Person Maria D, Lo Herng-Hsiang, Towndrow Kelly M, Jia Zhe, Monks Terrence J, Lau Serrine S
Center for Molecular and Cellular Toxicology, Division of Pharmacology and Toxicology, College of Pharmacy, The University of Texas at Austin, Austin, Texas 78712, USA.
Chem Res Toxicol. 2003 Jun;16(6):757-67. doi: 10.1021/tx020049d.
Protein identification by MS is well-established. Mixtures of proteins from cell extracts are separated by either one- or two-dimensional gel electrophoresis, and specific bands or spots are subjected to in-gel digestion and subsequent analysis by MS. The two most common types of ionization used in MS are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). When ESI is used, the sample is typically analyzed by inline HPLC-ESI-MS/MS with fragmentation of individual digest peptides, followed by database comparison between theoretical and experimental fragmentation patterns. MALDI-MS analysis is based on peptide mass mapping, with mass measurements of the digest peptides searched against a database of theoretical digests. We give here the results of a comparison between ESI-ion trap and MALDI-TOF (time-of-flight) analysis of 11-deoxy,16,16-dimethyl prostaglandin E(2) (DDM-PGE(2)) inducible proteins. Individual peptides identified by the two techniques differed, in general, but the resulting protein identification was the same. Slightly higher coverage of each protein was obtained by MALDI-TOF, but the MS/MS data were more definitive by requiring fewer peptides to assign a positive identification. Both methods effectively identified two proteins in the same gel band. The samples here are derived from a renal epithelial cell line (LLC-PK(1)) established from the New Hampshire minipig, a species poorly represented in the current database, and strategies and limitations for analyzing such species are discussed.
通过质谱进行蛋白质鉴定已经非常成熟。细胞提取物中的蛋白质混合物通过一维或二维凝胶电泳进行分离，特定的条带或斑点进行胶内消化，随后通过质谱进行分析。质谱中最常用的两种电离类型是电喷雾电离（ESI）和基质辅助激光解吸/电离（MALDI）。使用ESI时，样品通常通过在线HPLC-ESI-MS/MS进行分析，对单个消化肽进行碎片化处理，然后将理论和实验碎片化模式进行数据库比较。MALDI-MS分析基于肽质量图谱，对消化肽的质量测量结果与理论消化数据库进行比对。我们在此给出了对11-脱氧、16,16-二甲基前列腺素E(2)（DDM-PGE(2)）诱导蛋白进行ESI离子阱和MALDI-TOF（飞行时间）分析的比较结果。一般来说，两种技术鉴定出的单个肽段有所不同，但最终的蛋白质鉴定结果是相同的。MALDI-TOF对每种蛋白质的覆盖度略高一些，但通过ESI-MS/MS数据确定度更高，因为确定阳性鉴定所需的肽段数量更少。两种方法都有效地鉴定出了同一条凝胶带中的两种蛋白质。这里的样品来自于从新罕布什尔小型猪建立的肾上皮细胞系（LLC-PK(1)）——这是一种在当前数据库中代表性较差的物种，并讨论了分析此类物种的策略和局限性。
