Müller Markus, Gras Robin, Appel Ron D, Bienvenut Willy V, Hochstrasser Denis F
Swiss Institute of Bioinformatics, Geneva.
J Am Soc Mass Spectrom. 2002 Mar;13(3):221-31. doi: 10.1016/S1044-0305(01)00358-0.
The molecular scanner combines protein separation using gel electrophoresis with peptide mass fingerprinting (PMF) techniques to identify proteins in a highly automated manner. Proteins separated in a 2-dimensional polyacrylamide gel (2-D PAGE) are digested in parallel and transferred onto a membrane keeping their relative positions. The membrane is then sprayed with a matrix and inserted into a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometer, which measures a peptide mass fingerprint at each site on the scanned grid. First, visualization of PMF data allows surveying all fingerprints at once and provides very useful information on the presence of chemical noise. Chemical noise is shown to be a potential source for erroneous identifications and is therefore purged from the mass fingerprints. Then, the correlation between neighboring spectra is used to recalibrate the peptide masses. Finally, a method that clusters peptide masses according to the similarity of the spatial distributions of their signal intensities is presented. This method allows discarding many of the false positives that usually go along with PMF identifications and allows identifying many weakly expressed proteins present in the gel.
分子扫描仪将使用凝胶电泳的蛋白质分离技术与肽质量指纹图谱（PMF）技术相结合，以高度自动化的方式鉴定蛋白质。在二维聚丙烯酰胺凝胶（2-D PAGE）中分离的蛋白质被并行消化，并转移到保持其相对位置的膜上。然后将膜喷上基质，并插入基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱仪中，该质谱仪测量扫描网格上每个位点的肽质量指纹图谱。首先，PMF数据的可视化允许一次性查看所有指纹图谱，并提供有关化学噪声存在的非常有用的信息。化学噪声被证明是错误鉴定的潜在来源，因此从质量指纹图谱中清除。然后，利用相邻光谱之间的相关性对肽质量进行重新校准。最后，提出了一种根据肽信号强度空间分布的相似性对肽质量进行聚类的方法。该方法可以丢弃许多通常与PMF鉴定相关的假阳性结果，并能够鉴定凝胶中存在的许多低表达蛋白质。
