步确认肽段的特异性。最终筛选并确证了3种猪源性胶原蛋白特征肽段,4种牛源性胶原蛋白特征肽段,1种羊源性胶原蛋白特征肽段。所筛选的特征肽段具有良好的耐热性,可为动物源性胶原蛋白鉴定提供一种特异性强、准确可靠的检测方法。     

新型反相/强阴离子交换混合模式材料用于磷酸化肽富集

建立了新型反相/强阴离子交换混合模式材料(C18/SAX)的磷酸化肽富集方法.考察了流动相组成(乙腈浓度、甲酸浓度、缓冲盐浓度)对酪蛋白(α-Casein)酶解液中磷酸化肽分离选择性的影响.实验结果表明,磷酸化肽在C18/SAX上的保留行为受疏水和离子交换作用力的共同调控,单磷酸化肽先于多磷酸化肽从材料上洗脱出来.随着甲酸浓度增加,磷酸化肽的保留减弱;随着盐浓度增加,磷酸化肽保留变小.采用优化后的流动相,建立以20％ ACN/20 mmol/L NH4Ac作为上样溶液,20％ ACN/0.1％ FA和50％ ACN/100 mmol/L NH4Ac/2％ FA分别作为洗脱液分段洗脱单、多磷酸化肽的方法.以α-Casein和人血清白蛋白(HSA)酶解液的混合溶液(1∶20,n/n)作为模拟样品,实现了单、多磷酸化肽的同时富集和分段洗脱,分别检测到4条单磷酸化肽和14条多磷酸化肽的信号.将本方法用于牛奶中的磷酸化肽检测,共鉴定到4条单磷酸化肽和8条多磷酸化肽信号.结果表明,本富集方法选择性高,有良好的应用前景.     

质谱法分析大肠癌组织中亲核蛋白A的蛋白质变体

采用超滤、亲和层析和双向电泳分离并纯化了人大肠癌组织中的亲核蛋白,结合蛋白印迹分析,发现亲核蛋白存在几个蛋白质变体的现象。采用基质辅助激光解吸电离-飞行时间质谱以及电喷雾串联质谱技术,对亲核蛋白的主要蛋白质变体的胰酶水解肽段进行了一级结构的解析,发现大肠癌组织中的亲核蛋白主要存在N端乙酰化(Ac-1Met及脱去N端甲硫氨酸后的Ac-2Val)、5Thr的磷酸化等修饰状态,这些修饰形式单独或者相互组合构成了亲核蛋白的主要蛋白质变体。此外,在亲核蛋白中还发现一些氧化修饰和脱氨基修饰的情况。研究表明,利用质谱及串联质谱技术,能够快速、准确地鉴定人大肠癌组织中分离得到的亲核蛋白的几种蛋白质变体。     

液相色谱-串联质谱法测定牛奶中5种多肤类抗生素

建立了牛奶中杆菌肽、粘杆菌素A、粘杆菌素B、维吉尼霉素和万占霉素5种多肽类抗生素的反相液相色谱-串联质谱(HPLC-MS/MS)检测方法.牛奶样品经甲醇-0.1%甲酸水提取后,用4%三氯乙酸乙睛除蛋白,液-液萃取后,采用0.1%甲酸(A)和0.1%甲酸乙腈(B)作为流动相进行梯度洗脱.质谱(ESI+)采用多离子检测模式...     

分散固相萃取-液相色谱-串联质谱法测定水果中19种酸性农药

建立了同时检测葡萄、苹果和桔子等水果中2,4-滴等19种酸性农药的分散固相萃取-液相色谱-串联质谱(HPLC-MS/MS)方法。葡萄、苹果和桔子样品经乙酸-乙腈(1∶99,V/V)提取后,C18分散固相萃取净化,采用反相C18色谱柱分离。以0.1%甲酸和0.1%甲酸-乙腈溶液作为流动相进行梯度洗脱,采用多反应监测离子模式进行定性分析,基质标准曲线外标法进行定量分析,线性范围在0.01~0.2 mg/kg之间。在0.02,0.05和0.1 mg/kg添加水平下,19种酸性农药的回收率为70.3%～105.3%;相对标准偏差为0.37%～10.9%。本方法的定量限为0.005～0.02 mg/kg。     

