高效液相色谱–质谱法分析僵蚕蛋白酶解多肽

分析僵蚕蛋白酶解多肽类成分的相对分子质量和氨基酸组成。采用高效液相色谱–质谱联用正离子模式进行分析,以Hola C_(18)色谱柱(100 mm×2.1 mm,2.7μm)为分离色谱柱,以0.05%甲酸水溶液和0.05%甲酸–乙腈溶液为流动相,根据质谱一级、二级碎片离子信息,确定酶解多肽类相对分子质量信息和氨基酸组成。僵蚕样品经酶解后得到相对分子质量在500~1 000之间的多肽,经LC–MS分析,多肽由低于10个的氨基酸组成。高效液相色谱–质谱法分析平台可用于分析多肽化合物的相对分子质量和氨基酸组成,这有利于酶解多肽的生物活性分析。     

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。     

阴离子交换色谱-电感耦合等离子体质谱法测定长期汞暴露人群硒干预后血清中的小分子硒形态

建立了阴离子交换高效液相色谱-电感耦合等离子体质谱联用测定不同形态硒的在线分离检测方法,并将其应用于长期汞暴露人群血清中小分子硒的形态研究.流动相A为0.5 mmol/L柠檬酸铵-2%(V/V)甲醇(pH 3.7),流动相B为100 mmol/L柠檬酸铵-2%(V/V)甲醇(pH 8.0);采用93%A-7%B时,硒代...     

UltiMate 3000 DGLC双三元液相色谱的流动相在线除盐技术

近年来,LC-MS联用技术已被广泛应用于各个行业。液相色谱分离是联用技术的基础,然而现有的很多液相色谱分离方法为改善分离或检测经常会使用非挥发性缓冲盐流动相(如磷酸盐缓冲溶液或离子对试剂),这显然与质谱的ESI     

维生素E辛烯基琥珀酸酯的定性分析

通过比较不同高效液相色谱(HPLC)方法对维生素E与辛烯基琥珀酸酐反应产物分离效果的影响,最终建立了以Dionex Kromasil C18柱为分析柱、甲醇和0.033 mol/L H3PO4(100:1,体积比)为流动相的色谱分离条件;借助高效液相色谱-大气压化学电离源质谱联用(HPLC-APCI-MS)法检测分离得到的色谱峰,通过比较它们的紫外光谱和质谱特征,确定其中两种物质为维生素E辛烯基琥珀酸酯的异构体。     

人血清内源性多肽无标记定量分析新策略

多肽组学(Pepti domics)以其在疾病标志物发现中所起的重要作用正受到越来越多的关注.然而,目前尚缺少针对多肽组学的高效定量方法.本文建立了一种基于纳升液相色谱与四极杆飞行时间质谱联用(μLC-Q-TOF-MS/MS)系统的色谱峰面积无标记多肽定量方法.该方法首先对两组样品中的多肽进行相对定量,然后选取相对量大于2或小于0.5的多肽,用纳升液相色谱与串联二级质谱(μLC-MS/MS)的选择性数据依赖模式(DDA)进行序列鉴定.将不同量标准样品牛血清白蛋白(BSA)酶解液加入到相同量的正常人血清样品中,然后用该方法进行定量分析,得到的定量结果平均相对偏差为6.42%.该方法分别被用于健康人血清与肝细胞癌(HCC)以及乳腺癌病人血清中内源性多肽的定量比较分析,并成功鉴定了血清中一些与HCC和乳腺癌相关的内源性多肽.由于Q-TOF-MS/MS的高分辨率和宽质量检测范围,价态高达+5和分子量超过4000的多肽也可被可靠地进行定性和定量分析.实验结果进一步表明在多肽组学分析中μLC-Q-TOF-MS/MS比传统的MALDI-TOF-MS具有更高的检测灵敏度.     

酪蛋白多肽的制备和色谱分离方法

为了得到低成本的多肽,本文利用胰蛋白酶对酪蛋白进行了充分的酶解。采用分析级反相高效液相色谱-电喷雾质谱联用技术(RP-HPLC/ESI-MS)分析了酶解产物各组分的组成,并通过改变流动相的梯度洗脱程序,优化了分析级色谱条件以充分分离相对含量较高的多肽组分;将优化的分析级色谱条件直接放大到制备级RP-HPLC中,在程序控制下通过紫外吸收信号结合ESI-MS信号共同引导实现了多肽的全自动化分离和收集。整个过程方便快捷,经过这样一个单一的分离步骤,得到了多个纯度较高的多肽。除此之外,本文还考察了流动相的酸碱性、柱上样量等因素对该体系制备级分离的影响,并对一次分离中分辨率不好的亲水性多肽混合物进行了二次分离,得到了多个新的多肽。本文建立的多肽制备方法为多肽和多肽材料的广泛应用提供了一种选择。     

