纳升级电喷雾离子源-四极杆-飞行时间质谱中磷酸化肽的离子抑制作用

目前磷酸化蛋白质组学研究中的主要技术是蛋白质酶解产生的磷酸化肽的质谱检测。但是实际样品中的磷酸化肽(特别是多磷酸化肽)很难被检测到。其原因普遍认为是由于质谱检测时,非磷酸化肽抑制磷酸化肽。但也有认为非磷酸化肽对磷酸化肽没有抑制作用。另外磷酸化肽之间是否存在离子抑制作用还没有报道。本文采用相同氨基酸序列的标准磷酸化肽和非磷酸化肽,将其单独和混合进行质谱检测,通过对比混合前后磷酸化肽的信号强度,证明了非磷酸化肽对磷酸化肽有离子抑制作用;单磷酸化肽对二磷酸化肽有一定的抑制作用,但不太显著;单磷酸化肽对三磷酸化肽、二磷酸化肽对三磷酸化肽均有显著的离子抑制作用。该研究为今后单磷酸化肽和多磷酸化肽的分段富集和检测提供了有力的证明。     

混合模式色谱材料Click TE-GSH高效富集多磷酸化肽

多磷酸蛋白对于生物体适应内外环境具有重要意义,而明确多磷酸蛋白的磷酸位点功能及其信号转导机制尤为关键. 复杂生物样品中多磷酸化肽的低丰度、低电离的特性,以及非磷酸化肽的抑制作用,决定了质谱分析前进行多磷酸化肽富集是非常必要的步骤. 本工作采用基于巯基-烯烃点击化学法合成的混合模式材料Click TE-GSH进行单磷酸化肽和多磷酸化肽的选择性富集. 我们建立了单磷酸化肽、双磷酸化肽和多磷酸化肽的顺序分段富集方法. 该优化方法能抗干扰,应用于脱脂牛奶时富集到11条多磷酸化肽. 与商品化固化金属亲和色谱(IMAC)材料相比,Click TE-GSH富集多磷酸化肽的选择性更好. 本工作所建立的富集方法为高效富集多磷酸化肽提供新方法和新技术.     

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.     

合成受体与模型磷酸化肽的相互作用机理

为了研究基于Zn2+-二甲基吡啶胺及胍羰基吡咯基团的配位型受体Zn Dpa G与磷酸化肽的相互作用机制,选取具有不同序列的磷酸化肽作用模型,采用等温滴定微量热法考察了Zn Dpa G与磷酸化肽的结合常数,研究了模型肽中磷酸基团的数量、密度及位置等因素对多肽与受体间结合强度的影响.结果表明,Zn Dpa G受体对双磷酸化肽结合能力显著高于单磷酸化肽,其结合常数可提高10~40倍,2个磷酸基团的距离越近,结合作用越强;而磷酸基团的位置显著影响受体与单磷酸化肽的结合强度.本研究结果为进一步优化磷酸化肽受体结构设计,实现肽与受体间高选择性识别提供了一定的理论依据.     

季铵化磁性壳聚糖复合材料对磷酸化肽的富集研究

蛋白质磷酸化是最重要和最普遍的翻译后修饰之一,蛋白质磷酸化的测定对于全面了解生物过程中的磷酸化途径至关重要。质谱技术是分析蛋白质磷酸化的重要手段,但磷酸化肽固有的低丰度、低电离效率以及与非磷酸化肽共存等特性严重影响质谱对其进行直接分析。为解决此问题,需在质谱分析前对磷酸化肽进行选择性富集。本研究制备了一种基于季铵化磁性壳聚糖的复合材料用于磷酸化肽的富集。此磁性材料具有快速的磁响应性、良好的生物相容性、正电性以及廉价易得等优点。采用β-酪蛋白作为模型蛋白质,结果表明此材料对磷酸化肽具有良好的富集选择性。经过富集后,结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）检测手段,方法的检出限为0.4 fmol。本方法被成功用于脱脂牛奶中磷酸化肽的检测,表明其对复杂样品中磷酸化肽的富集和检测具有良好的应用潜力。     

