Ahmed Farid E
General, Environmental and Molecular Toxicology (GEM Tox) Consultants & Labs, Inc., 2607 Calvin Way, Greenville, NC 27834, USA.
Expert Rev Proteomics. 2008 Dec;5(6):841-64. doi: 10.1586/14789450.5.6.841.
This article is the first in a series of reviews intended as a tutorial providing the inexperienced, as well as the experienced, reader with an overview of principles of peptide and protein fragmentation in mass spectrometers for protein identification, surveying of the different types of instrument configurations and their combinations for protein identification. The first mass spectrometer was developed in 1899, but it took almost a century for the instrument to become a routine analytical method in proteomic research when fast atom bombardment ionization was developed, followed shortly by soft desorption/ionization methods, such as MALDI and electrospray ionization, to volatize biomolecules with masses of tens of kiloDaltons into the gas phase under vacuum pressure without destroying them. Thereafter, other soft ionization techniques that offered ambient conditions were also introduced, such as atmospheric pressure MALDI, direct analysis in real time, atmospheric-pressure solid analysis probe and hybrid ionization, sources of MALDI and electrospray ionization (e.g., two-step fused droplet electrospray ionization, laser desorption atmospheric-pressure chemical ionization, electrosonic spray ionization, desorption electrospray ionization, and electrospray-assisted laser desorption/ionization). The five basic types of mass analyzers currently used in proteomic research are the quadrupole, ion trap, orbitrap, Fourier transform ion cyclotron resonance and TOF instruments, which differ in how they determine the mass-to-charge ratios of the peptides. They have very different design and performance characteristics. These analyzers can be stand alone or, in some cases, put together in tandem or in conjunction with ion mobility mass spectrometry to take advantage of the strengths of each. Several singly or multiply charged fragment ion types, such as b, y, a, c, z, v, y and immonium ions are produced in the gas phase of the spectrometer. In the bottom-up sequencing approach for protein identification in a shotgun proteomic experiment, proteolytic digestion of proteins is accomplished by cleavage of the different bonds along the peptide backbone and/or side chain through a charge-directed transfer to the vicinity of the cleavage side. These various mass spectrometers and the types of ions produced have become important analytical tools for studying and analyzing proteins, peptides and amino acids.
本文是系列综述中的第一篇，旨在作为教程，为缺乏经验以及经验丰富的读者提供质谱仪中肽和蛋白质片段化原理的概述，用于蛋白质鉴定，探讨不同类型的仪器配置及其用于蛋白质鉴定的组合方式。第一台质谱仪于1899年研制出来，但直到快原子轰击电离技术的出现，该仪器才在蛋白质组学研究中成为一种常规分析方法，随后不久又出现了软解吸/电离方法，如基质辅助激光解吸电离（MALDI）和电喷雾电离（ESI），能够在真空压力下将质量达数十千道尔顿的生物分子挥发到气相中而不破坏它们。此后，还引入了其他提供环境条件的软电离技术，如大气压MALDI、实时直接分析、大气压固体分析探头和混合电离、MALDI和ESI源（如两步融合液滴电喷雾电离、激光解吸大气压化学电离、电声喷雾电离、解吸电喷雾电离以及电喷雾辅助激光解吸/电离）。目前蛋白质组学研究中使用的五种基本类型的质量分析器是四极杆、离子阱、轨道阱、傅里叶变换离子回旋共振和飞行时间（TOF）仪器，它们在确定肽的质荷比的方式上有所不同。它们具有非常不同的设计和性能特点。这些分析器可以单独使用,在某些情况下,也可以串联在一起或与离子淌度质谱联用，以发挥各自的优势。在质谱仪的气相中会产生几种单电荷或多电荷的碎片离子类型，如b、y、a、c、z、v、y和亚铵离子。在鸟枪法蛋白质组学实验中用于蛋白质鉴定的自下而上测序方法中，蛋白质的蛋白酶解消化是通过沿着肽主链和/或侧链的不同键的断裂来完成的，通过电荷导向转移到裂解侧附近。这些各种质谱仪以及产生的离子类型已成为研究和分析蛋白质、肽和氨基酸的重要分析工具。
