用于人血浆中蛋白质分离的在线阵列式二维常规柱液相色谱系统的建立

构建了一种在线阵列式二维常规柱液相色谱系统,并将其应用于分离血浆中的完整蛋白质。该系统以1根强阴离子交换柱作为第一维分离柱,8根阵列式反相色谱柱作为第二维分离柱。强阴离子交换柱分离的馏分通过十通阀被依次转移到第二维预柱上并得到保留富集,随后第二维流动相通过分流器同时将预柱上的蛋白质反冲至分析柱上进行分离。二维之间以及第二维阵列色谱柱之间均相互独立,从而可以提高系统分离的通量和总峰容量。采用该系统对血浆中的蛋白质进行了完整蛋白质水平上的分离。该系统具有高通量和高分辨率的特点,为血浆样品中高丰度蛋白质的去除以及血浆样品的深入研究提供了一种有效的手段。     

IEX/RP二维液相色谱中弱保留组分收集模式的构建及系统评价

以十通阀和捕集柱接口形式,构建了弱阴离子交换/反相(WAX/RP)二维液相色谱分离系统.通过将第一维离子交换色谱分析中的前部集中洗脱出的弱保留组分收集后单独分析,剩余样品进一步采用二维液相色谱分析,可以有效避免第二维色谱柱对特殊样品局部集中流出导致的第二维分离超柱容量问题,提高了系统的整体分离能力.使用4种蛋白胰蛋白酶酶解后的多肽样品评价该系统,在不分流的系统中共检测到115个峰.对第一维分离的前15 min分流后得到的组分单独分析,共识别出58个峰,后续的二维分离中共得到78个峰.2种方法相比,第二种方法检测峰数增加了21个,一些低丰度的组分在弱保留组分收集后被识别.     

二维液相色谱接口的改进及其在蛋白质组学研究中的应用

随着蛋白质组学、本草物质组学等组学概念的提出,所需分析的样品的成分越来越复杂,因此具有强大分离能力的多维液相色谱技术受到人们越来越多的关注。二维液相色谱中第二维的分离性能和速度是整个分离系统性能的关键。基于捕集柱模式,我们采用经特殊设计的流路系统,使得双捕集柱型接口具有预分离的功能。样品从第一维流出以后被富集在捕集柱1的柱头,经过脱盐后,正冲捕集柱,捕集柱1与第二维色谱柱联用对富集的样品进行分离,增加了第二维分离效率。当捕集柱上的样品全部被洗脱到第二维色谱柱上时,捕集柱2已经完成对第一维洗脱液中样品的捕集和脱盐,此时将阀进行切换,捕集柱2与第二维色谱柱直接相连进行洗脱。循环切换捕集柱1和捕集柱2,维持较高的阀切换频率,实现了第二维色谱柱的连续洗脱。因此保证了第二维分离具有较快速度,同时具有较高的分离效率。使用35 mm长捕集柱和十通阀为接口,以弱阴离子交换(WAX)色谱为第一维分离模式,以反相(RP)色谱为第二维分离模式,构建了WAX-RP二维液相色谱系统(2D-LC system)。以小鼠血清为样品对系统进行了初步评价。色谱流出曲线出现了明显的界面现象,这是由于捕集柱流动相中含有的较多盐分流出时的背景吸收造成的。同时,由于界面两侧的流动相黏度不同产生了黏性指进(VF)现象。当第二维色谱柱长度为50 mm时,理论上可将第二维分离效能提高70%。该接口可以应用于多种二维液相色谱模式,适用于蛋白质组学和本草物质组学研究中对于复杂样品的分离分析。     

高温二维液相色谱系统的构建及其评价

在二维液相色谱中, 第二维的分离速度是制约其发展的重要因素. 升高色谱柱温度可以有效降低流动相粘度, 加快溶质在两相间的传质速率, 有效加快分析速度. 以离子交换色谱法(WAX)为第一维分离模式和反相色谱法(RP)为第二维分离模式, 十通阀和两个捕集柱为接口, 通过将第二维色谱柱温度升高到80 ℃和提高流量到3 mL/min, 构建了高温WAX/RP二维液相色谱系统. 以4种标准蛋白的酶解物为样品评价系统的分离性能, 第一维共有33个馏分被捕集并导入到第二维分析, 高温二维液相色谱系统识别出187个色谱峰.     

