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Improved 2D Nano-LC/MS for Proteomics Applications: A Comparative Analysis Using Yeast Proteome
用于蛋白质组学应用的改进 2D 纳米 LC/MS:使用酵母蛋白质组的比较分析
Abstract 抽象的
The most commonly used method for protein identification with two-dimensional (2D) online liquid chromatography-mass spectrometry (LC/MS) involves the elution of digest peptides from a strong cation exchange column by an injected salt step gradient of increasing salt concentration followed by reversed phase separation. However, in this approach ion exchange chromatography does not perform to its fullest extent, primarily because the injected volume of salt solution is not optimized to the SCX column. To improve the performance of strong cation exchange chromatography, we developed a new method for 2D online nano-LC/MS that replaces the injected salt step gradient with an optimized semicontinuous pumped salt gradient. The viability of this method is demonstrated in the results of a comparative analysis of a complex tryptic digest of the yeast proteome using the injected salt solution method and the semicontinuous pump salt method. The semicontinuous pump salt method compares favorably with the commonly used injection method and also with an offline 2D-LC method.
最常用的二维 (2D) 在线液相色谱-质谱 (LC/MS) 蛋白质鉴定方法涉及通过增加盐浓度的注射盐梯度梯度从强阳离子交换柱中洗脱消化肽,然后反相分离。然而,在这种方法中,离子交换色谱法并未发挥最大程度的作用,主要是因为盐溶液的注入量并未针对 SCX 柱进行优化。为了提高强阳离子交换色谱的性能,我们开发了一种二维在线纳流 LC/MS 的新方法,用优化的半连续泵送盐梯度取代了注入盐步骤梯度。使用注射盐溶液法和半连续泵盐法对酵母蛋白质组进行复杂胰蛋白酶消化的比较分析结果证明了该方法的可行性。半连续泵盐法与常用的注射法以及离线 2D-LC 法相比具有优势。
关键词:蛋白质组学,在线 2D-LC/MS,2D-LC/MS,多维 LC,酵母蛋白质组
In order to identify proteins from a complex mixture of 5 × 103 to 5 × 104 with a dynamic range of at least 105, it is crucial to develop technologies that have extremely good resolving power as well as extraordinary sensitivity. The most frequently used high-performance liquid chromatographic approach for the separation of peptides from protein digests in complex proteomics applications is two-dimensional (2D) nano-liquid chromatography–mass spectrometry (LC/MS). In this approach, a strong cation exchange (SCX) column is used for the first dimension and a reversed phase (RP) column for the second.1,2 The sample peptides bound on the SCX columns are then eluted by injected salt solution plugs of increasing concentration, trapped on a short enrichment column, and subsequently analyzed on a nano-RP column.
为了从动态范围至少为 10 5的 5 × 10 3至 5 × 10 4的复杂混合物中识别蛋白质,开发具有极好的分辨率和非凡灵敏度的技术至关重要。在复杂的蛋白质组学应用中,最常用的从蛋白质消化物中分离肽的高效液相色谱方法是二维 (2D) 纳米液相色谱-质谱 (LC/MS)。在此方法中,强阳离子交换 (SCX) 柱用于第一维,反相 (RP) 柱用于第二维。 1 2结合在 SCX 柱上的样品肽随后通过浓度不断增加的注射盐溶液塞进行洗脱,捕获在短富集柱上,随后在 nano-RP 柱上进行分析。
This method is capable of delivering valuable results for proteomics research. For instance, it has been used successfully in elucidating the yeast proteome and the proteome of other microorganisms.3,4 Even the comprehensive analysis of subproteomes consisting of few proteins in a background of several hundred has recently been achieved.5,6 However, because the injected volume of salt solution is not optimized to the SCX column, the SCX column is cannot work to its fullest potential. This lack of optimization results in the distribution of the peptides over more than one fraction, which can dilute them below their detection level or suppress their ionization in nanoelectrospray by higher abundant peptides in the mass spectrometric analysis.
