Edman降解中异硫氰酸苯酯新修饰位点的发现和验证

Edman降解是最早建立的一种用于多肽和蛋白质氨基端测序的方法,该方法现在仍被广泛用于生物化学领域。随着高通量蛋白质组学技术的发展和应用,该方法中的异硫氰酸苯酯反应被用于修饰蛋白质氨基端,并用于检测蛋白质水解位点。但还没有异硫氰酸苯酯是否可以修饰其他氨基酸侧链并影响多肽序列分析的研究。为了探究其修饰其他氨基酸的可能性,本文利用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS）和液相色谱-串联质谱（LC-MS/MS）研究了异硫氰酸苯酯对一个模型肽的化学修饰。质谱数据解析后发现在高浓度异硫氰酸苯酯的反应条件下,组氨酸上可以引入一个新的异硫氰酸苯酯修饰位点。这一修饰位点的发现预示着通过改变实验条件或分析方法,可以更准确地利用Edman降解和蛋白质组学技术分析多肽和蛋白质。     

牡蛎源类蛋白反应修饰肽的分离纯化及肽锌螯合物的结构表征

以牡蛎为原料制备了类蛋白反应修饰肽,利用Sephadex G-15凝胶层析柱和反向高效液相色谱(RP-HPLC)等分离技术得到1条锌离子螯合活性为161 mg/g的多肽(M_w=835),多肽序列为EVPPEEH.以测得的肽序列为模板合成多肽,将纯肽与锌离子进行螯合反应制备肽锌螯合物.螯合物的红外光谱和圆二色光谱表征结果表明,锌离子主要与多肽链上的羰基氧发生相互作用.与多肽的空间结构相比,螯合物的无规则卷曲结构减少,β转角增加而β折叠减少.由肽锌螯合物的分子模拟和二级质谱结果可知,多肽与锌离子螯合后有2种空间构象:一种通过六配位的方式螯合1个锌离子,其中主要的螯合位点为多肽Val-2和Pro-3或者Glu-5和Glu-6之间的羰基氧;另一种是通过四配位的方式螯合1个锌离子,主要的螯合位点为多肽Glu-5和Glu-6之间的羰基氧.     

含有28个氨基酸的复杂多肽的串级质谱全序列分析研究

利用MALDI-TOF/TOF MS和ESI-MS/MS对一种含有多达28个氨基酸的复杂合成多肽成功进行了全序列测定. 通过调节激光强度、碰撞诱导解离(CID)能量等质谱参数以及依据不同序列分析软件, 获得了涵盖所有b型和y型碎片离子的串级质谱图. 结果显示这种方法可以有效地解决de novo测序方法遇到的谱峰过于复杂导致运算死机等问题. 通过讨论如何对含有超过20个氨基酸片断的多肽进行合格串级质谱实验, 为蛋白质组学肽段序列测定提供了新的方法和思路.     

同位素稀释质谱法测定多肽含量

利用Leu-Phe作为模型肽,建立了水解后同位素稀释质谱测定多肽含量的方法。在实验中优化了酸水解的时间,采用高效液相色谱法和质谱法证明模型肽已完全水解。水解后的氨基酸经过HPLC分离,质谱检测采用选择离子监测模式,分别检测苯丙氨酸(m/z=166)和标记苯丙氨酸(m/z=174)的离子。根据苯丙氨酸的含量计算模型肽的含量,并评定了测定结果的不确定度。     

反相高效液相色谱-电喷雾质谱法分析食源血管紧张素转换酶抑制肽

利用反相高效液相色谱/电喷雾离子阱质谱法,直接分析从牦牛乳酪蛋白中酶水解得到的血管紧张素转换酶抑制肽粗产物。RP-HPLC显示具有活性的多肽粗产物含有3个主要成分,质谱同步测定各组分的分子量(m/z)分别为815.2,1680.1,962.2,然后选择[M H] 离子通过串联质谱(MS/MS)得到碎片离子,利用b离子和y离子互补的方法鉴定了多肽序列。三条肽分别为Leu-Pro-Tyr-Tyr,Pro-Leu-Pro-Leu-Leu-Gln,Phe-Leu-Pro-Pro-Tyr-Tyr。结果显示,所获得的多肽序列与牛乳酪蛋白一级结构中相应肽段的序列一致。     

