Baldwin M A, Medzihradszky K F, Lock C M, Fisher B, Settineri T A, Burlingame A L
Mass Spectrometry Facility, Department of Pharmaceutical Chemistry, University of California, San Francisco, California 94143-0446, USA.
Anal Chem. 2001 Apr 15;73(8):1707-20. doi: 10.1021/ac0011080.
The design and operation of a novel UV-MALDI ionization source on a commercial QqoaTOF mass spectrometer (Applied Biosystem/MDS Sciex QSTAR Pulsar) is described. Samples are loaded on a 96-well target plate, the movement of which is under software control and can be readily automated. Unlike conventional high-energy MALDI-TOF, the ions are produced with low energies (5-10 eV) in a region of relatively low vacuum (8 mTorr). Thus, they are cooled by extensive low-energy collisions before selection in the quadrupole mass analyzer (Q1), potentially giving a quasi-continuous ion beam ideally suited to the oaTOF used for mass analysis of the fragment ions, although ion yields from individual laser shots may vary widely. Ion dissociation is induced by collisions with argon in an rf-only quadrupole cell, giving typical low-energy CID spectra for protonated peptide ions. Ions separated in the oaTOF are registered by a four-anode detector and time-to-digital converter and accumulated in "bins" that are 625 ps wide. Peak shapes depend upon the number of ion counts in adjacent bins. As expected, the accuracy of mass measurement is shown to be dependent upon the number of ions recorded for a particular peak. With internal calibration, mass accuracy better than 10 ppm is attainable for peaks that contain sufficient ions to give well-defined Gaussian profiles. By virtue of its high resolution, capability for accurate mass measurements, and sensitivity in the low-femotomole range, this instrument is ideally suited to protein identification for proteomic applications by generation of peptide tags, manual sequence interpretation, identification of modifications such as phosphorylation, and protein structural elucidation. Unlike the multiply charged ions typical of electrospray ionization, the singly charged MALDI-generated peptide ions show a linear dependence of optimal collision energy upon molecular mass, which is advantageous for automated operation. It is shown that the novel pulsing technique of this instrument that increases the sensitivity for precursor ions scans is applicable to the identification of peptides labeled with isotope-coded affinity tags.
本文描述了一种新型紫外基质辅助激光解吸电离（UV-MALDI）源在商用四极杆-线性离子阱飞行时间质谱仪（Applied Biosystem/MDS Sciex QSTAR Pulsar）上的设计与操作。样品加载在96孔靶板上，靶板的移动由软件控制，且易于实现自动化。与传统的高能MALDI-TOF不同，离子在相对低真空（8 mTorr）区域以低能量（5 - 10 eV）产生。因此，在四极杆质量分析器（Q1）中进行选择之前，它们通过大量的低能量碰撞而冷却，这有可能产生一个准连续离子束，非常适合用于碎片离子质量分析的oaTOF，尽管单次激光照射产生的离子产率可能有很大差异。离子解离是通过在仅含射频的四极杆池中与氩气碰撞诱导产生的，从而给出质子化肽离子典型的低能量碰撞诱导解离（CID）谱。在oaTOF中分离的离子由四阳极探测器和时间数字转换器记录，并累积在宽度为625 ps的“bin”中。峰形取决于相邻bin中的离子计数数量。正如预期的那样，质量测量的准确性显示取决于为特定峰记录的离子数量。通过内部校准，对于包含足够离子以给出明确高斯分布的峰，质量准确度可优于10 ppm。凭借其高分辨率、精确质量测量能力以及在低飞摩尔范围内的灵敏度，该仪器非常适合通过生成肽标签、手动序列解析、鉴定诸如磷酸化等修饰以及蛋白质结构阐明来进行蛋白质组学应用中的蛋白质鉴定。与电喷雾电离典型的多电荷离子不同，MALDI产生的单电荷肽离子显示出最佳碰撞能量对分子量的线性依赖性，这有利于自动化操作。结果表明，该仪器增加前体离子扫描灵敏度的新型脉冲技术适用于鉴定用同位素编码亲和标签标记的肽。