该方法能够为蛋白质组学研究提供有价值的结果。例如,它已成功用于阐明酵母蛋白质组和其他微生物的蛋白质组。 3 4最近甚至还实现了对数百种背景中由少量蛋白质组成的亚蛋白质组的综合分析。 5 6然而,由于盐溶液的进样量并未针对 SCX 色谱柱进行优化,因此 SCX 色谱柱无法充分发挥其潜力。这种优化的缺乏导致肽分布在多个级分上,这可以将它们稀释到检测水平以下,或者通过质谱分析中丰度较高的肽来抑制它们在纳电喷雾中的电离。
To overcome these limitations we developed an improved method for online 2D-LC. In this method the optimized semicontinuous salt solution gradient for the elution of the peptides from the SCX column is delivered very precisely with a capillary pump, and the SCX column is always kept under conditions very close to the optimum state. The eluted peptides are trapped rotatory on two enrichment columns and are subjected to RP separation followed by MS/MS analysis. The principle of this method is illustrated in Figure 11.
为了克服这些限制,我们开发了一种改进的在线二维液相色谱方法。在此方法中,通过毛细管泵非常精确地输送用于从 SCX 柱洗脱肽的优化半连续盐溶液梯度,并且 SCX 柱始终保持在非常接近最佳状态的条件下。洗脱的肽被旋转捕获在两个富集柱上,并进行反相分离,然后进行 MS/MS 分析。该方法原理如图1 1所示。
In this paper, the improved method for 2D nano-LC/MS is explained in detail. To show the full performance of the method, a complex tryptic digest of the yeast proteome was analyzed and the results compared with those obtained by analysis using the injected salt solution method in current use as well as an offline 2D LC method.
本文详细阐述了二维纳米LC/MS 的改进方法。为了显示该方法的全部性能,对酵母蛋白质组的复杂胰蛋白酶消化进行了分析,并将结果与使用当前使用的注入盐溶液方法以及离线二维液相色谱方法进行分析所获得的结果进行了比较。
MATERIALS AND METHODS 材料和方法
Equipment: Agilent (Walbronn, Germany) 1100 Series nanoflow pump with micro-vacuum degasser, 1100 Series thermostatted micro well-plate autosampler, micro 2-position/10-port switching valve box with holder, 1100 Series capillary pump with micro vacuum degasser, 1100 Series LC/MSD Trap XCT with orthogonal nanospray ion source. Software: ChemStation A10.01, Ion Trap Software 4.2, Spectrum Mill MS Proteomics Workbench. Chromatography columns: Agilent BioSCX Series II, 0.30 × 35 mm, 3.5 μm particles; Zorbax 300 SB C18, 75 μm × 150 mm, 3.5 μm particles; Zorbax 300 SB C18, 0.3 mm × 5 mm, 5.0 μm particles.
设备:安捷伦(Walbronn,德国)带微量真空脱气器的 1100 系列纳流泵、1100 系列恒温微量孔板自动进样器、带支架的微型 2 位/10 通切换阀箱、带微量真空脱气器的 1100 系列毛细管泵、 1100 系列 LC/MSD Trap XCT 配备正交纳喷雾离子源。软件:ChemStation A10.01、离子阱软件 4.2、Spectrum Mill MS 蛋白质组学工作台。色谱柱:Agilent BioSCX 系列 II,0.30 × 35 mm,3.5 µm 颗粒; Zorbax 300 SB C18,75 μm × 150 mm,3.5 μm 颗粒; Zorbax 300 SB C18,0.3 mm × 5 mm,5.0 μm 颗粒。
System Description 系统说明
The micro 2-position/10-port valve included in the LC-System (Fig. 22)) is connected with two enrichment columns. This valve is also connected directly to the nanoflow pump and to the nanocolumn. The second pump used in the system, the capillary pump, is connected to the micro 2-position/6-port valve in the micro well-plate autosampler, which is also connected with the micro 10-port valve (Fig. 33).). The sample peptides retained on the SCX column are eluted stepwise with a semicontinuous salt solution gradient pumped by the capillary pump and subsequently trapped on the enrichment column currently inline with the SCX column (Fig. 3A-13A-1).). Each step of the semicontinuous salt solution gradient starts with the end concentration of the foregoing step and ends with the starting concentration of the following step. After each step the SCX column is bypassed by switching the micro valve in the autosampler (Fig. 3A-23A-2)) to retain the current salt concentration in the SCX column. In this state the enrichment column is still inline with the capillary pump, which starts to pump water to wash out salt residues from the capillaries and the enrichment column prior to the RP separation and the MS analysis. The salt-free enrichment column is then switched into the nanoflow path and exchanged with the second enrichment column, also located at the micro 10-port valve, and the cycle starts over again (Fig. 3B3B).
