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1.
为了提高二氧化钛富集磷酸肽法对磷酸肽的富集效率,以6种标准蛋白酶切肽段混合物为研究对象,对二氧化钛富集磷酸肽过程中的乙腈比例、三氟乙酸比例、二氧化钛用量等条件分别进行了优化。结果表明在乙腈含量为80%(v/v),三氟乙酸含量为1%(v/v),二氧化钛用量与需要富集肽段的质量比为40:1的条件下,可以取得较好的富集效果。将优化后的富集方法应用于腾冲嗜热厌氧菌磷酸化蛋白质的分析,初步鉴定到25个磷酸化蛋白质,为进一步研究这种极端环境下生存的低等生物的生命活动提供了参考信息。 相似文献
2.
蛋白质磷酸化是最重要和最普遍的翻译后修饰之一。基于质谱的技术已成为分析蛋白质磷酸化的重要手段。然而,磷酸化肽固有的低丰度和电离效率以及由非磷酸化肽共存引起的严重抑制使得直接质谱分析仍然是一个挑战。为解决此问题,需在质谱分析前对磷酸化蛋白质进行选择性富集。磁性纳米材料具有良好的磁响应性,可以在外界磁铁的帮助下实现与溶液的迅速分离。功能化磁性纳米材料作为一种新型的分析技术已在蛋白质组学研究中得到广泛的应用。该文就近年来对磁性纳米粒子进行各种功能化修饰以提高其特异性吸附能力的吸附材料在磷酸化肽的富集方面的应用予以综述,并展望了功能化磁性纳米材料在磷酸化肽富集领域的应用前景。 相似文献
3.
为了在短时间内获得相对含量高的磷酸化肽段,以标准磷酸化蛋白质为模型对强阳离子交换色谱(SCX)分离磷酸化肽段体系的缓冲溶液和梯度设置进行了研究,并用酵母酶切肽段混合物考察了该路线在较复杂的样品中的应用。实验结果表明优化后的体系能够在30 min内分离出磷酸肽段,而且非磷酸化肽段的干扰很少,这样便相对提高了磷酸化肽段在质谱仪中的响应强度,重要的是该体系可以对复杂样品进行很好的分离。这说明SCX用于规模化磷酸化肽段富集的策略是可行的。本研究为磷酸化蛋白质组学规模化分析提供了实用技术。 相似文献
4.
酪氨酸磷酸化及其相应激酶活性的研究在抗肿瘤药物靶点的研发中具有重要意义.由于酪氨酸磷酸化仅占蛋白质总磷酸化含量的不足0.1%,因此规模化的酪氨酸磷酸化鉴定面临着重大技术挑战.本研究构建了TiO2串联C18反相填料的离心式富集装置,结合抗体免疫沉淀法,建立了酪氨酸磷酸肽的富集策略.此新型富集装置由吸头、适配器和离心(EP)管组成,将TiO2富集磷酸肽和C18填料反相分离磷酸肽有机结合,以离心的方式进行样品的上样、清洗、洗脱和分离,再通过抗酪氨酸磷酸化抗体进一步特异性富集酪氨酸磷酸肽,从而实现了酪氨酸磷酸肽的高效富集和大规模质谱鉴定.通过离心式富集装置简化了实验步骤,减少了样品损失和人为因素干扰;而且离心式、平行化的样品处理方式可显著提高分析通量.将此策略成功用于小鼠肝脏蛋白质酪氨酸磷酸化肽段的富集和质谱鉴定,在5 mg鼠肝蛋白中共鉴定出967个酪氨酸磷酸化位点,对应545个蛋白质,显示了其在蛋白质组学研究中的应用潜力. 相似文献
5.
蛋白质的磷酸化修饰广泛存在于细胞生命活动进程中,在细胞的分化、繁殖、代谢及信号转导等方面均发挥着重要作用.MALDI-TOF-MS技术因其高灵敏度等优点,已成为蛋白质研究的重要分析工具之一.由于磷酸化蛋白的天然丰度和离子化效率较低、检测信号被抑制等缺点,因此需要发展新的分析方法以提高其分析灵敏度和选择性.本文建立了基于介孔氧化钛的多磷酸化肽段高效分析方法,同时利用了TiO2对磷酸化肽的选择性吸附作用和介孔材料特殊空间结构所形成的对多磷酸肽的动力学及热力学吸附效应,结合MALDI-TOF-MS技术,成功地实现了对混合溶液中多磷酸化肽的选择性富集和高灵敏度分析鉴定,与其他方法相比,分析性能得到明显的提高,可实现对低量磷酸化肽段的识别与检测. 相似文献
6.