毛细管反相液相色谱-串联质谱用于肽的鉴定和相对定量分析

建立了一种基于毛细管反相液相色谱-串联质谱联用技术和质谱峰强度数据处理的肽段鉴定和相对定量分析方法。该方法无需对样品中的肽进行化学标记,在对样品进行反相色谱分离和串联质谱分析后,将二级质谱扫描数据进行蛋白质数据库搜索,获得所鉴定肽段的序列、保留时间、质荷比、带电荷数等定性信息;再以此为定位依据,在全扫描质谱数据中提取该肽段对应的离子峰并以该离子峰的峰强度作为定量信息,从而实现对不同样品中的共有肽段进行差异比较分析。以标准蛋白酶解混合肽段为实验对象,以肽段相对强度的相对标准偏差为指标,考察了该方法用于肽段相对定量分析的重现性、检测动态范围以及浓度标准曲线等,为将该方法用于生物样品中内源性肽的差异分析奠定了基础。     

反相高效液相色谱法测定蟾酥中的3种蟾毒内酯

建立了一种基于毛细管反相液相色谱-串联质谱联用技术和质谱峰强度数据处理的肽段鉴定和相对定量分析方法。该方法无需对样品中的肽进行化学标记,在对样品进行反相色谱分离和串联质谱分析后,将二级质谱扫描数据进行蛋白质数据库搜索,获得所鉴定肽段的序列、保留时间、质荷比、带电荷数等定性信息;再以此为定位依据,在全扫描质谱数据中提取该肽段对应的离子峰并以该离子峰的峰强度作为定量信息,从而实现对不同样品中的共有肽段进行差异比较分析。以标准蛋白酶解混合肽段为实验对象,以肽段相对强度的相对标准偏差为指标,考察了该方法用于肽段相对定量分析的重现性、检测动态范围以及浓度标准曲线等,为将该方法用于生物样品中内源性肽的差异分析奠定了基础。。     

多金属氧酸盐/壳聚糖磁性复合材料的制备及其用于磷酸化肽的富集

建立了一种基于多金属氧酸盐磁性材料富集磷酸化肽的方法。采用层层自组装技术制备多金属氧酸盐/壳聚糖磁性材料,结合基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）检测手段,用于磷酸化肽的富集。该磁性材料具有快速磁响应、亲水性、正电性等优点,对磷酸化肽具有高的富集选择性。实验用β-酪蛋白作为模型蛋白质,通过富集后,方法的检出限为0.02 fmol,说明合成的磁性材料对微量蛋白样品分析具有很高的应用潜力。     

蛋白质组衍生肽段文库用于鉴定酪蛋白激酶2的底物模体

模体是蛋白质二级结构上的一种特征序列,激酶底物通常具有一类特征性的模体,识别底物模体对鉴定激酶底物具有重要意义。为了快速鉴定激酶底物模体,将全细胞蛋白质酶解液作为肽段文库的来源,采用碱性磷酸酶去除肽段的固有磷酸化后构建了用于筛选激酶底物模体的肽段文库。该肽段文库是大量非磷酸化肽段的混合物,将此混合肽段与酪蛋白激酶2和三磷酸腺苷作用30 min后,通过固定化金属离子亲和色谱法富集磷酸化肽段,采用反相液相色谱-串联质谱分析,成功地鉴定到472条非冗余底物肽段,包含451个非冗余磷酸化位点,并由此得到底物模体S/T-D/E-x-D/E。该法能够快速准确地筛选出激酶底物模体,对研究激酶-底物识别以及信号转导过程具有重要意义。     

基于保留时间和质荷比匹配的液相色谱-质谱联用技术用于非标记肽段的差异分析

建立了一种无需化学标记的,基于纳升级毛细管液相色谱-电喷雾离子阱质谱联用技术和质谱数据处理的肽段差异分析方法。本方法采用定量差异分析与肽序列鉴定分析分别进行的策略,首先对样品进行质谱全扫描的液质全谱式分析,在全扫描质谱数据中提取肽特征点信息,通过保留时间和质荷比参数匹配不同样品中的共有肽特征点,比较其相对峰强度有无差异。最后对样品中存在丰度差异的肽特征点进行选择性二级质谱分析和序列鉴定,从而实现复杂样品中肽段的差异比较分析。以血浆蛋白酶解混合物为实验对象,考察了本方法用于肽段相对定量分析的重现性以及浓度信号响应曲线等。结果表明:提取的肽特征点峰强度相对标准偏差的中值<22%,肽段离子强度动态范围达3个数量级,在5～1000fmol范围内对肽段定量具有良好线性关系。本方法可用于不同条件样品中具有倍数差异的内源性肽的比较分析。     

Characterization of multi-phosphopeptides by muHPLC-ESI-MS/MS with alkaline phosphatase treatment