LC 系统中包含的微型 2 位/10 通阀(图 2 2) )与两个富集柱连接。该阀门还直接连接到纳流泵和纳流柱。系统中使用的第二个泵,即毛细管泵,连接到微孔板自动进样器中的微型2位/6通阀,该微孔板自动进样器还连接到微型10通阀(图3 3) )。 )。保留在 SCX 柱上的样品肽通过毛细管泵泵送的半连续盐溶液梯度逐步洗脱,随后捕获在当前与 SCX 柱串联的富集柱上(图 3A-1 3A-1)。 )。半连续盐溶液梯度的每一步都以前一步骤的最终浓度开始,并以下一步骤的起始浓度结束。每个步骤后,通过切换自动进样器中的微阀绕过 SCX 色谱柱(图 3A-2 3A-2) ) 以保留 SCX 柱中当前的盐浓度。在此状态下,富集柱仍与毛细管泵串联,毛细管泵开始抽水以在反相分离和 MS 分析之前从毛细管和富集柱中冲洗掉盐残留物。然后将无盐富集柱切换到纳流路径并与同样位于微型十通阀处的第二个富集柱交换,然后再次开始循环(图3B 3B) )。
Chromatographic Method 色谱法
For the chromatographic separation in the first- and second dimension it is necessary to set up two different methods, one for the SCX chromatography and another one for the RP separation. The semicontinuous salt solution gradient for the elution of the peptides from the SCX column is delivered from the capillary pump and the gradient for the RP separation is delivered from the nanoflow pump (Fig. 44).). The nanoflow gradient starts with 5% acetonitrile and increases up to 65% acetonitrile with a slope of 1%/min for each RP analysis. The salt gradient is pumped in steps beginning at 0% to 2.5% of a 500- mM NaCl solution for the first step. The following steps start with the end concentration of the foregoing step and end with the starting concentration of the following step. The salt solution gradient is developed for 15 min in each step and then, prior to the washing step, the SCX column is switched to bypass with the micro 6-port valve in the autosampler to retain the current condition. Therefore, each step contributes to a semicontinuous salt gradient on the SCX column (Fig. 44).
对于第一维和第二维的色谱分离,有必要建立两种不同的方法,一种用于SCX色谱,另一种用于RP分离。用于从 SCX 柱洗脱肽的半连续盐溶液梯度由毛细管泵输送,用于 RP 分离的梯度由纳流泵输送(图 4 4)。 )。每次 RP 分析的纳流梯度从 5% 乙腈开始,逐渐增加至 65% 乙腈,斜率为 1%/分钟。第一步从 0% 至 2.5% 的 500 mM NaCl 溶液开始分步泵入盐梯度。以下步骤以上一步骤的最终浓度开始,并以下一步骤的起始浓度结束。每个步骤中形成盐溶液梯度 15 分钟,然后在洗涤步骤之前,将 SCX 柱切换至自动进样器中的微型 6 通阀旁路以保持当前状态。因此,每个步骤都有助于 SCX 柱上的半连续盐梯度(图 4 4 )。
To obtain a good separation for the majority of peptides eluting at lower salt concentration, the slope is shallower in this area and steeper in the area of higher salt concentrations. The micro 6-port valve in the autosampler switches the SCX column into the salt solution flow at the beginning of each step and switches the SCX column to bypass at the and of each salt step (Figure 44).). At the starting point of each RP analysis cycle the charged enrichment column is exchanged against the empty one by a switch of the micro 10-port valve (Fig. 33).). The detailed gradient settings for the SCX and the RP chromatography, the valve switching points for the autosampler micro 6-port valve, and the micro 10-port valve are outlined in Table 11.