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。 相似文献
7.
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。 相似文献
8.
磷酸化是蛋白质翻译后修饰的重要形式之一,其异常往往会导致细胞内信号通路的紊乱和疾病的发生。固定化金属离子亲和色谱(IMAC)是磷酸化肽段的高效富集技术,在磷酸化蛋白质组研究方面应用广泛。该研究以金属钛离子(Ti4+)螯合IMAC材料(Ti4+-IMAC)为载体,进行磷酸化肽段富集。比较了10 μm Ti4+-IMAC通过振荡法和固相萃取法(SPE)富集磷酸肽的效果,发现振荡法可以富集到更多的磷酸肽;对比了两种尺寸(10 μm和30 μm)Ti4+-IMAC在磷酸化肽段富集中的差异,发现小尺寸材料富集效果更佳。进一步采用优化的策略比较了不同转移能力肺癌细胞的磷酸化蛋白质组,免标记定量蛋白质组学结果表明,优化的Ti4+-IMAC方法可以从正常的肺成纤维细胞MRC5、低转移肺癌细胞95C和高转移肺癌细胞95D中分别鉴定到510、863和1108种磷酸化蛋白质,其中317种为3组所共有。该研究共鉴定到1268种磷酸化蛋白质上的7560个磷酸化位点,其中1130个为差异磷酸化位点,文献报道显示部分异常表达的激酶与癌症转移密切相关。通过生信对比分析发现,异常表达的磷酸化蛋白质主要与细胞侵袭、迁移和死亡等细胞迁移方面的功能有关。通过优化磷酸化肽富集策略,初步阐明了磷酸化蛋白质网络的异常与肺癌转移之间的相关性,该方法有望用于肺癌进展相关的磷酸化位点、磷酸化蛋白质及其信号通路研究。 相似文献
9.
蛋白质的磷酸化是一种可逆性的翻译后修饰,在细胞的增值、分化、信号转导以及转录与翻译调控、蛋白质复合体的形成、蛋白质降解等方面发挥着极为重要的作用.因此磷酸化蛋白的鉴定成为翻译后修饰研究的重要内容.但由于磷酸化蛋白的丰度较低, 难以用质谱直接检测.为了解决这个问题,改善质谱对磷酸肽的信号响应, 需要对磷酸化蛋白质或磷酸肽进行富集.本文系统地介绍了磷酸化蛋白组学研究中应用较为广泛和最新建立的各种分离富集方法的原理、特点、应用研究进展,包括抗体富集法、激酶特异富集法、亲和富集法、化学修饰法、多种色谱分离富集方法以及MALDI靶盘富集法. 相似文献
10.
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。 相似文献
11.
The selective enrichment of specific proteins or peptides on micropipette tips prior to mass spectrometry analysis, which can minimize non-specific interferences as well as sample loss, has been an important issue in current proteomics field. In this paper, we have developed an easy-to-use phosphopeptide-selective pipette tip in which titanium dioxide nanoparticles were embedded in monolithic structure photopolymerized from ethylene glycol dimethacrylate. The simple and convenient fabrication was feasible in a commercial polypropylene pipette tip. Phosphorylated peptides were isolated from non-phosphopeptides by TiO(2) nanoparticle and eluted by 100 mM ammonium phosphate (pH 8.5), which was compatible with 2,5-dihydroxybenzoic acid (DHB)/1% phosphoric acid matrix and allowed for direct analysis of the elution fraction by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) without the necessity of desalting pretreatment. Tryptic digested alpha-casein and beta-casein spiked into bovine serum albumin (BSA) nonphosphorylated peptides (molar ratio 1:1:10) were used to assess the selectivity of TiO(2) tips. The effect of 50 mM ammonium hydrogencarbonate, pH 8 in 50% acetonitrile used as a wash buffer in reduction of nonspecific bound peptide to TiO(2) tip was dramatic. Almost all non-phosphopeptides were not detected by MALDI-MS analysis. The lowest detectable amount of phosphopeptide was estimated at low femtomole level. The easy-to-use TiO(2)-embeded tips operated in combination with the modified wash and elution conditions enable an efficient phosphopeptide enrichment for mass spectrometric analysis. 相似文献
12.
