Müller Markus, Gras Robin, Binz Pierre-Alain, Hochstrasser Denis F, Appel Ron D
Swiss Institute of Bioinformatics, Geneva, Switzerland.
Proteomics. 2002 Oct;2(10):1413-25. doi: 10.1002/1615-9861(200210)2:10<1413::AID-PROT1413>3.0.CO;2-P.
The development of high throughput utilities to identify proteins is a major challenge in present research in the field of proteomics. One such utility, the molecular scanner, uses proteins separated by two-dimensional polyacrylamide gel electrophoresis that are digested in the gel and during transfer onto a collecting membrane. After adding a matrix, the membrane is inserted into a matrix-assisted laser desorption/ionization-time of flight mass spectrometer and a peptide mass fingerprint (PMF) is measured for every scanned site. Since the spacing between scanned sites is much smaller than the size of the most abundant protein spots, there is a certain redundancy in the data that was used in an earlier experiment with Escherichia coli [1] to improve mass calibration and PMF identification results. It was observed that the signal intensity of a peptide mass as a function of the position on the membrane showed similar patterns if peptides stemmed from the same protein. Taking account of these similarities a clustering algorithm was used to find lists of experimental masses with similar intensity distributions, which provided clearer identification of the corresponding proteins. Here, these methods are applied to a human plasma scan, where proteins were highly modified and less separated. The presence of very abundant proteins like albumin and immunoglobulins added another difficulty. The calibration of the initial PMFs was not satisfactory and masses had to be recalibrated. After discarding chemical noise, the membrane was partitioned into regions and for each region protein identification was carried out separately. A new scoring method was used, where the PMF score was multiplied by a factor that measures the similarity of matching peptides. This method proved to be more robust than the method developed in [1] if the region where a protein was found had an extended, nonspherical shape and strong overlap with regions of other proteins. Many proteins annotated on the SWISS-2D PAGE human plasma master gel could be clearly identified and many interesting properties were observed.
开发用于鉴定蛋白质的高通量工具是目前蛋白质组学领域研究的一项重大挑战。其中一种工具，即分子扫描仪，它利用二维聚丙烯酰胺凝胶电泳分离出的蛋白质，这些蛋白质在凝胶中以及转移到收集膜的过程中会被消化。添加基质后，将膜插入基质辅助激光解吸/电离飞行时间质谱仪，对每个扫描位点测量肽质量指纹（PMF）。由于扫描位点之间的间距远小于最丰富蛋白质斑点的大小，在早期对大肠杆菌的实验中所使用的数据存在一定冗余，该实验用于改进质量校准和PMF鉴定结果。据观察，如果肽来自同一蛋白质，肽质量的信号强度作为膜上位置的函数会呈现相似的模式。考虑到这些相似性，使用了一种聚类算法来查找具有相似强度分布的实验质量列表，这使得对相应蛋白质的鉴定更加清晰。在此，这些方法被应用于人体血浆扫描，其中蛋白质高度修饰且分离度较低。像白蛋白和免疫球蛋白等非常丰富的蛋白质的存在又增加了另一难度。初始PMF的校准并不令人满意，必须重新校准质量。在去除化学噪声后，将膜划分为多个区域，并对每个区域分别进行蛋白质鉴定。使用了一种新的评分方法，其中PMF分数乘以一个衡量匹配肽相似性的因子。如果发现蛋白质的区域具有扩展的、非球形形状且与其他蛋白质的区域有强烈重叠，那么该方法被证明比文献[1]中开发的方法更稳健。在SWISS - 2D PAGE人体血浆主凝胶上注释的许多蛋白质能够被清晰鉴定，并且观察到了许多有趣的特性。