为了使大多数在较低盐浓度下洗脱的肽获得良好的分离,该区域的斜率较浅,而在较高盐浓度的区域则较陡。自动进样器中的微型 6 通阀在每个步骤开始时将 SCX 色谱柱切换到盐溶液流中,并在每个盐步骤结束时将 SCX 色谱柱切换为旁路(图 4 4)。 )。在每个 RP 分析周期的起始点,通过微型 10 通阀的开关将带电的富集柱与空的富集柱交换(图 3 3)。 )。 SCX 和 RP 色谱的详细梯度设置、自动进样器微型 6 通阀和微型 10 通阀的阀门切换点如表 1 1所示。
TABLE 1 表1
NANOFLOW PUMP 纳流泵 | |||||||||||||||||||||
Time [min] 时间[分钟] | 0 | 10 | 70 | 70.01 | 85 | 145 | 145.01 | 160 | 220 | 220.01 | 235 | 295 | 295.01 | 310 | 370 | 370.01 | 385 | ||||
% Solvent B % 溶剂 B | 5 | 5 | 65 | 5 | 5 | 65 | 5 | 5 | 65 | 5 | 5 | 65 | 5 | 5 | 65 | 5 | 5 | ||||
Time [min] 时间[分钟] | 445 | 445.01 | 460 | 520 | 520.01 | 535 | 595 | 595.01 | 610 | 675 | 675.01 | 685 | 745 | 745 | 760 | 820 | 820.01 | ||||
% Solvent B % 溶剂 B | 65 | 5 | 5 | 65 | 5 | 5 | 65 | 5 | 5 | 65 | 5 | 5 | 65 | 5 | 5 | 65 | 5 | ||||
CAPILLARY PUMP 毛细管泵 | |||||||||||||||||||||
Time [min] 时间[分钟] | 0 | 15 | 15.01 | 30 | 30.01 | 90 | 90.01 | 105 | 105.01 | 165 | 165.01 | 180 | 180.01 | 240 | 240.01 | 255 | 255.01 | 315 | 315.01 | 330 | 330.01 |
% Solvent B % 溶剂 B | 0 | 0 | 0 | 2.5 | 0 | 0 | 2.5 | 5 | 0 | 0 | 5 | 7.5 | 0 | 0 | 7.5 | 10 | 0 | 0 | 10 | 15 | 0 |
Time [min] 时间[分钟] | 390 | 390.01 | 405 | 405.01 | 465 | 465.01 | 480 | 480.01 | 540 | 540.0 | 555 | 555.01 | 615 | 615.01 | 630 | 630.01 | 690 | 690.01 | 705 | 705.01 | 820.01 |
% Solvent B | 0 | 15 | 20 | 0 | 0 | 20 | 30 | 0 | 0 | 30 | 50 | 0 | 0 | 50 | 100 | 0 | 0 | 100 | 100 | 0 | 0 |
10-PORT VALVE | |||||||||||||||||||||
Switch Position | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | 1 | 2 | |||||||||
Time [min] | 0 | 10 | 85 | 160 | 235 | 310 | 385 | 460 | 535 | 610 | 685 | 760 | |||||||||
MICRO WELL-PLATE AUTOSAMPLER 6-PORT VALVE | |||||||||||||||||||||
Mainpass time [min] | 0 | 90 | 165 | 240 | 315 | 390 | 465 | 540 | 615 | 695 | 765 | ||||||||||
Bypass time [min] | 30 | 105 | 180 | 255 | 330 | 405 | 480 | 555 | 630 | 705 |
Nanoflow Pump 纳流泵
The solvents are A = water + 0.1% formic acid and B= acetonitrile + 0.1% formic acid. The primary flow is 200–500 μL/min, and the column flow is 300 nL/min. The stop time is 825 min and post time is 15 min.
溶剂为A=水+0.1%甲酸和B=乙腈+0.1%甲酸。主流量为 200–500 μL/min,柱流量为 300 nL/min。停靠时间为825分钟,发车时间为15分钟。
Capillary Pump 毛细管泵
The solvents are A = water + 3% acetonitrile + 0.1% formic acid and B = 500 mM NaCl + 3% acetonitrile + 0.1% formic acid. The primary flow is 500–800 μL/min, and the column flow 10 μL/min.
溶剂为 A = 水 + 3% 乙腈 + 0.1% 甲酸,B = 500 mM NaCl + 3% 乙腈 + 0.1% 甲酸。初级流量为 500–800 μL/min,柱流量为 10 μL/min。
MS Conditions 多发性硬化症条件
The ionization mode is positive nanoelectrospray with an Agilent orthogonal source. Drying gas flow is 5 L/min and drying gas temperature is 300°C. Vcap is typically 1800–2000 V, skim 1 is 30 V, and capillary exit offset is 75 V. The trap drive is 85 V with averages of 1 or 2. ICC is on; maximum accumulation time is 150 ms, smart target is 125,000, and MS scan range is 300–2200.