T Keough M P Lacey R J Strife 《Rapid communications in mass spectrometry : RCM》2001,15(23):2227-2239
Atmospheric pressure matrix-assisted laser desorption/ionization (AP-MALDI) and ion trap mass spectrometry have been used to study the fragmentation behavior of native peptides and peptide derivatives prepared for de novo sequencing applications. Sulfonic acid derivatized peptides were observed to fragment more extensively and up to 28 times more efficiently than the corresponding native peptides. Tandem mass spectra of native peptides containing aspartic or glutamic acids are dominated by cleavage on the C-terminal side of the acidic residues. This significantly limits the amount of sequence information that can be derived from those compounds. The MS/MS spectra of native tryptic peptides containing oxidized Met residues show extensive loss of CH(3)SOH and little sequence-specific fragmentation. On the other hand, the tandem mass spectra of derivatized peptides containing Asp, Glu and oxidized Met show much more uniform fragmentation along the peptide backbone. The AP-MALDI tandem mass spectra of some derivatized peptides were shown to be qualitatively very similar to the corresponding vacuum MALDI postsource decay mass spectra, which were obtained on a reflector time-of-flight instrument. However, the ion trap mass spectrometer offers several advantages for peptide sequencing relative to current reflector time-of-flight instruments including improved product ion mass measurement accuracy, improved precursor ion selection and MS(n). These latter capabilities were demonstrated with solution digests of model proteins and with in-gel digests of 2D-gel separated proteins. 相似文献
13.
Matthew B. Gates Kenneth B. Tomer Leesa J. Deterding 《Journal of the American Society for Mass Spectrometry》2010,21(10):1649-1659
Several affinity resins consisting of ionic metals or metal oxides were investigated for their phosphopeptide enrichment capabilities
with subsequent mass spectrometric analyses. Commercially-available enrichment metal oxide affinity chromatography (MOAC)
resins using manufacturer’s and/or published protocols were compared and evaluated for the most efficient and selective method
that could be implemented as a standard enrichment procedure. From these comparative analyses, using a tryptic digest of casein
proteins, it was determined that in our hands, two of the resins out-performed the others based on a variety of criteria,
including the number of phosphorylation sites identified during MS analyses, the lower numbers of nonspecifically bound peptides
observed, and the limits of detection. Applicability of these enrichment resins to a complex biological mixture was investigated.
For this work, a mixture of avian histones was digested, subjected to titanium dioxide phosphopeptide enrichment, and analyzed
by mass spectrometry. Eight phosphorylated tryptic peptides were observed following enrichment and subsequent LC/MS/MS analyses.
Of note, seven of the eight phosphopeptides were not observed without titanium dioxide enrichment. From these analyses, four
sites of phosphorylation were unequivocally determined, two of which have not been reported previously. Four additional phosphopeptides
were observed; however, the site of phosphorylation could not be distinguished but was localized to one of two possible amino
acids. These methods should aid in the investigation of proteins post-translationally modified with phosphate, especially
those present at low concentrations as was demonstrated by successful enrichment at the femtomole level. 相似文献
14.
Deprotonated peptides containing C-terminal glutamic acid, aspartic acid, or serine residues were studied by sustained off-resonance irradiation collision-induced dissociation (SORI-CID) in a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer with ion production by electrospray ionization (ESI). Additional studies were performed by post source decay (PSD) in a matrix-assisted laser desorption ionization/time-of-flight (MALDI/TOF) mass spectrometer. This work included both model peptides synthesized in our laboratory and bioactive peptides with more complex sequences. During SORI-CID and PSD, [M - H]- and [M - 2H]2- underwent an unusual cleavage corresponding to the elimination of the C-terminal residue. Two mechanisms are proposed to occur. They involve nucleophilic attack on the carbonyl carbon of the adjacent residue by either the carboxylate group of the C-terminus or the side chain carboxylate group of C-terminal glutamic acid and aspartic acid residues. To confirm the proposed mechanisms, AAAAAD was labelled by 18O specifically on the side chain of the aspartic acid residue. For peptides that contain multiple C-terminal glutamic acid residues, each of these residues can be sequentially eliminated from the deprotonated ions; a driving force may be the formation of a very stable pyroglutamatic acid neutral. For peptides with multiple aspartic acid residues at the C-terminus, aspartic acid residue loss is not sequential. For peptides with multiple serine residues at the C-terminus, C-terminal residue loss is sequential; however, abundant loss of other neutral molecules also occurs. In addition, the presence of basic residues (arginine or lysine) in the sequence has no effect on C-terminal residue elimination in the negative ion mode. 相似文献
15.
Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE‐ESI‐MS/MS
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Rabah Gahoual Alain Beck Yannis‐Nicolas François Emmanuelle Leize‐Wagner 《Journal of mass spectrometry : JMS》2016,51(2):150-158
Amino acids residues are commonly submitted to various physicochemical modifications occurring at physiological pH and temperature. Post‐translational modifications (PTMs) require comprehensive characterization because of their major influence on protein structure and involvement in numerous in vivo process or signaling. Mass spectrometry (MS) has gradually become an analytical tool of choice to characterize PTMs; however, some modifications are still challenging because of sample faint modification levels or difficulty to separate an intact peptide from modified counterparts before their transfer to the ionization source. Here, we report the implementation of capillary zone electrophoresis coupled to electrospray ionization tandem mass spectrometry (CZE‐ESI‐MS/MS) by the intermediate of a sheathless interfacing for independent and highly sensitive characterization of asparagine deamidation (deaN) and aspartic acid isomerization (isoD). CZE selectivity regarding deaN and isoD was studied extensively using different sets of synthetic peptides based on actual tryptic peptides. Results demonstrated CZE ability to separate the unmodified peptide from modified homologous exhibiting deaN, isoD or both independently with a resolution systematically superior to 1.29. Developed CZE‐ESI‐MS/MS method was applied for the characterization of monoclonal antibodies and complex protein mixture. Conserved CZE selectivity could be demonstrated even for complex samples, and foremost results obtained showed that CZE selectivity is similar regardless of the composition of the peptide. Separation of modified peptides prior to the MS analysis allowed to characterize and estimate modification levels of the sample independently for deaN and isoD even for peptides affected by both modifications and, as a consequence, enables to distinguish the formation of l ‐aspartic acid or d ‐aspartic acid generated from deaN. Separation based on peptide modification allowed, as supported by the ESI efficiency provided by CZE‐ESI‐MS/MS properties, and enabled to characterize and estimate studied PTMs with an unprecedented sensitivity and proved the relevance of implementing an electrophoretic driven separation for MS‐based peptide analysis. Copyright © 2016 John Wiley & Sons, Ltd. 相似文献
16.
Klemm C Otto S Wolf C Haseloff RF Beyermann M Krause E 《Journal of mass spectrometry : JMS》2006,41(12):1623-1632
The affinity of titanium dioxide for phosphate groups has been successfully used for enrichment of phosphopeptides from complex mixtures. This paper reports the relationship between the occurrence of some amino acids and the phospho-specific and nonspecific binding of peptides that occurs during titanium dioxide enrichment. In order to perform a systematic study, two well-characterized peptide mixtures consisting of either 33 or 8 synthetic phosphopeptides and their nonphosphorylated analogs, which differed in charge and hydrophobicity, were synthesized and analyzed by ESI-MS and MALDI-MS. The titanium dioxide procedure was also evaluated for comprehensive detection of phosphopeptides in phosphoproteomics. In summary, our results clearly confirm the high selectivity of titanium dioxide for phosphorylated sequences. Drastically reduced recovery was observed for phosphopeptides with multiple basic amino acids. Nonspecific binding of nonphosphorylated peptides and sample loss of phosphopeptides must also be taken into account. 相似文献
17.