色谱最新研究亮点

1 多维分离技术新进展 一维色谱是目前最常用的分离分析方法,然而对于复杂体系如蛋白质组,采用一维分离模式其分离度远远不能满足要求.Giddings理论告诉我们:对于分离机理相互正交的二维分离系统(如色谱),峰的容量应该为两个色谱柱峰容量的乘积.因此,多维分离系统是解决复杂分离体系的一个最佳选择.在多维色谱中二维气相色谱发展较快,目前全二维气相色谱仪业已商品化,其峰的容量达到104以上.而二维液相色谱,尤其是正相/反相二维液相色谱技术发展较为缓慢,其主要的技术瓶颈在于第一维色谱(正相)分离后的流动相严重干扰第二维色谱(反相)的分离.     

蛋白质高效分离两维色谱柱的选择优化

为了构建高效的离子交换/反相二维液相色谱(IEC/RPLC)分离平台系统,提高复杂蛋白质样品的分离效率,对色谱柱进行了评价与筛选。通过对实际人肝蛋白质样品的分离效果的比较,选择确定了TSKgel DEAE-5PW弱阴离子交换色谱柱(WAX)作为第一维色谱分离柱;考察了同一规格的10支代表性反相色谱柱(250 mm×4.6 mm, 5 μm, 30 nm, C4、C8或C18),通过评价其对尿嘧啶、硝基苯、萘和芴的分离性能以及对3种标准蛋白质样品的非特异性吸附、对人肝蛋白质样品的WAX馏分的分离效果,最终确定以Jupiter 300 C4反相色谱柱作为第二维色谱分离柱。对两维色谱柱的选择优化为蛋白质高效分离二维液相色谱平台的搭建提供了可靠基础。     

凝胶过滤/离子交换全二维液相色谱系统的构建与评价

基于蛋白质的尺寸及带电性质,将凝胶过滤色谱(GFC)与离子交换色谱(IEC)两种分离模式结合,采用双捕集柱接口构建了GFC/2×IEC二维液相色谱(2-D LC)分离系统,同时考虑离子交换色谱分离蛋白质对等电点范围的限制,进一步结合中心切割平行柱的方法实现对蛋白质的全二维分离。为与后续蛋白质在线酶解、多肽分离及质谱鉴定匹配,系统中采用常规柱以保证蛋白质质谱鉴定对样品量的要求,3种常规分离柱分别选用凝胶过滤色谱柱TSK-GEL G3000SW_(XL)(300 mm×7.8 mm,5μm)、强阴离子交换色谱柱Hypersil SAX(100 mm×4.6 mm,10μm)和强阳离子交换色谱柱Hypersil SCX(100 mm×4.6 mm,10μm)。最终以酵母细胞蛋白质提取液为样品,对构建的二维系统加以评价,在总蛋白质浓度13.5 mg/mL、上样体积100μL的条件下,将第一维分离等时间切割17次,并将切割馏分全部导入第二维继续分离,二维系统在148 min内获得的总峰容量达到884。说明所构建的系统可以用于蛋白质的在线全二维分离。     

六味地黄丸组分的二维液相色谱分离

以1个常规六通阀直接连接两支常规尺寸的色谱柱(250 mm×4.6 mm i.d.),构建简单的SCX/RP在线二维液相色谱系统,对中成药六味地黄丸组分进行了优化分离。样品经过第一维阳离子交换色谱(Hypersil SCX),洗脱产物分离后通过六通阀直接富集到反相分析柱(C18)顶端,被转移到第二维色谱柱上继续进行分离。经过11步不连续的线性梯度洗脱,二维分离系统出峰数量达到550多个,峰容量达到2266。构建的二维液相色谱系统结构简单,与一维色谱相比,具有分辨率高、峰容量大的特点。     