电离模式是采用安捷伦正交源的正纳电喷雾。干燥气体流量为5L/min,干燥气体温度为300℃。 Vcap 通常为 1800–2000 V,撇去 1 为 30 V,毛细管出口偏移为 75 V。陷阱驱动为 85 V,平均值为 1 或 2。ICC 打开;最大累积时间为150 ms,智能目标为125,000,MS扫描范围为300-2200。
Automatic MS/MS is in peptide scan mode, with the number of parents 3 or 4, averages of 2, fragmentation amplitude of 1.3 V, SmartFrag on (30–200%), active exclusion on (after 2 spectra for 1 min), prefer +2 on, MS/MS scan range of 100–1800, and ultra scan on.
自动 MS/MS 处于肽扫描模式,亲本数量为 3 或 4,平均值为 2,碎片幅度为 1.3 V,SmartFrag 打开 (30–200%),主动排除打开(2 个谱图 1 分钟后),首选 +2 打开,MS/MS 扫描范围为 100–1800,以及超扫描打开。
Sample Preparation 样品制备
Lyophilized yeast cells (Saccharomyces cerevisiae), resuspended in cooled 50 mM NH4HCO3 containing 8 M urea, were disrupted in a bead beater with 0.5 mm glass beads (Bead beater, BioSpec Products, Bartlesville, OK). After centrifugation to remove cell debris, proteins in the supernatant were reduced with 1 mM DTT at 37°C for 1 h, alkylated in the dark with 10 mM iodoacetamide for 30 min at RT, ultrafiltrated for buffer exchange, and tryptically digested with TPCK trypsin at 37°C for 16 h. Finally, the sample was lyophilized in a SpeedVac (Bachofer, Reutingen, Germany) and dissolved in 5% acetonitrile, 0.03% formic acid prior to analysis.
将冻干的酵母细胞(酿酒酵母)重悬于含有8M尿素的冷却的50mM NH 4 HCO 3中,并在具有0.5mm玻璃珠的珠式搅拌器(珠式搅拌器,BioSpec Products,Bartlesville,OK)中破碎。离心去除细胞碎片后,用 1 mM DTT 在 37°C 还原上清液中的蛋白质 1 小时,在黑暗中用 10 mM 碘乙酰胺在室温下烷基化 30 分钟,超滤以进行缓冲液交换,并用 TPCK 胰蛋白酶进行胰蛋白酶消化37°C 16 小时。最后,在分析前将样品在 SpeedVac(Bachofer,Reutingen,德国)中冻干并溶解在 5% 乙腈、0.03% 甲酸中。
RESULTS AND DISCUSSION 结果与讨论
For the regular online 2D LC method, which is working with an injected salt step gradient, the peptide MS/MS spectra obtained from a single salt step elution are stored individually. Hence, a complete 2D LC/MS run yields as many data files as fractions are taken. In contrast, during the newly developed semicontinuous 2D LC run, all MS/MS data are saved into one single data file. Therefore, the whole 820-min run can be displayed in one chromatogram (Fig. 55).). As an example, a whole yeast proteome digest was subjected to analysis with this technique. The obtained base peak chromatogram (Fig. 55)) indicates that, besides the peptides in the unbound fraction, the majority of the peptides are eluted in the semicontinuous salt gradient up to a salt concentration of 100 mM. Therefore, this concentration range was divided into smaller salt elution steps and the region from 100 mM up to 500 mM into larger salt elution intervals. There are only a few remaining strongly binding peptides that require high salt concentrations for elution.