高效液相色谱法测定食品添加剂中水杨酸

提出了高效液相色谱法测定食品添加剂中水杨酸含量的方法。样品采用含0.1%(体积分数)甲酸的甲醇-水(9+1)混合溶剂溶解,超声提取后经0.22μm滤膜过滤,滤液供高效液相色谱荧光仪测定。采用ZORBAX SB-C18色谱柱(4.6mm×250mm,3.5μm)分离,用不同配比的(A)甲酸-乙腈(0.1+99.9)和(B)甲酸-水(0.1+99.9)的混合溶液为流动相梯度洗脱,在激发波长为290nm、发射波长为400nm处检测。水杨酸的质量浓度在41.60～1 664μg·L-1范围内与其对应的峰面积呈线性关系,检出限(3S/N)为1.55μg·L-1,方法的回收率在90.4%～101.7%。     

液相色谱-串联质谱法测定水产品中磺胺类及喹诺酮类药物残留量

水产品样品(5.00g)经乙腈-甲酸(99+1)混合液20mL提取,无水乙醇除水,浓缩并加正己烷2mL脱脂。所得溶液进行液相色谱分离。以ACQUITY UPLC BEH HILIC色谱柱为分离柱,以不同体积比的甲醇和0.1%(体积分数)甲酸溶液的混合液为流动相进行梯度洗脱。质谱分析中,采用电喷雾正离子源多反应监测模式检测。采用内标法定量。所涉11种药物的线性范围均为5~200μg·L~(-1),方法的测定下限(10S/N)在0.07~0.20μg·kg~(-1)之间。在1.0,4.0,20.0μg·kg-1等3个浓度水平进行加标回收试验,回收率在80.3%~119%之间,测定值的相对标准偏差(n=6)在1.3%~12%之间。     

新型固定化铁离子亲和色谱整体柱的制备及评价

以N-丙烯酰氧基琥珀酰亚胺为功能单体,制备了含有高活性基团的整体材料基质.以次氨基三乙酸为配体,通过固载Fe3+,发展了一种固定化Fe3+亲和色谱(Fe-IMAC)整体柱的制备方法.该整体柱不仅对磷酸化肽具有很好的选择性,而且富集容量大、回收率高、重现性好.此外,利用该整体柱实现了牛奶蛋白质酶解产物中磷酸化肽段的选择性富集.本实验研制的Fe-IMAC整体柱有望用于磷酸化蛋白质组研究.     

Profiling of Ubiquitination Modification Sites in Talin in Colorectal Carcinoma by Mass Spectrometry

Talin protein was partially purified from human colorectal carcinoma tissues, which was subject to tryptic digestion. Immunoaffinity precipitation with specific antibodies that recognize diglycyl-lysine(Lys) remnants from tryptic digestion of ubiquitinated peptides was used to enrich ubiquitinated sites in talin. Mass spectrometry coupled with capillary reverse-phase high-performance liquid chromatography was used to analyze tlie enriched peptides. Specifically, four peptides containing diglycyl-Lys remnants from talin, namely, TAK(ub)VLVEDTK, QQQYK(ub) FLPSELRDEH, K(ub)STVLQQQYNR, and EGILK(ub)TAK can be determined using mass spectrometric data. This study provides an analytical method for further study in tlie relationship between ubiquitination modification of talin and its biological activity in colorectal cancer tissues with different pathological processes.     

Quantitative comparison of IMAC and TiO<Subscript>2</Subscript> surfaces used in the study of regulated,dynamic protein phosphorylation