基于在线二维色谱的不同生长时期鹿茸的比较蛋白质组分析

建立了基于在线二维弱阴离子交换色谱-反相液相色谱(2D-WAX-RPLC)的蛋白质分离系统,并用于不同生长时期鹿茸的比较蛋白质组分析。以5种标准蛋白质的混合物为样品,考察了系统的重现性。通过改变标准蛋白质之间的浓度比,研究了该系统进行蛋白质相对定量的能力。在此基础上,针对4个不同生长时期的鹿茸样品,分别采用5种不同的方法进行蛋白质提取,经2D-WAX-RPLC分离后,收集各生长时期对应蛋白质的峰高最大比超过2的组分。酶解后,采用微柱反相液相色谱-串联质谱(μRPLC-MS/MS)进行肽段的分离鉴定;共从9个差异蛋白质峰中鉴定到了22个差异蛋白质。研究结果表明,利用基于蛋白质水平的在线二维液相色谱分离技术找寻差异蛋白质,具有重现性好、自动化程度高等优点,可用于开展比较蛋白质组学的研究。     

六昧地黄丸组分的二维液相色谱分离

以1个常规六通阀直接连接两支常规尺寸的色谱柱(250 mm×4.6 mm i.d.),构建简单的SCX/RP在线二维液相色谱系统,对中成药六味地黄丸组分进行了优化分离.样品经过第一维阳离子交换色谱(Hypersil SCX),洗脱产物分离后通过六通阀直接富集到反相分析柱(C18)顶端,被转移到第二维色谱柱上继续进行分离.经过11步不连续的线性梯度洗脱,二维分离系统出峰数量达到550多个,峰容量达到2266.构建的二维液相色谱系统结构简单,与一维色谱相比,具有分辨率高、峰容量大的特点.     

Proteomic analysis of rat plasma by two-dimensional liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry

The proteomic analysis of plasma and serum samples represents a formidable challenge due to the presence of a few highly abundant proteins such as albumin and immunoglobulins. Detection of low abundance protein biomarkers therefore requires either the specific depletion of high abundance proteins using immunoaffinity columns and/or optimized protein fractionation methods based on charge, size or hydrophobicity. Here we describe a two-dimensional (2D) liquid chromatography separation method for the fractionation of rat plasma. In the first dimension proteins were separated by chromatofocusing according to their isoelectric point (pI). In the second dimension, proteins were further fractionated by non-porous, reversed-phase chromatography according to their hydrophobicity. The data from both separations was displayed as a 2D protein expression map of pI versus retention time (relative hydrophobicity). Both separations were carried out on the ProteomeLab PF 2D system (Beckman Coulter), an instrument platform that provides a high degree of automation and real-time monitoring of the separation process. The reproducibility of the first-dimension separation was evaluated in terms of pH gradient formation. The second-dimension separation was evaluated in terms of peak retention times on the reversed-phase column. We found in four consecutive chromatofocusing separations that the pH gradient differed by less than 0.2 pH units at any time during the elution step. Second dimension retention times of peaks from identical pI fractions differed by less than 7 s in six consecutive separations. Each 2D separation generated a total of 540 fractions which were analyzed by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). We detected approximately 275 peptides and proteins with molecular masses ranging from 3 to 225 kDa. Most fractions were found to contain multiple low and high molecular weight proteins. Differential display of 2D protein expression maps from retinol-sufficient and -deficient rat plasma samples identified a fraction with several proteins that appeared to be down-regulated in the vitamin A-deficient animal. Quantitative proteomic analysis of complex samples such as plasma is still a difficult task. We discuss the potential of this approach for biomarker discovery and address the experimental challenges that remain.     

多维色谱串联质谱分析肝癌患者血浆中的低丰度差异蛋白

建立了分析肝癌患者血浆中低丰度差异蛋白的新的多维色谱串联质谱方法.以5例肝细胞癌(HCC)患者血浆为研究对象,选用免疫亲和色谱Agilent MARS Spin Cartridge去除血浆中6种主要高丰度蛋白;离线RP-HPLC预分级血浆低丰度蛋白并收集差异表达蛋白组;液相色谱-芯片(HPLC-CHIP,包含一支纳升级Zorbax 300SB-C18富集柱和一支纳升级Zorbax 300SB-C18分析柱)富集、分离差异表达蛋白组胰酶酶解肽混合物,线性离子阱质谱在线分析,以Spectrum Mill MS Proteomics Workbench自动分析MS和MS/MS数据后在UniProtKB/SWISS-PORT数据库中进行搜索,鉴定得到27种差异蛋白,其中23种蛋白与各种疾病或肿瘤相关,IMP3、ARNT2和GRIP1可能成为诊断HCC的潜在生物标志物.     