对于使用注入盐步梯度的常规在线 2D LC 方法,从单盐步洗脱获得的肽 MS/MS 谱图会单独存储。因此,完整的 2D LC/MS 运行会产生与获取的分数一样多的数据文件。相比之下,在新开发的半连续 2D LC 运行期间,所有 MS/MS 数据都保存到一个数据文件中。因此,整个 820 分钟的运行可以显示在一张色谱图中(图 5 5)。 )。例如,使用该技术对整个酵母蛋白质组消化进行了分析。得到的基峰色谱图(图5 5) ) 表明,除了未结合级分中的肽之外,大多数肽在盐浓度高达 100 mM 的半连续盐梯度中洗脱。因此,该浓度范围被分为较小的盐洗脱步骤,并且从 100 mM 到 500 mM 的区域被分为较大的盐洗脱间隔。仅剩下少数强结合肽需要高盐浓度进行洗脱。
Figure 66 shows the magnified base peak chromatograms obtained after RP separation for all semicontinuous salt elution steps up to 100 mM. The peptides resulting from each salt step are separated within 30 min in subsequent RP gradient runs. In the same time frame the associated MS/MS spectra necessary for the database search are acquired.
图6 6显示了所有半连续盐洗脱步骤(最高 100 mM)的 RP 分离后获得的放大基峰色谱图。每个盐步骤产生的肽在随后的 RP 梯度运行中在 30 分钟内分离。在同一时间范围内,获取数据库搜索所需的相关 MS/MS 谱图。
During the entire run time more than 30,000 MS/MS spectra were recorded. From those spectra only the peptide MS/MS spectra were extracted with the spectra extractor of the Spectrum Mill software within a few minutes. After the subsequent database search and validation of the obtained protein hits, 122 proteins were identified with high confidence. The 21 top score proteins are presented in Table 22.. Their resulting score indicates high-quality MS/MS spectra and a high percentage of sequence coverage. Despite of the fact that only a few peptides were identified for proteins at the end of the list (positions 120–222), the quality of the corresponding MS fragmentation spectra are still sufficiently convincing for the unequivocal identification of the corresponding proteins. To provide an estimate of the reliability of the identified proteins obtained from database search, exemplary MS/MS spectra are shown for peptides identified for the first and last protein from the score hit list. The tryptic peptide (GVE)VVLPVDFIIADAFSADA(NTK), which according to the top score is protein phosphoglycerate kinase, shows a complete fragmentation pattern with all y- and b-series ions (Fig. 7A7A).). The same holds true for the fragmentation pattern of a tryptic peptide (SQ)LAQQIQAR from protein hit number 120, polyadenylate-binding protein (Fig. 7B7B).
在整个运行时间内,记录了超过 30,000 个 MS/MS 谱图。使用 Spectrum Mill 软件的光谱提取器在几分钟内仅从这些光谱中提取肽 MS/MS 光谱。经过随后的数据库搜索和对获得的蛋白质命中进行验证后,以高置信度鉴定了 122 个蛋白质。表 2 2 列出了 21 种得分最高的蛋白质。 。他们的得分表明高质量的 MS/MS 谱图和高百分比的序列覆盖率。尽管事实上仅在列表末尾(位置 120-222)仅鉴定出少量蛋白质的肽,但相应 MS 碎片谱的质量对于明确鉴定相应蛋白质而言仍然足够令人信服。为了提供对从数据库搜索中获得的已鉴定蛋白质的可靠性的估计,显示了针对得分命中列表中的第一个和最后一个蛋白质鉴定的肽的示例性MS/MS谱图。胰蛋白酶肽 (GVE)VVLPVDFIIADAFSADA(NTK) 根据最高分是蛋白磷酸甘油酸激酶,显示出所有 y 系列和 b 系列离子的完整断裂模式(图 7A 7A)。 )。对于来自蛋白质命中编号 120 的多聚腺苷酸结合蛋白的胰蛋白酶肽 (SQ)LAQQIQAR 的片段化模式也是如此(图 7B 7B )。
TABLE 2 表2
Hit no. 打不。 | Protein name 蛋白质名称 | Score 分数 | Peptides 肽 | Spectra (no.) 光谱(数量) | % AA coverage % AA 覆盖率 | Protein MW (Da) 蛋白质分子量 (Da) | Protein pI 蛋白质等电点 |
1 | Phosphopyruvate hydratase 磷酸丙酮酸水合酶 | 142.37 | 8 | 78 | 29 | 46802.3 | 6.16 |