电喷雾质谱动力学方法测定磷酰化丙丙二肽的气相质子亲和能

自 1 98 4年 Yamashita等[1] 提出电喷雾质谱技术 ( ESI-MS)以来 ,它已成为现代生物学研究的重要工具 ,已被用于蛋白 [2 ]和寡聚核苷酸序列 [3 ]的测定、蛋白折叠途径表征 [4 ]、非共轭复合物检测 [5]及酶催化反应的定量研究 [6 ]等 .前文 [7]研究发现 ,氨基酸和小肽的氮端磷酰化使其在快原子轰击质谱和电喷雾质谱正离子模式下峰强明显增强 ,表现出很强的增敏效应 .这一现象可能在蛋白质组学质谱小肽序列的测定中得到应用 .因此 ,阐明其产生机理非常重要 .一个可能的机理是磷酰化基团的引入增加了氨基酸和小肽的气相质子亲和能 ( Proton …     

无机磷试剂辅助均环肽库的建立及其电喷雾质谱研究

在无机磷试剂辅助下建立了氨基酸自组装成均环肽的方法, 得到了相应的均环肽库. 均环肽库的建立增加了肽库的多样性, 为药物筛选提供了新的选择性. 采用电喷雾多级质谱技术, 对系列均环多肽 [M＋H]＋离子和[M＋Na]＋离子的质谱裂解规律进行了系统研究, 两种系列的离子具有不同的质谱裂解特征, 分别提出了其可能的质谱裂解机制. 该研究丰富了环多肽化合物的电喷雾多级质谱研究, 结果表明环肽化合物的加钠离子较加氢离子的质谱图可以更容易地用于环多肽的序列测定. 本研究为其它类似环肽化合物结构的分析鉴定及利用电喷雾质谱推测环肽序列提供了有效的质谱方法.     

纳升电喷雾串联质谱法确证阿托西班的一级结构

利用电喷雾串联质谱对合成八肽阿托西班进行了二硫键还原前后的精确分子量测定和一级结构的确证.首先通过全扫描模式测定了其还原前后的精确分子量,然后选择母离子m/z 498.73(双电荷)通过串联质谱(MS/MS)得到碎片离子,采用Y离子的方法测定了阿托西班的序列并对其的修饰位点进行了确证.本方法具有灵敏度高、速度快、样品无需纯化等特点,在多肽类药物一级结构分析方面具有独特的优势.     

纳升电喷雾串联质谱在阿托西班一级结构确证中的应用

利用电喷雾串联质谱对合成八肽阿托西班进行了二硫键还原前后的精确相对分子质量测定和一级结构的确证.首先通过全扫描模式测定了其还原前后的精确相对分子质量,然后通过串联质谱(MS/MS)得到母离子m/z 498.73(双电荷)的碎片离子,采用y离子的方法测定了阿托西班的序列并对其修饰位点进行了确证.该方法具有灵敏度高、速度快、样品无需纯化等特点,在多肽类药物一级结构分析方面具有独特的优势.     

基于准确质量数对肽族[γ-(Glu-Cys)_n-Gly]色谱-质谱分析研究

基于多肽γ-(Glu-Cys)n-Gly系列化合物合成的递增规律性,利用Trap-MS离子筛选功能实现多肽的定性定量分析。首先利用多肽分子中巯基与单溴二胺(mBBr)发生缩合反应,生成具有荧光信号的多肽衍生物,比较衍生化前后分子量发现多肽与mBBr的反应比例为1∶1,荧光信号强度1周内稳定性较好。优化了色谱分离及质谱条件,建立了HPLC-Trap-MS定性定量分析方法,离子提取结果显示:多肽化合物基于其理论分子量定性分析准确快速,化合物分离效果明显,PC_2,PC_3,PC_4,PC_5和PC_6系列标准溶液的线性范围宽,相关系数(r)大于0.999,各化合物的方法检出限分别为0.13,0.02,0.17,0.39,0.82 mg/L,化合物的回收率为89.0%~99.5%;相对标准偏差(RSD)小于3.5%;多肽化合物的荧光标记和Trap-MS筛选可实现荧光与质谱双重检测器的定量分析,该方法可对多肽系列化合物进行快速、准确的测定。检测了Zn和Cd诱导后藻体中多肽的含量变化,结果显示藻体中的多肽对金属具有诱导响应,其中小分子肽变化相对明显,随着碳链的增加,多肽对重金属的诱导响应滞后。     

The extent and effects of peptide sequence scrambling via formation of macrocyclic <Emphasis Type="Italic">b</Emphasis> ions in model proteins

The extent and effects of sequence scrambling in peptide ions during tandem mass spectrometry (MS/MS) have been examined using tryptic peptides from model proteins. Sequencescrambled b ions appeared in about 35% of 43 tryptic peptides examined under MS/MS conditions. In general, these ions had relatively low abundances with averages of 8% and 16%, depending on the instrumentation used. A few tryptic peptides gave abundant scrambled b ions in MS/MS. However, peptide and protein identifications under proteomic conditions with Mascot were not affected, even for these peptides wherein scrambling was prominent. From the 43 tryptic peptides that have been investigated, the conclusion is that sequence scrambling is unlikely to impact negatively on the accuracy of automated peptide and protein identifications in proteomics.     