聚羧酸系减水剂的大单体组分的分离分析

采用高效液相色谱-电喷雾式检测器分离分析聚羧酸系减水剂的大单体组分-脂肪醇聚氧乙烯基醚类。以Agilent ZORBAX SB-C8柱为分离柱,甲醇-水溶液为流动相进行梯度洗脱,采用电喷雾式检测器。在优化的色谱条件下,样品各组分间分离效果良好。高效液相色谱-质谱联用分析结果表明,各组分依据大单体中EO加合数的不同进行分离。     

基于液相色谱-质谱技术的代谢组学分析方法新进展

液相色谱-质谱联用技术是代谢组学研究领域的主要技术平台之一,近年来基于液相色谱-质谱联用技术的代谢组学分析方法获得了巨大发展。本文结合本研究组在代谢组学方面的研究成果,综述了近年来液相色谱-质谱联用技术在代谢组学分析方法方面的新进展,并对其发展前景进行了展望。综述引用文献41篇。     

天然维生素E制品中杂质的分离与鉴定

利用高效液相色谱、气相色谱-质谱联用与高分辨质谱对天然维生素E制品中的杂质进行了分离分析与结构鉴定。采用正相高效液相色谱法分离天然维生素E的4种异构体及2种杂质,并对杂质馏分进行富集纯化。将气相色谱-质谱联用与高分辨质谱检测相结合,用于获得杂质的结构信息。通过比较杂质精确相对分子质量和解析质谱碎片离子,推断杂质为芝麻素及其同分异构体表芝麻素。经与芝麻素对照品保留时间及碎片离子数据比对,确证了对杂质结构的推断。所建立的杂质鉴定方法快捷、有效,可应用于天然维生素E制品的食品安全控制。     

Separation of small molecular peptides with the same amino acid composition but different sequences by high performance liquid chromatography-electrospray ionization-mass spectrometry

Peptidomics has emerged as a new discipline in recent years. Mass spectrometry (MS) is the most universal and efficient tool for structure identification of proteins and peptides. However, there is a limitation for the identification of peptides with the same amino acid composition but different sequences because these peptides have identical mass spectra of molecular ions. This paper presents a high performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS) method for the separation of small molecular peptides with the same amino acid composition but different sequences. Two tripeptides of Gly-Ser-Phe and Gly-Phe-Ser were used as a model sample. The separation behavior has been investigated and the separation conditions have been optimized. Under the optimum conditions, good repeatability was achieved. The developed method could provide a helpful reference for the separation of other peptides with the same amino acid composition but different sequences in the study of proteomics and peptidomics.     

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。     

C60-fullerene bound silica for the preconcentration and the fractionation of multiphosphorylated peptides

Phosphorylation of proteins is an important cellular regulatory process. The analysis of protein phosphorylation is challenging due to the high dynamic range and low abundance natures of phosphorylated species. Mass spectrometry (MS) of phosphopeptides obtained from tryptic protein digests is the method-of-choice for characterization of phosphorylated proteins. However, determination of phosphopeptides by MS represents a major challenge, especially in the presence of unmodified peptides. Due to lower ionization efficiency of phosphopeptides, as well as the fact that the stoichiometry of phosphorylation is often present at low relative abundance, efficient enrichment of the phosphorylated peptides prior to MS analysis is therefore of high demand. In addition, successful identification of peptides with different phosphorylation grades still remains challenging.     

High-throughput one-bead-one-compound approach to peptide-encoded combinatorial libraries: MALDI-MS analysis of single TentaGel beads

The identification of pharmacologically promising compounds (lead compounds) from combinatorial libraries is frequently limited by the throughput of the analytical technique employed. Fourier transform mass spectrometry (FTMS) offers high sensitivity, mass accuracy (m/Deltam > 500 000), and sequencing capabilities. A rapid and efficient method for high-throughput analysis of single beads from peptide-encoded combinatorial libraries with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is presented. Encoding peptides on single beads are identified and structurally characterized by MALDI time-of-flight (TOF) and ultrahigh-resolution MALDI Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry. A strategy of on-probe sample preparation is developed to minimize handling of the beads.     

Separation with zwitterionic hydrophilic interaction liquid chromatography improves protein identification by matrix‐assisted laser desorption/ionization‐based proteomic analysis

Comprehensive proteomic analyses necessitate efficient separation of peptide mixtures for the subsequent identification of proteins by mass spectrometry (MS). However, digestion of proteins extracted from cells and tissues often yields complex peptide mixtures that confound direct comprehensive MS analysis. This study investigated a zwitterionic hydrophilic interaction liquid chromatography (ZIC‐HILIC) technique for the peptide separation step, which was verified by subsequent MS analysis. Human serum albumin (HSA) was the model protein used for this analysis. HSA was digested with trypsin and resolved by ZIC‐HILIC or conventional strong cation exchange (SCX) prior to MS analysis for peptide identification. Separation with ZIC‐HILIC significantly improved the identification of HSA peptides over SCX chromatography. Detailed analyses of the identified peptides revealed that the ZIC‐HILIC has better peptide fractionation ability. We further demonstrated that ZIC‐HILIC is useful for quantitatively surveying cell surface markers specifically expressed in undifferentiated embryonic stem cells. These results suggested the value of ZIC‐HILIC as a novel and efficient separation method for comprehensive and quantitative proteomic analyses. Copyright © 2009 John Wiley & Sons, Ltd.     

