Merck

蛋白质谱

用于蛋白质鉴定、表征和定量的蛋白质谱仪

蛋白质谱广泛用于生物样品的分析,适用于生物标记物发现、蛋白质组学研究和临床应用。与其他蛋白大规模鉴定的技术相比,质谱仪可进行复杂的分析,已经成为蛋白质组学的主要工具。

质谱已用于基于蛋白质结构、翻译后修饰和相互作用分析,以定量鉴定蛋白质。

  • 蛋白质鉴定 通常涉及将蛋白质进行化学或酶消化成肽,然后通过质谱进行分析并使用计算方法或测序进行鉴定。
  • 翻译后修饰 可以通过氨基酸残基质量的变化来进行确定。修饰位点可以通过测序或相关计算方法来进行定位。
  • 对于聚糖分析与分析来说,需使用酶或化学方法从糖蛋白释放聚糖基团,然后将释放的聚糖衍生化以进行质谱分析。
  • 蛋白质相互作用 通过特异性靶蛋白与任何相互作用的蛋白的亲和共纯化来进行确定,或在进行质谱分析之前使用尺寸排阻或离子交换色谱进行更全面的研究。

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相关实验方案


对于定量蛋白质组学而言,可以使用串联质量标记(TMT)和iTRAQ对具有稳定同位素的蛋白质或肽进行化学标记,或者通过掺入标记的氨基酸(SILAC)在代谢上进行蛋白质标记。通过将质谱峰强度与蛋白质丰度进行关联,可以对重同位素和轻同位素的掺入量进行比较,从而实现相对定量。为了进行绝对定量,可以在样品中掺入同位素标记的合成肽或蛋白质标准品,以进行选定的反应监测(SRM)分析

在蛋白质谱中,不同蛋白质和多肽的质量是通过测量其气相离子的m/z(质荷比)来进行确定。质谱仪首先会使用离子源将蛋白质分子转换为气相离子。接下来,质量分析器根据m/z比对离子化的分析物进行分离。然后,检测器会记录下每个m/z值处的离子数。MALDI和电喷雾电离(ESI)通常被用于对肽或蛋白质进行电离。

MALDI-TOF质谱

MALDI是一种电离方法,它使用激光能量吸收基质来产生离子,并保证蛋白质分子最少的片段化。首先将样品与合适的基质材料进行混合。接下来,使用脉冲激光照射样品,从而触发样品和基质材料的烧蚀和解吸。然后分析物分子在烧蚀气体中通过质子化或去质子化进行电离,从而再进行质谱分析。

电喷雾电离质谱

电喷雾电离(ESI)使用电喷雾产生离子,在电喷雾中向液体样品施加高电压以产生气溶胶,从而产生肽和蛋白质片段化最少的离子。由于液相色谱洗脱液可直接加入电喷雾中进行串联处理,因此当需将质谱联用到液相色谱仪(LC-MS)时,通常使用电喷雾电离。


工作流程

Sample preparation for protein analysis by mass spectrometry

蛋白质样品的制备

Protein sample preparation for mass spectrometry requires cell lysis or protein solubilization and stabilization. For proteomic analysis, cells are lysed using buffers that break down the cell membrane with protease inhibitors to prevent protein degradation. High-abundance proteins are removed as necessary. Recovery and enrichment of select fractions facilitate proteomic analysis.

Site-specific proteolysis of samples for protein mass spectrometry

Site-specific proteases, such as trypsin, are used to cleave proteins into small fragments to enable identification by matching experimental spectra to theoretical spectra from protein databases or comparing to run standards. For relative quantitation, cell cultures can be metabolically labeled using stable isotopes incorporated through amino acid feeding or samples can be labeled with stable isotopes using chemical methods. Spiking samples with isotopically labeled synthetic peptides permits absolute quantitation through selected reaction monitoring (SRM) analysis.

Use of calibrators and standards for protein mass spectrometry

Calibration standards can serve as controls for sample analysis and be used to determine protein identity, experimental sensitivity, digestion efficiency, and aid in chromatographic separation and quantitative analysis.

Use of tandem chromatography in protein mass spectrometry

Chromatography allows separation of proteins and peptides into more manageable samples for analysis. Because multiple distinct peptides can have similar masses, HPLC is commonly used to prevent the simultaneous addition of peptides with very similar or identical masses to the mass spectrometer, increasing the overall dynamic range of measurements.

Protein detection and analysis in mass spectrometry workflows

Proteins and peptides are ionized by MALDI or ESI prior to detection and analysis. A mass analyzer distinguishes ions based on their m/z values. Resulting fragmentation patterns can be used for identification, and samples can be quantitated relatively by evaluating peak intensity ratios from samples differentiated by isotopic labeling or quantitated absolutely using SRM analysis with labeled internal standards.




产品亮点

Mass spectrometry workflow and tools
ISOTEC®

细胞培养中氨基酸稳定同位素标记(SILAC)入门指南

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Primer for stable isotope labeling by amino acids in cell culture (SILAC)
质谱分析工具

质谱工作流程和质谱法蛋白监测工具

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