Protein phosphorylation regulates many aspects of cellular function, including cell proliferation, migration, and signal transduction. An efficient strategy to isolate phosphopeptides from a pool of unphosphorylated peptides is essential to global characterization using mass spectrometry. We describe an approach employing isotope tagging reagents for relative and absolute quantification (iTRAQ) labeling to compare quantitatively commercial and prototypal immobilized metal affinity chelate (IMAC) and metal oxide resins. Results indicate a prototype iron chelate resin coupled to magnetic beads outperforms either the Ga(3+)-coupled analog, Fe(3+), or Ga(3+)-loaded, iminodiacetic acid (IDA)-coated magnetic particles, Ga(3+)-loaded Captivate beads, Fe(3+)-loaded Poros 20MC, or zirconium-coated ProteoExtract magnetic beads. For example, compared with Poros 20MC, the magnetic metal chelate (MMC) studied here improved phosphopeptide recovery by 20% and exhibited 60% less contamination from unphosphorylated peptides. With respect to efficiency and contamination, MMC performed as well as prototypal magnetic metal oxide-coated (TiO(2)) beads (MMO) or TiO(2) chromatographic spheres, even if the latter were used with 2,5-dihydroxybenzoic acid (DHB) procedures. Thus far, the sensitivity of the new prototypes reaches 50 fmol, which is comparable to TiO(2) spheres. In an exploration of natural proteomes, tryptic (phospho)peptides captured from stable isotopic labeling with amino acids in cell culture (SILAC)-labeled immunocomplexes following EGF-treatment of 5 x 10(7) HeLa cells were sufficient to quantify stimulated response of over 60 proteins and identify 20 specific phosphorylation sites.     

Enrichment and analysis of phosphopeptides under different experimental conditions using titanium dioxide affinity chromatography and mass spectrometry

Titanium dioxide metal oxide affinity chromatography (TiO₂‐MOAC) is widely regarded as being more selective than immobilized metal‐ion affinity chromatography (IMAC) for phosphopeptide enrichment. However, the widespread application of TiO₂‐MOAC to biological samples is hampered by conflicting reports as to which experimental conditions are optimal. We have evaluated the performance of TiO₂‐MOAC under a wide range of loading and elution conditions. Loading and stringent washing of peptides with strongly acidic solutions ensured highly selective enrichment for phosphopeptides, with minimal carryover of non‐phosphorylated peptides. Contrary to previous reports, the addition of glycolic acid to the loading solution was found to reduce specificity towards phosphopeptides. Base elution in ammonium hydroxide or ammonium phosphate provided optimal specificity and recovery of phosphorylated peptides. In contrast, elution with phosphoric acid gave incomplete recovery of phosphopeptides, whereas inclusion of 2,5‐dihydroxybenzoic acid in the eluant introduced a bias against the recovery of multiply phosphorylated peptides. TiO₂‐MOAC was also found to be intolerant of many reagents commonly used as phosphatase inhibitors during protein purification. However, TiO₂‐MOAC showed higher specificity than immobilized gallium (Ga³⁺), immobilized iron (Fe³⁺), or zirconium dioxide (ZrO₂) affinity chromatography for phosphopeptide enrichment. Matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS) was more effective in detecting larger, multiply phosphorylated peptides than liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI‐MS/MS), which was more efficient for smaller, singly phosphorylated peptides. Copyright © 2009 Crown in the right of Canada. Published by John Wiley & Sons, Ltd.     

Detection and sequencing of phosphopeptides

Consecutive enzymatic reactions of analytes which are affinity bound to immobilized metal ion beads with subsequent direct analysis of the products by matrix-assisted laser desorption/ionization mass spectrometry have been used for detecting phosphorylation sites. The usefulness of this method was demonstrated by analyzing two commercially available phosphoproteins, beta-casein and alpha-casein, as well as one phosphopeptide from a kinase reaction mixture. Agarose loaded with either Fe3+ or Ga3+ was used to isolate phosphopeptides from the protein digest. Results from using either metal ion were complementary. Less overall suppression effect was achieved when Ga3+-loaded agarose was used to isolate phosphopeptides. The selectivity for monophosphorylated peptides, however, was better with Fe3+-loaded agarose. This technique is easy to use and has the ability to analyze extremely complicated phosphopeptide mixtures. Moreover, it eliminates the need for prior high-performance liquid chromatography separation or radiolabeling, thus greatly simplifying the sample preparation.     

Facile preparation of titanium phosphate‐modified chitosan for selective capture of phosphopeptides

A facile two‐step method for preparing chitosan‐based immobilized metal ion affinity chromatography was developed. First, chitosan was phosphorylated by esterification with phosphoric acid, and then titanium was chelated onto the phosphorylated chitosan. The obtained chitosan‐based titanium immobilized metal ion affinity chromatography was ultrafine microparticles and had good dispersibility in acidic buffer. The selectivity and sensitivity were evaluated by phosphopeptide enrichment of mixtures of α‐casein and bovine serum albumin. The enriched peptides were analyzed by mass spectrum. Enrichment protocols were optimized and the optimum‐loading buffer was 80% acetonitrile with 1% trifluoroacetic acid. With α‐casein concentration as low as 2 pmol, 12 phosphopeptides were detected with considerably high intensity from the digest mixtures of α‐casein and bovine serum albumin with molar ratio of 1:200. The microparticles was also applied in real biological samples, 29 phosphoproteins containing 40 phosphorylated sites were identified from salt‐stressed Arabidopsis thaliana leaves.     