The detection of phosphopeptides, especially multi-phosphopeptides, by tandem electrospray ionization mass spectrometry (ESI-MS/MS) is a great challenge due to their low abundance and the poor ionization efficiency of samples. In our recent study, a strategy was proposed for the analysis of trace multi-phosphopeptides which combined selective enrichment of phosphorylated peptides by TiO2 and dephosphorylation by alkaline phosphatase (AP). After separation by muHPLC, the profiles of enriched peptides before and after AP treatment were compared, and the additional peaks appearing in the latter case hinted at the existence of multi-phosphopeptides. Subsequently, an incomplete dephosphorylation reaction was performed to partially remove the phosphate groups so that the phosphorylation sites of the multi-phosphopeptides might be estimated. Through analysis of the digests of beta-casein and extracted proteins of bovine milk, more information on the multi-phosphopeptides was obtained by muHPLC-ESI-MS/MS than that obtained without AP treatment, which demonstrated that such a strategy might supply some potential information about trace multi-phosphopeptides lost in shotgun analysis.     

Enhanced Multi-phosphopeptide Enrichment and Nano LC-ESI-qTOF-MS Detection Strategy Using Click OEG-CD Matrix

Detection and determination of multi-phosphopeptides from protein digestion products are difficult due to the low abundance and ion-suppression effect arised from the existence of their high-abundance counterparts. Click OEG-CD[olio(ethylene glycol)(OEG) linked β-cyclodextrin(CD)] matrix has shown excellent performance in phosphopeptide enrichment. However, few multi-phosphopeptides were detected previously. In this investigation, an improved method aiming at enhancing the enrichment selectivity and mass spectrometry(MS) detection of multi-phosphopeptide was developed via optimizing the sample loading amount on per mg of matrix. Mass spectra were obtained on a Nano liquid chromatography-electrospray ionization-quadrupole time of flight-mass spectrometry (LC-ESI-qTOF-MS). The enrichment selectivity of double-phosphopeptide could be enhanced with the increase of loading amount on per mg of matrix when taking the mixture of mono-, double-, and non-phosphopeptides as probe. Furthermore, the multi-phosphopeptide enrichment selectivity was enhanced under the condition of optimized loading amount when taking the product of typtic digestion α-casein as test sample. When the loading amount was 573 pmol/mg matrix, up to 20 α-casein phosphopeptide signals(including 15 multi-phosphopeptides) were detected. The result is much better than that of our previous report. The reduction of ion-suppression effect arised from the existence of high-abundance non-or mono-phosphopeptides and the stronger interactions between multi-phosphopepides and the matrix were contributed to the result. The study could be helpful to the better utilization of Click OEG-CD matrix in the enrichment of multi-phosphopeptide from complex biosamples in the subsequent investigation.     

On-membrane digestion of beta-casein for determination of phosphorylation sites by matrix-assisted laser desorption/ionization quadrupole/time-of-flight mass spectrometry

This article discusses the features of a newly developed matrix-assisted laser desorption/ionization quadrupole/time-of-flight (MALDI-QqTOF) mass spectrometer that is useful in the analysis of phosphorylated peptides. Aliquots of beta-casein, a commonly used phosphorylated protein standard, were digested with trypsin directly on a non-porous polyurethane membrane used as sample support in MALDI-QqTOF mass spectrometry (MS) experiments. Although a complete peptide map was obtained, it was difficult to obtain sequence information for some of the tryptic fragments, in particular T1-2, which bears four phosphate groups and is thus difficult to ionize in positive mode. This article focuses on the sequencing of this particular fragment by comparing MS/MS spectra obtained using different precursor ions. These precursors associated with T1-2 were [M + H](+), [M + H](2+), and [M + H - nH(3)PO(4)](+) ions. Typically, phosphorylated ions showed facile unimolecular losses of phosphoric acid moieties, and produced limited backbone fragmentation. The abundance of [M + H](2+) ions of T1-2 in the full mass spectrum was low relative to that of [M + H](+). [M + H - 4H(3)PO(4)](+) ions as MS/MS precursors underwent backbone fragmentations, with phosphoserine residues transformed into dehydroalanines or serines. Unusual b + 18 u fragments were observed, although only for segments with previously phosphorylated serines. These partly interfered with c-ions, and were noticeable due to overlapping isotopic envelopes. It was possible to establish the sequence of phosphorylated tryptic fragment T1-2 and the location of phosphate groups using the mass of dehydroalanine residues (69 Da) and b + 18 u fragments as markers. All MS and MS/MS spectra obtained with fully phosphorylated beta-casein were compared with spectra acquired with dephosphorylated beta-casein obtained commercially. These comparisons helped assess the spectral differences caused by the presence of phosphate groups. Also, they highlighted the potential usefulness of conducting dephosphorylation directly on the probe prior to MALDI analysis in future studies.     