疏水型色谱饼对人血清蛋白质组学样品的快速分离与制备

建立了疏水型色谱饼(10 mm×20 mm i.d.)与反相色谱(RPLC)离线二维色谱快速分离制备人血清蛋白质组学样品,并用基体辅助激光解吸电离飞行时间质谱(MALDI-TOF MS)进行检测的方法。以4种标准蛋白质的稀溶液为模型进行分离富集,得到细胞色素c(Cyt-c)与肌红蛋白(Myo)的检出限均为1 pmol/μL,溶菌酶(Lys)和胰岛素（Ins）的检出限为0.1 pmol/μL。将此方法用于人血清蛋白质组学样品的分离与制备,随着血清处理量的增大,质谱可检出的组分数目与信号强度均增加,当血清处理量达到1.0 mL时,可检出低丰度蛋白质或多肽285个(相对分子质量均在15000以下)。研究中将1 μg Cyt-c加入到0.5 mL血清中,用上述方法在分离富集低丰度Cyt-c上取得了很好的效果。结果表明,采用疏水型色谱饼与反相色谱联用技术不仅可对血清样品中低丰度蛋白质进行有效的分离和富集,而且一次样品的处理量大,可显著提高低丰度蛋白质的分析、检测水平。     

Selectivity of monolithic supports under overloading conditions and their use for separation of human plasma and isolation of low abundance proteins

Human serum albumin (HSA) and immunoglobulin G (IgG) represent over 75% of all proteins present in human plasma. These two proteins frequently interfere with detection, determination and purification of low abundance proteins that can be potential biomarkers and biomarker candidates for various diseases. Some low abundance plasma proteins such as clotting factors and inhibitors are also important therapeutic agents. In this paper, the characterization of ion-exchange monolithic supports under overloading conditions was performed by use of sample displacement chromatography (SDC). If these supports were used for separation of human plasma, the composition of bound and eluted proteins in both anion- and cation-exchange mode is dependent on column loading. Under overloading conditions, the weakly bound proteins such as HSA in anion-exchange and IgG in cation-exchange mode are displaced by stronger binding proteins, and this phenomenon was not dependent on column size. Consequently, small monolithic columns with a column volume of 100 and 200 μL are ideal supports for high-throughput screening in order to develop new methods for separation of complex mixtures, and for sample preparation in proteomic technology.     

一种新的人血浆中含硒蛋白测定方法

提出一个综合聚丙酰胺凝胶电泳分离和２,３－二氨基萘柱前衍生正相液体色谱测定硒的方法（ＤＡＮ－ＨＰＬＣ－ＦＬＤ）,分离测定了高硒地区人血浆中的含硒蛋白。在所鉴定的５种含硒蛋白中,其中至少有３种是从未明确鉴定过的含硒蛋白。     

An analysis of protein abundance suppression in data dependent liquid chromatography and tandem mass spectrometry with tryptic peptide mixtures of five known proteins

Reverse phase liquid chromatography (RPLC) has been widely used in proteomics research for peptide separation. When protein samples are separated by RPLC and identified with electrospray ion trap mass spectrometry (ESI-MS), the signals of high-abundance proteins may suppress those of low-abundance proteins, a phenomenon known as abundance suppression. To what degree the abundance suppression correlates to the number of tryptic peptides in the high-abundance proteins has not been carefully investigated. We tried to answer this question by studying the mixtures digested from five known proteins. The numbers of identified tryptic peptides (longer than five amino acids) of the five proteins ranged from 12 to 47. Four different peptide mixtures with 10- to 100-fold abundance differences of five known proteins were separated by RPLC and identified by ESI-MS. Our results showed that abundance suppression was related to the tryptic peptide numbers in the high-abundance protein. Within a 100-fold protein abundance difference range, tryptic peptide number in the low-abundance proteins could be suppressed up to seven times by high-abundance proteins. The procedure we suggest here can help to identify low-abundance proteins co-purified with their high-abundance binding protein. The result can also help to identify specific high-abundance proteins for removal by immunoaffinity.     