2 | Phosphoglycerate kinase 磷酸甘油酸激酶 | 137.35 | 9 | 29 | 33 | 44738.6 | 7.11 |
3 | Pyruvate kinase 丙酮酸激酶 | 115.12 | 7 | 9 | 28 | 54599 | 8.00 |
4 | Pyruvate decarboxylase 丙酮酸脱羧酶 | 112.05 | 7 | 8 | 21 | 61495.7 | 5.80 |
5 | Reading frame 阅读框 | 84.38 | 4 | 114 | 28 | 35731.9 | 6.46 |
6 | Heat-shock protein 26 kDa 热休克蛋白 26 kDa | 84.12 | 5 | 9 | 30 | 23879.7 | 5.32 |
7 | Aldehyde dehydrogenase 乙醛脱氢酶 | 72.17 | 5 | 6 | 18 | 56723.9 | 6.31 |
8 | Hexokinase A 己糖激酶A | 71.71 | 4 | 6 | 14 | 53738.7 | 5.28 |
9 | Alcohol dehydrogenase I 乙醇脱氢酶I | 57.18 | 4 | 12 | 16 | 36823.3 | 6.26 |
10 | Triosephosphate isomerase 磷酸丙糖异构酶 | 49.69 | 4 | 4 | 30 | 26795.6 | 5.74 |
11 | Ketol-acid reductoisomerase 酮酸还原异构酶 | 46.82 | 3 | 3 | 12 | 44368.7 | 9.11 |
12 | Phosphoglycerate mutase 磷酸甘油酸变位酶 | 44.71 | 3 | 4 | 19 | 27608.7 | 8.81 |
13 | 60S ribosomal protein L4-B 60S核糖体蛋白L4-B | 38.32 | 3 | 3 | 17 | 39062.2 | 10.64 |
14 | Glucose kinase 葡萄糖激酶 | 35.38 | 2 | 2 | 7 | 55377.7 | 5.80 |
15 | Hexokinase PII 己糖激酶PII | 33.32 | 2 | 2 | 14 | 27485.6 | 5.19 |
16 | Pyrophosphatase 焦磷酸酶 | 32.77 | 3 | 3 | 11 | 32315.8 | 5.36 |
17 | 60S ribosomal protein L5 60S核糖体蛋白L5 | 31.08 | 2 | 2 | 13 | 33743 | 6.36 |
18 | BMH1 | 29.17 | 2 | 4 | 15 | 30176.6 | 4.87 |
19 | Translation elongation factor eEF-1 平移延伸因子 eEF-1 | 28.39 | 2 | 10 | 7 | 50032.9 | 9.14 |
20 | Citrate (si)-synthase 柠檬酸(si)-合酶 | 25.16 | 2 | 2 | 5 | 53360.3 | 8.23 |
21 | Superoxide dismutase 超氧化物歧化酶 | 24.66 | 2 | 2 | 27 | 15854.7 | 5.62 |
120 | Polyadenylate-binding protein 聚腺苷酸结合蛋白 | 9.10 | 1 | 1 | 1 | 64344.5 | 5.71 |
121 | Ribosomal protein S15 核糖体蛋白S15 | 9.06 | 1 | 1 | 13 | 16001.9 | 10.70 |
122 | Ribosomal protein S14 核糖体蛋白S14 | 9.03 | 1 | 1 | 7 | 14649.8 | 10.54 |
To evaluate this new methodology, the results obtained by the semicontinuous salt gradient approach were compared with those obtained by the conventional injected salt step gradient as well as with those obtained by high resolution off-line 2D methodology, which also works with a linear continuous salt gradient elution in the first dimension.7 For this comparison, sample and columns used were identical. Salt steps for the online 2D LC, the semicontinuous 2D LC, and the linear gradients with fraction collection for the offline 2D LC were adjusted so that they covered comparable salt concentration ranges. The results of these experiments are summarized in Table 33.. The number of identified proteins as well as the number of corresponding peptides increased from the injected step gradient approach to the semicontinuous gradient approach. It is also evident that the off-line methodology is superior to the on-line variants, which is related to higher investment efforts for instrumentation. These results demonstrate clearly the significant improvement which can be achieved with slight modifications of the instrumental system setup and the elaborated 2D LC protocol.2