咪唑键合硅胶固定相微柱液相色谱分离酚类和胺类化合物

由于微柱液相色谱(μ-LC)具有高检测灵敏度、低溶剂消耗、可以与质谱等多种检测器联用的优点,近年来受到广泛关注。将咪唑键合硅胶固定相填充到毛细管中,在自制的微柱液相色谱系统下利用此键合相具有的弱疏水作用,采用不同的流动相对酚类和胺类化合物进行了分离。结果表明,流动相中只需添加少量的有机溶剂就可以实现对一些有机化合物的分离,甚至可以只用纯水作流动相就能分离一些弱疏水性化合物,如酚类。微柱液相色谱的流动相用量少,避免或大大减少了对环境的污染。自制微柱液相色谱系统为下一步微柱液相色谱-质谱联用奠定了一定的基础。     

Improved detection of multi-phosphorylated peptides in the presence of phosphoric acid in liquid chromatography/mass spectrometry

In contrast to lower phosphorylation states (e.g. the tryptic monophosphopeptide FQpSEEQQQTEDELQDK from bovine beta-casein), the specific detection of multi-phosphorylated peptides (e.g. the tetraphosphopeptide RELEELNVPGEIVEpSLpSpSpSEESITR from tryptic digestion of bovine beta-casein) has often been problematic for liquid chromatographic mass spectrometric (LC/MS) analysis owing to their high affinity for adsorption to exposed surfaces. We observed an enhancement in the overall detection of phosphopeptides on addition of phosphoric acid (0.1-1.0%) to the sample solution; a 10-fold increase in sensitivity was determined for the detection of two tryptic phosphopeptides and also a significant improvement in the detection of the tetraphosphopeptide. Using capillary LC with ion trap tandem MS for detection and identification, the achievable detection limits were 50 fmol and 50 pmol for the monophosphopeptide and the tetraphosphopeptide, respectively. Phosphoric acid is believed to act as a blocking agent to available silanol groups on both the silica capillary surface and the C(18)-bonded stationary phase silica surface.     

Detection and characterization by high-performance liquid chromatography and mass spectrometry of a goat beta-casein associated with a CSN2 null allele

The identification and characterization of a truncated goat beta-casein, associated with a null beta-casein allele (CSN2(O')), is reported. The truncated beta-casein predicted at the DNA level (NCBI Acc. No. CAB39313) but never observed at the protein level, here named beta-casein O, was detected as a minor component in a goat milk sample from an autochthonous breed from southern Italy, 'Rossa Mediterranea', by reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry (RP-HPLC/ESI-MS). The ESI mass spectrum of the intact beta-casein O determined an M(r) value of 18 780 Da (calculated 18 781.5). Characterization of the amino acid sequence, performed by coupling trypsin digestion with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), RP-HPLC/ESI-MS and tandem mass spectrometry (MS/MS), demonstrated that the amino acid sequence corresponds to the 1-166 sequence of mature beta-casein variant A (Acc. No. P33048), thus confirming that the protein is coded by the null allele CSN2(O'), characterized by a transition (C --> T) at the 373rd nucleotide of the 7th exon of the gene, which generates a premature stop codon in position 182.     

Mapping the phosphorylation sites of proteins using on-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization multiple stage tandem mass spectrometry

On-line immobilized metal affinity chromatography/capillary electrophoresis/electrospray ionization-mass spectrometry (IMAC/CE/ESI-MS) offers selective preconcentration of phosphorylated peptides with identification of the phosphorylated amino acid(s). The preconcentration provides low concentration limits of detection and capillary electrophoresis separates the peptides. Recently, we reported a fast, simple, and sensitive on-line IMAC/CE/ESI-MS/MS method for the determination of phosphopeptides at low-pmole levels. That work is expanded here by use of multiple stage tandem mass spectrometry (MS(n), n = 2,3) to isolate and fragment target ions to provide more reliable assignments of phosphorylated residues. The application of IMAC/CE/ESI-MS(n) is demonstrated by the analysis of tryptic digests of alpha- and beta-casein and in-gel tryptic digests of beta-casein.     