人肝癌细胞系HLE细胞表面抗原多肽的纳升电喷雾串联质谱从头测序分析

肿瘤细胞表面的抗原多肽能够被细胞毒T淋巴细胞特异性识别而引起免疫应答,因此有可能用于研制基于多肽的抗肿瘤疫苗。用弱酸将人肝癌细胞系HLE细胞表面抗原多肽和人正常肝细胞表面多肽洗脱后,经RP-HPLC分离,选择HLE细胞表面特异性多肽进行纳升电喷雾串联质谱(nanoESI-MS/MS)测序,共测定5个色谱峰中的20个多肽序列,分子量分布范围为1000~2000 Da。借助M asSeq软件分析出其中12个多肽的序列。经数据库查寻,其中的3个肽段分别来自钙调节蛋白、核蛋白S19和伴侣蛋白10。这些多肽的生物学功能及与肿瘤的关系值得深入研究。该研究表明nanoESI-MS/MS是测定微量混合多肽序列的最有效方法。     

Bovine serum albumin as a universal suppressor of non-specific peptide binding in vials prior to nano-chromatography coupled mass-spectrometry analysis

Non-specific binding (NSB) is a well-known problem for any application that deals with ultralow analyte quantities. The modern nano-flow chromatography coupled tandem mass-spectrometry (nanoLC-MS/MS) works with the lowest conceivable analyte concentrations. However, while the NSB problem is widely accepted and investigated for metabolomics and single-peptide medicine-related assays, its impact is not studied for complex peptide mixtures in proteomic applications. In this work peptide NSB to a common plastic autosampler vial was studied for a model mixture of 46 synthetic peptides. A significant NSB level was demonstrated for total peptide concentrations of up to 1 mg mL⁻¹. Different agents were tried for NSB suppression and compatibility with nanoLC-MS/MS analysis: a chaotropic agent, an amino acid mixture, a peptide mixture and a protein solution. The first two were inefficacious. The peptide matrix blocked NSB, however, it also led to analyte ionization suppression in nanoLC-MS/MS. The protein solution (0.1% BSA) efficiently eliminated NSB, while a trap-elute nanoHPLC configuration together with a small-pore reverse-phased sorbent effectively and quantitatively extracted the model peptides and depleted protein material from the sample. Higher protein concentration partially impeded peptide extraction. Thus, the 0.1% BSA solution might be regarded as an effective non-interfering blockader of NSB for sample resuspension and storage in an autosampler prior to LC-MS/MS analysis.     

Use of an integrated MS--multiplexed MS/MS data acquisition strategy for high-coverage peptide mapping studies

Liquid chromatography/mass spectrometry (LC/MS) peptide maps have become a basic tool for characterizing proteins of biological and pharmaceutical interest. The ability to generate reproducible maps with high protein sequence coverage is a central goal of methods development. We have applied a recently developed analytical approach (termed LC/MS(E)) to LC/MS peptide mapping. Using the LC/MS(E) approach, the mass detector alternates between a low-energy scanning mode (MS) for accurate mass peptide precursor identification, and an elevated-energy mode (MS(E)) for generation of accurate mass multiplex peptide fragmentation data. In this paper, we evaluate this analytical approach against a tryptic digest of yeast enolase. From the low-energy data, high peptide map coverage (98% of sequence from peptides >3 amino acids) was reproducibly obtained. The MS signal for essentially equimolar peptides varied over 2 orders of magnitude in intensity, and peptide intensities could be precisely and reproducibly measured. Using the temporal constraint that MS(E) peptide fragment ions exhibit chromatographic profiles that parallel the precursor ions that generated them, we were able to produce accurate mass time-resolved MS/MS information for all enolase peptides with sufficient abundance to produce a detectable fragment ion.     

Independent highly sensitive characterization of asparagine deamidation and aspartic acid isomerization by sheathless CZE‐ESI‐MS/MS

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.     