为了评估这种新方法,将半连续盐梯度方法获得的结果与传统注入盐梯度方法获得的结果以及高分辨率离线二维方法获得的结果进行了比较,该方法也适用于线性连续盐第一维梯度洗脱。 7在此比较中,使用的样品和色谱柱是相同的。调整在线 2D LC、半连续 2D LC 的盐步骤以及离线 2D LC 的带有馏分收集的线性梯度,以便它们覆盖可比的盐浓度范围。这些实验的结果总结在表 3 3 中。 。从注射步进梯度方法到半连续梯度方法,鉴定的蛋白质数量以及相应肽的数量有所增加。同样明显的是,离线方法优于在线方法,这与仪器设备的更高投资有关。这些结果清楚地表明,通过对仪器系统设置和详细的 2D LC 协议进行轻微修改即可实现显着的改进。 2
TABLE 3 表3
Method 方法 | Online 2D LC with injected salt steps 带注入盐步骤的在线二维液相色谱 | Online 2D LC with pumped semicontinuous salt gradient 具有泵送半连续盐梯度的在线二维液相色谱 | Off-line 2D LC with pumped continuous salt gradient 具有泵送连续盐梯度的离线二维液相色谱 |
Identified proteins 已鉴定的蛋白质 | 101 | 122 | 144 |
Assigned peptides 指定肽 | 179 | 207 | 269 |
CONCLUSION 结论
In this work we demonstrate that the performance of the classical approach of 2D LC/MS for protein identification, which works with injected salt solution plugs of increasing concentration to elute peptides of digested proteome samples from the first dimension SCX column, can be improved significantly. This improvement can be attributed mainly to the semicontinuous salt solution gradient, which keeps the SCX column in an optimum state during peptide elution of the first dimension. In this state, peptides elute in much sharper peaks and therefore lower abundant proteins are identified more easily. To utilize this effect only a minor hardware investment is necessary—mainly a precise second pump and a micro 10-port valve with two enrichment columns. Table 44 shows a summary of the semicontinuous gradient online 2D LC method in comparison to the injected step gradient on-line 2D LC method and to the offline 2D LC method.
在这项工作中,我们证明了用于蛋白质鉴定的 2D LC/MS 经典方法的性能可以得到显着改善,该方法使用浓度不断增加的注射盐溶液塞,从第一维 SCX 柱中洗脱消化蛋白质组样品的肽。这种改进主要归功于半连续盐溶液梯度,它使 SCX 柱在第一维肽洗脱过程中保持最佳状态。在这种状态下,肽以更尖锐的峰洗脱,因此更容易识别丰度较低的蛋白质。为了利用这种效应,只需要少量的硬件投资——主要是一个精确的第二泵和一个带有两个浓缩柱的微型 10 通阀。表4 4显示了半连续梯度在线 2D LC 方法与注入阶跃梯度在线 2D LC 方法和离线 2D LC 方法的比较总结。
TABLE 4 表4
Method 方法 | Online 2D LC with injected salt steps 带注入盐步骤的在线二维液相色谱 | Online 2D LC with pumped semicontinuous salt gradient 具有泵送半连续盐梯度的在线二维液相色谱 | Off-line 2D LC with pumped continuous salt gradient 具有泵送连续盐梯度的离线二维液相色谱 |
SCX Resolution SCX分辨率 | low 低的 | high 高的 | very high 非常高 |
Automation 自动化 | high 高的 | high 高的 | medium 中等的 |
Complexity of set-up 设置的复杂性 | medium 中等的 | high 高的 | medium 中等的 |
Investment 投资 | low 低的 | medium 中等的 | high 高的 |
Effort 努力 | medium 中等的 | medium 中等的 | high 高的 |
Access to SCX fractions 获取 SCX 馏分 | no 不 | no 不 | yes 是的 |
Flexibility 灵活性 | low 低的 | low 低的 | high 高的 |
The described method offers the opportunity to analyze real-life proteomics samples with high complexity in a highly automated manner with maximum results. Moreover, this approach has the potential for further improvement by the adaptation of the method to a true continuous salt gradient for the first dimension SCX chromatography.
所述方法提供了以高度自动化的方式分析具有高复杂性的现实蛋白质组学样本的机会,并获得最大的结果。此外,通过将该方法适应第一维 SCX 色谱的真正连续盐梯度,该方法具有进一步改进的潜力。
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生物分子技术杂志:JBT中的文章由生物分子资源设施协会提供