液相色谱-质谱法比较分析正常人与胃癌患者的胃组织蛋白质

通过蛋白质组学技术筛选胃癌相关标志物是目前胃癌研究的热点,也是早期诊断的关键。针对组织蛋白质提取物非常复杂的特点,并根据疏水性的差异,采用反相液相色谱对正常及癌症组织提取蛋白质进行分离。通过比较正常及癌症组织提取蛋白质的谱图差异,收集并酶解差异最大的保留时间为45～47 min的馏分,采用液相色谱-多级质谱联用(LC-MS/MS)鉴定其酶解产物。鉴定结果显示,正常及癌症组织中的共有蛋白质为9个,正常组织中有6个特异蛋白质,而癌症组织中有17个特异蛋白质。通过进一步分析,筛选出胃癌组织中含有的丰度较高的两个蛋白质。应用生物信息学方法分析这些蛋白质,能为将来的药物靶点、药物作用通路研究提供更多的信息。     

Analysis of peptides and protein digests by reversed phase high performance liquid chromatography–electrospray ionisation mass spectrometry using neutral pH elution conditions

In this study, the advantages of carrying out the analysis of peptides and tryptic digests of proteins under gradient elution conditions at pH 6.5 by reversed-phase liquid chromatography (RP-HPLC) and in-line electrospray ionisation mass spectrometry (ESI-MS) are documented. For these RP separations, a double endcapped, bidentate anchored n-octadecyl wide pore silica adsorbent was employed in a capillary column format. Compared to the corresponding analysis of the same peptides and protein tryptic digests using low pH elution conditions for their RP-HPLC separation, this alternative approach provides improved selectivity and more efficient separation of these analytes, thus allowing a more sensitive identification of proteins at different abundance levels, i.e. more tryptic peptides from the same protein could be confidently identified, enabling higher sequence coverage of the protein to be obtained. This approach was further evaluated with very complex tryptic digests derived from a human plasma protein sample using an online two-dimensional (2D) strong cation-exchange (SCX)-RP-HPLC-ESI-MS/MS system. Again, at pH 6.5, with mobile phases of different compositions, improved chromatographic selectivities were obtained, concomitant with more sensitive on-line electrospray ionisation tandem mass spectrometric (ESI-MS/MS) analysis. As a consequence, more plasma proteins could be confidently identified, highlighting the potential of these RP-HPLC methods with elution at pH 6.5 to extend further the scope of proteomic investigations.     

Two-dimensional capillary liquid chromatography: pH gradient ion exchange and reversed phase chromatography for rapid separation of proteins

In the present work, an orthogonal two-dimensional (2D) capillary liquid chromatography (LC) method for fractionation and separation of proteins using wide range pH gradient ion exchange chromatography (IEC) in the first dimension and reversed phase (RP) in the second dimension, is demonstrated. In the first dimension a strong anion exchange (SAX) column subjected to a wide range (10.5-3.5) descending pH gradient was employed, while in the second dimension, a large pore (4,000 A) polystyrene-divinylbenzene (PS-DVB) RP analytical column was used for separation of the protein pH-fractions from the first dimension. The separation power of the off-line 2D method was demonstrated by fractionation and separation of human plasma proteins. Seventeen pH-fractions were manually collected and immediately separated in the second dimension using a column switching capillary RP-LC system. Totally, more than 200 protein peaks were observed in the RP chromatograms of the pH-fractions. On-line 2D analysis was performed for fractionation and separation of ten standard proteins. Two pH-fractions (basic and acidic) from the first dimension were trapped on PS-DVB RP trap columns prior to back-flushed elution onto the analytical RP column for fast separation of the proteins with UV/MS detection.