Nanoliter chemistry combined with mass spectrometry for peptide mapping of proteins from single mammalian cell lysates.
Nanoliter chemistry combined with mass spectrometry for peptide mapping of proteins from single mammalian cell lysates.
复制标题
纳升化学与质谱分析相结合，对单个哺乳动物细胞裂解物中的蛋白质进行肽图谱分析。
作者：
R. Whittal;B. Keller;L. Li
纳米化学站结合基质辅助激光解吸/电离飞行时间（MALDI-TOF）质谱法在原子水平上对蛋白质进行了表征。化学反应包括在纳升或亚纳升的体积中进行蛋白质消化，然后将消化的微点样品沉积到MALDI-TOF质谱仪上。从酶消化中精确测定肽的质量，结合蛋白质数据库搜索，可以在蛋白质数据库中鉴定蛋白质。这种方法特别适用于处理小体积样品，如单细胞分析。这种方法的高灵敏度和特异性被证明是通过多肽定位和识别单个红细胞镰状细胞病的血红蛋白变异。将纳米升化学与高灵敏度质谱分析相结合的方法应该在只有有限数量的材料可用于询问的生物系统中表征蛋白质方面得到普遍应用。
A nanoliter-chemistry station combined with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry was developed to characterize proteins at the attomole level. Chemical reactions including protein digestion were carried out in nanoliter or subnanoliter volumes, followed by microspot sample deposition of the digest to a MALDI-TOF mass spectrometer. Accurate mass determination of the peptides from the enzyme digest, in conjunction with protein database searching, allowed the identification of the proteins in the protein database. This method is particularly useful for handling small-volume samples such as in single-cell analysis. The high sensitivity and specificity of this method were demonstrated by peptide mapping and identifying hemoglobin variants of sickle cell disease from a single red blood cell. The approach of combining nanoliter chemistry with highly sensitive mass spectrometric analysis should find general use in characterizing proteins from biological systems where only a limited amount of material is available for interrogation.