An integrated analytical strategy for enrichment, detection and sequencing of phosphorylated peptides by matrix-assisted laser desorption/ionization (MALDI) tandem mass spectrometry (MS/MS) is reported. o-Phosphoric acid was found to enhance phosphopeptide ion signals in MALDI-MS when used as the acid dopant in 2,5-dihydroxybenzoic acid (2,5-DHB) matrix. The effect was largest for multiply phosphorylated peptides, which exhibited an up to ten-fold increase in ion intensity as compared with standard sample preparation methods. The enhanced phosphopeptide response was observed during MALDI-MS analysis of several peptide mixtures derived by proteolytic digestion of phosphoproteins. Furthermore, the mixture of 2,5-DHB and o-phosphoric acid was an excellent eluant for immobilized metal affinity chromatography (IMAC). Singly and multiply phosphorylated peptide species were efficiently recovered from Fe(III)-IMAC columns, reducing sample handling for phosphopeptide mapping by MALDI-MS and subsequent phosphopeptide sequencing by MALDI-MS/MS. The enhanced response of phosphopeptide ions in MALDI facilitates MS/MS of large (>3 kDa) multiply phosphorylated peptide species and reduces the amount of analyte needed for complete characterization of phosphoproteins. 相似文献
18.
Ahn YH Ji ES Lee JY Cho K Yoo JS 《Rapid communications in mass spectrometry : RCM》2007,21(24):3987-3994
Titanium dioxide (TiO2)-mediated phosphopeptide enrichment has been introduced as an effective method for extracting phosphopeptides from highly complex peptide mixtures. Chemical labeling by beta-elimination/Michael addition is also useful for increasing mass intensity in phosphopeptide analysis. Both of these methods were coupled in order to simultaneously enrich phosphopeptides and allow for detection and sequencing of the enriched peptides with high mass sensitivity. Phosphopeptides were successfully enriched on TiO2 beads without the use of any hydroxy acid additives like 2,5-dihydroxybenzoic acid. Labeling was accomplished on-bead with a guanidinoethanethiol (GET) tag containing a guanidine moiety. These GET-labeled derivatives were detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). GET labeling converted phosphoserine into guanidinoethylcysteine, a structural arginine-mimic. In particular, GET-labeled lysine-terminated phosphopeptides showed dramatically increased peak intensities compared to those of the corresponding intact phosphopeptides. Additionally, the on-bead labeling minimized manipulation steps and sample loss. The coupled technique was also further validated by applying to the analysis of phosphopeptides from complex tryptic digests of phosphoprotein mixtures. 相似文献
19.
Wai Yi Kelly Chan T. W. Dominic Chan Peter B. O’Connor 《Journal of the American Society for Mass Spectrometry》2010,21(6):1012-1015
Electron-transfer dissociation (ETD) with supplemental activation of the doubly charged deamidated tryptic digested peptide
ions allows differentiation of isoaspartic acid and aspartic acid residues using the c + 57 or z • − 57 peaks. The diagnostic peak clearly localizes and characterizes the isoaspartic acid residue. Supplemental activation
in ETD of the doubly charged peptide ions involves resonant excitation of the charge reduced precursor radical cations and
leads to further dissociation, including extra backbone cleavages and secondary fragmentation. Supplemental activation is
essential to obtain a high quality ETD spectrum (especially for doubly charged peptide ions) with sequence information. Unfortunately,
the low-resolution of the ion trap mass spectrometer makes detection of the diagnostic peak, [M-60], for the aspartic acid
residue difficult due to interference with side-chain loss from arginine and glutamic acid residues. 相似文献
20.
A MALDI TOF-TOF instrument was optimized and evaluated for intact protein analysis by tandem mass spectrometry. Ion source voltages and delay times were adjusted to affect an up to a 10-fold improvement in fragment ion yield compared to data obtained using default settings employed in peptide analysis. For large peptides (3-4.5 kDa), up to 90% of all possible b- and y-fragment ions were observed, which provides sufficient information for de novo sequencing and unambiguous protein identification. Product ion signals associated with preferential cleavages C-terminal to aspartic acid and glutamic acid residues and N-terminal to proline residues became dominant with increased protein molecular weight. Matrix effects were also evaluated and, among the eight matrices examined, alpha-cyano-4-hydroxycinnamic acid (CHCA) was found to produce the best intact protein tandem mass spectra for proteins up to 12 kDa. Optimized performance yielded detection limits of 50-125 fmol for proteins of 4 and 12 kDa, respectively. This improved performance has yielded an instrument with potential to be a useful tool in proteomic investigations via analysis of intact proteins. 相似文献