Improving peptide identification using an empirical peptide retention time database

Peptide retention time (RT) is independent of tandem mass spectrometry (MS/MS) parameters and can be combined with MS/MS information to enhance peptide identification. In this paper, we utilized peptide empirical RT and MS/MS for peptide identification. This new approach resulted in the construction of an Empirical Peptide Retention Time Database (EPRTD) based on peptides showing a false‐positive rate (FPR) ≤1%, detected in several liquid chromatography (LC)/MS/MS analyses. In subsequent experiments, the RT of peptides with FPR >1% was compared with empirical data derived from the EPRTD. If the experimental RT was within a specified time range of the empirical value, the corresponding MS/MS spectra were accepted as positive. Application of the EPRTD approach to simple samples (known protein mixtures) and complex samples (human urinary proteome) revealed that this method could significantly enhance peptide identification without compromising the associated confidence levels. Further analysis indicated that the EPRTD approach could improve low‐abundance peptides and with the expansion of the EPRTD the number of peptide identifications will be increased. This approach is suitable for large‐scale clinical proteomics research, in which tens of LC/MS/MS analyses are run for different samples with similar components. Copyright © 2008 John Wiley & Sons, Ltd.     

Direct ampholyte-free liquid-phase isoelectric peptide focusing: application to the human serum proteome

In this study, we utilized a multidimensional peptide separation strategy combined with tandem mass spectrometry (MS/MS) for the identification of proteins in human serum. After enzymatically digesting serum with trypsin, the peptides were fractionated using liquid-phase isoelectric focusing (IEF) in a novel ampholyte-free format. Twenty IEF fractions were collected and analyzed by reversed-phase microcapillary liquid chromatography (microLC)-MS/MS. Bioinformatic analysis of the raw MS/MS spectra resulted in the identification of 844 unique peptides, corresponding to 437 proteins. This study demonstrates the efficacy of ampholyte-free peptide autofocusing, which alleviates peptide losses in ampholyte removal strategies. The results show that the separation strategy is effective for high-throughput characterization of proteins from complex proteomic mixtures.     

Intermolecular condensation products formed during the pyrolysis of peptides

Mass Spectrometry (MS) analysis of pyrolysis products of simple peptides has revealed several non-volatile thermal degradation products at masses lower than the precursor peptide. In addition to these products, many other signals were also observed at higher masses than the precursor peptide, and their characterization is the focus of this study. Here we report on the observation of homo and hetero condensation peptide products formed during the pyrolysis of peptides. The observed peptide condensation products are formed between two, three or even four peptides. Tandem MS (MS/MS) analyses of these products showed that C-terminal to N-terminal intermolecular bonding is preferred during pyrolysis when combining two peptides, rather than involving crosslinking between basic and acidic side chain groups like arginine and aspartic acid. These observations are rationalized by steric hindrance effect and known pK_a values of the peptide C- and N-termini and amino acid side groups like aspartic acid and arginine. Pyrolysis of a standard N-acetylated peptide showed no detectable condensation and/or crosslinked products, even in peptides with basic side groups, providing further evidence for the C-terminus to N-terminus intermolecular bonding between peptides under pyrolytic conditions.     

Combining solid-phase preconcentration, capillary electrophoresis and off-line matrix-assisted laser desorption/ionization mass spectrometry: intracerebral metabolic processing of peptide E in vivo

The in vivo metabolism of peptide E was studied in the anesthetized rat using a combination of microdialysis sampling, solid-phase preconcentration capillary electrophoresis and imaging matrix-assisted laser desorption/ionization mass spectrometry (MALDI/MS). The metabolic profile of peptides identified by MALDI/MS showed that the primary enzymatic activity for degradation of peptide E was due to carboxypeptidases and, to a lesser extent, aminopeptidases and some trypsin-like endopeptidases. Over 75 metabolic fragments were detected from the action of these enzymes in vivo.     

A method for rapidly confirming protein N-terminal sequences by matrix-assisted laser desorption/ionization mass spectrometry

The N-terminal sequence is important for the identification of a protein and the confirmation of its N-terminal processing. Although mass spectrometry (MS) is a sensitive and high-throughput method to sequence and identify peptides and proteins, N-terminal peptides, diluted among most of the peptides that do not originate at the N-termini, are not easy to identify directly with MS. To develop a simple and rapid method to identify and sequence the N-terminal peptide of a protein, a new strategy based on specific sulfonation of terminal amino groups and selective monitoring of the sulfonated peptide was introduced. After a protein had been guanidinated, 2-sulfobenzoylated, and reduced, it was digested with trypsin and analyzed by MS. Because of the strong acidity of sulfonic groups and the specific sulfonation of alpha-amino groups, the sulfonated N-terminal peptide dominated as base peak in the negative mode peptide mass fingerprint (PMF) and was easy to identify. The N-terminal peptide was then selected as precursor ion for tandem mass spectrometric (MS/MS) analysis. Four proteins were tested with this method and their N-terminal peptides were successfully recognized and sequenced. The results suggest that the addition of a sulfonic acid group facilitates the identification and de novo sequencing of N-terminal peptides.