Selective detection and identification of phosphorylated proteins by simultaneous ligand-exchange fluorescence detection and mass spectrometry

A ligand-exchange method for the detection and identification of phosphorylated peptides in complex mixtures is presented that is based on the characterization of phosphorylated species by solution-phase interactions with Fe(III) ions and subsequent fluorescence readout. After the separation of the peptides and digest products on a reversed-phase LC column, the flow is split between the two detection systems. One part is directed towards an electrospray mass spectrometer for direct detection and identification of all the peptides present in the sample. The other part of the flow is directed towards a ligand-exchange detection system. This system relies on the specific release of a fluorescent reporter ligand from a Fe(III)-complex in the presence of phosphorylated peptides. To recognize false positive signals due to high-affinity non-phosphorylated high-acidic peptides and other compounds which are known to be a problem in for instance immobilized metal affinity chromatography (IMAC), a second run is performed after incubation of the sample with alkaline phosphatase. A positive signal in this second run indicates a high-affinity non-phosphorylated compound. The method is illustrated using digest from a phosphorylated alpha-casein. Automated switching between MS and MS-MS was performed to obtain additional information about the compounds present in the sample. The linearity of the method was tested in the range of 0.5-80 microM of phosphorylated peptides. A limit of detection (LOD) of 0.5 microM was obtained for a mono-phosphorylated peptide. The interday (n=4) and intraday precision (n=3) expressed as relative standard deviation was better than 10%.     

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.     

人神经母细胞瘤的磷酸化膜蛋白质组分析

针对人神经母细胞瘤SH-SY5Y细胞系的磷酸化膜蛋白质组,发展了基于多酶酶解法结合杂化硅胶基质固定化钛离子亲和色谱(Ti4+-IMAC)整体柱富集的分析策略。该方法通过对细胞裂解液进行超速离心,以及1 mol/L NaCl和0.1 mol/L Na2CO3顺序清洗,获得膜蛋白质组分。所提取的蛋白质分别经胰蛋白酶、胰凝乳蛋白酶和胃蛋白酶平行酶解,产生的肽段经Ti4+-IMAC整体柱选择性富集磷酸肽后,采用纳升级反相液相色谱分离和质谱鉴定,成功鉴定到43个磷酸化蛋白质,其中有14个定位于膜上。研究结果表明,采用该策略开展SH-SY5Y细胞系磷酸化膜蛋白质组学分析有望加速对该肿瘤的研究和相关潜在标记物的筛选。     

Highly specific enrichment of phosphopeptides by zirconium dioxide nanoparticles for phosphoproteome analysis

Large-scale characterization of phosphoproteins requires highly specific methods for the purification of phosphopeptides because of the low abundance of phosphoproteins and substoichiometry of phosphorylation. A phosphopeptide enrichment method using ZrO2 nanoparticles is presented. The high specificity of this approach was demonstrated by the isolation of phosphopeptides from the digests of model phosphoproteins. The strong affinity of ZrO2 nanoparticles to phosphopeptides enables the specific enrichment of phosphopeptides from a complex peptide mixture in which the abundance of phosphopeptides is two orders of magnitude lower than that of nonphosphopeptides. Superior selectivity of ZrO2 nanoparticles for the enrichment of phosphorylated peptides than that of conventional immobilized metal affinity chromatography was observed. Femtomole phosphopeptides from digestion products could be enriched by ZrO2 nanoparticles and can be well detected by MALDI mass spectrometric analysis. ZrO2 nanoparticles were further applied to selectively isolate phosphopeptides from the tryptic digestion of mouse liver lysate for phosphoproteome analysis by nanoliter LC MS/MS (nano-LC-MS/MS) and MS/MS/MS. A total of 248 defining phosphorylation sites and 140 phosphorylated peptides were identified by manual validation using a series of rigid criteria.