气相色谱-串联质谱法测定皮革和纺织品中的富马酸二甲酯

建立了气相色谱-串联质谱(GC-MS/MS)准确、快速测定皮革和纺织品中富马酸二甲酯(DMF)的分析方法。样品经乙酸乙酯提取、浓缩及固相萃取柱净化处理,通过VF-5ms色谱柱分离、串联质谱对DMF进行定性和定量分析。实验结果表明: 优化样品预处理条件、固相萃取条件、质谱分析条件后,该方法可进一步消除杂质干扰,结果准确可靠,灵敏度、精密度、线性关系良好,回收率较高。3种浓度水平的加标回收率(n=6)保持在84%～93%之间,相对标准偏差小于7.2%;DMF在8种皮革和纺织品中的检出限 (S/N=3)为0.012～0.039 mg/kg, 0.05～100 mg/L质量浓度范围内线性相关系数为0.9990,适用于皮革和纺织品中DMF的日常检测。     

Facile synthesis of gallium ions immobilized and adenosine functionalized magnetic nanoparticles with high selectivity for multi-phosphopeptides

Despite recent advances in phosphoproteome research, detection and characterization of multi-phosphopeptides have remained a challenge. Here we present a novel IMAC strategy for effective extracting multi-phosphopeptides from complex samples, through Ga³⁺ chelation to the adenosine tri-phosphate (ATP)-functionalized magnetic nanoparticles (Ga³⁺-ATP-MNPs). The high specificity of Ga³⁺-ATP-MNPs was demonstrated by efficient enriching multi-phosphopeptides from the digest mixture of β-casein and BSA with molar ratio as low as 1:5000. Ga³⁺-ATP-MNPs were also successfully applied for the phosphoproteome analysis of rat liver mitochondria, resulting in the identification of 193 phosphopeptides with 331 phosphorylation sites from 158 phosphoproteins. In other words, 54.4% of the phosphopeptides trapped by Ga³⁺-ATP-MNPs were observed with more than one phosphorylated sites, resulting in significant improvement on the identification of peptides with multi-phosphorylated sites. The high specificity of Ga³⁺-ATP-MNPs towards multi-phosphopeptides may be due to the synergistic effect of the strong hydrophilic surface functionalized by ATP and the proper chelating strength provided by Ga³⁺. Moreover, the unique magnetic core of Ga³⁺-ATP-MNPs also facilitates the isolation process and on-plate enrichment for direct MALDI MS analysis with limit of detection as low as 30 amol. This new affinity-based protocol is expected to provide a powerful approach for characterizing multiple phosphorylation sites on proteins in complex and dilute analytes, which may be explored as complementary technique for improving the coverage of phosphoproteome.     

On‐chip solid‐phase extraction pre‐concentration/focusing substrates coupled to atmospheric pressure matrix‐assisted laser desorption/ionization ion trap mass spectrometry for high sensitivity biomolecule analysis

Atmospheric pressure matrix‐assisted laser desorption/ionization (AP‐MALDI) has proven a convenient and rapid method for ion production in the mass spectrometric (MS) analysis of biomolecules. AP‐MALDI and electrospray ionization (ESI) sources are easily interchangeable in most mass spectrometers. However, AP‐MALDI suffers from less‐than‐optimal sensitivity due to ion losses during transport from the atmosphere into the vacuum of the mass spectrometer. Here, we study the signal‐to‐noise ratio (S/N) gains observed when an on‐chip dynamic pre‐concentration/focusing approach is coupled to AP‐MALDI for the MS analysis of neuropeptides and protein digests. It was found that, in comparison with conventional AP‐MALDI targets, focusing targets showed (1) a sensitivity enhancement of approximately two orders of magnitude with S/N gains of 200–900 for hydrophobic substrates, and 150–400 for weak cation‐exchange (WCX) substrates; (2) improved detection limits as low as 5 fmol/µL for standard peptides; (3) significantly reduced matrix background; and (4) higher inter‐day reproducibility. The improved sensitivity allowed successful tandem mass spectrometric (MS/MS) sequencing of dilute solutions of a derivatized tryptic digest of a protein standard, and enabled the first reported AP‐MALDI MS detection of neuropeptides from Aedes aegypti mosquito heads. Copyright © 2009 John Wiley & Sons, Ltd.