Rapid characterization of Bacillus spores targeting species-unique peptides produced with an atmospheric pressure matrix-assisted laser desorption/ionization source

New and improved strategies are eagerly sought for the rapid identification of microorganisms, particularly in mixtures. Mass spectrometry remains a powerful tool for this purpose. Small acid-soluble proteins (SASPs), which are relatively abundant in Bacillus spores, represent potential biomarkers for species characterization. Despite sharing extensive sequence homology, these proteins differ sufficiently in sequence for discrimination between species. This work focuses on the differences in sequence between SASPs from various Bacillus species. Compilation of SASP sequences from protein database searches, followed by in silico trypsin digestion and analysis of the resulting fragments, identified several species-specific peptides that could be targeted for analysis using mass spectrometry. This strategy was tested and found to be successful in the characterization of Bacillus spores both from individual species and in mixtures. Analysis was performed using an ion trap mass spectrometer with an atmospheric pressure MALDI source. This instrumentation offers the advantage of increased speed of analysis and accurate precursor ion selection for tandem mass spectrometric analysis compared with vacuum matrix-assisted laser desorption/ionization and time-of-flight instruments. The identification and targeting of species-specific peptides using this type of instrumentation offers a rapid, efficient strategy for the identification of Bacillus spores and can potentially be applied to different microorganisms.     

Sequit: software for de novo peptide sequencing by matrix-assisted laser desorption/ionization post-source decay mass spectrometry

Peptide sequencing by mass spectrometry is gaining increasing importance for peptide chemistry and proteomics. However, available tools for interpreting matrix-assisted laser desorption/ionization post-source decay (MALDI-PSD) mass spectra depend on databases, and identify peptides by matching experimental data with spectra calculated from database sequences. This severely obstructs the identification of proteins and peptides not listed in databases or of variations, e.g. mutated proteins. The development of a new computer program for database-independent peptide sequencing by MALDI-PSD mass spectrometry is reported here. This computer program was validated by the determination of the correct sequences for various peptides including sequences listed in the sequence databases, but also for peptides that deviate from database sequences or are completely artificial. This strategy should substantially facilitate the identification of novel or variant peptides and proteins, and increase the power of MALDI-PSD analyses in proteomics.     

Mass spectrometric analysis of enzymatic digestion of denatured collagen for identification of collagen type

Collagen type II and I from bovine were thermally denatured and digested with trypsin. The digest mixture was analyzed with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS). Peptides in the digest mixture were identified by mass spectrometry/mass spectrometry (MS/MS) sequencing. The results indicated that the digest mixtures of collagen type II and I contained lots of specific peptides and common peptides. Specific peptides could be used as index for identifying collagen type. Articular cartilage from bovine was pretreated and analyzed with the same method to determine the collagen types. The result indicated that the method developed was effective for identification of collagen types. The research provided a possible approach for collagen identification in particular tissues.     

Assessing matrix assisted laser desorption/ ionization-time of flight-mass spectrometry as a means of rapid embryo protein identification in rice.

Rice embryo proteins were separated by two-dimensional gel electrophoresis (2-DE). A total of 105 spots were digested with trypsin and the resultant peptides were analyzed by matrix assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF-MS). Raw mass spectra were fully-automatically processed and searched with selected monoisotopic masses against SWISS-PROT/TrEMBL and NCBInr databases. High quality mass spectra were obtained from 53 spots, of which 36 spots were identified including 29 not registered in databases. Fifty percent of the rice embryo proteins resolved in 2-DE could not be identified, indicating more efficient sample preparation techniques need to be developed in the future. At least four to five matching peptides were found to be essential for unambiguous identification of rice embryo proteins; peptide matching of less than four lead to ambiguous results. The suitability of peptide mass fingerprinting method as a means of rapid embryo protein identification in rice was discussed.     

Mass spectrometric analysis of head ganglia and neuroendocrine tissue of larval Galleria mellonella (Arthropoda, Insecta)

A brain-retrocerebral complex-subesophageal ganglion acidified methanolic extract of 100 larval Galleria mellonella (greater wax moth) was prepared for the isolation and identification of (neuro)peptides. To reduce sample complexity, the isolated peptides were roughly separated using a single, conventional chromatographic separation step. Subsequently, screening of these fractions with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in combination with nanoflow electrospray ionization quadrupole time-of-flight tandem mass spectrometry resulted in the identification of 12 lepidopteran peptides. None of these had been previously isolated or characterized within this species. VIFTPKLamide encoded by the diapause hormone-pheromone biosynthesis activating neuropeptide precursor was for the first time isolated and biochemically identified in a tissue extract, providing irrefutable evidence of its expression in larval nervous tissue. Another pentapeptide, AMVRFamide, with no resemblance to other lepidopteran peptides, was de novo sequenced and is most related to the neuropeptide F peptide family.