Mass spectrometric analysis of protein sequences and posttranslational modifications.

摘要

Mass spectrometry (MS) has increasingly become an important and indispensable tool for proteomic studies, especially with the availability of DNA database and technical or conceptual advances in many fields. However, identification of novel proteins not encoded in DNA database, low abundance proteins with incomplete MS/MS spectrum, co/posttranslational modifications not predicted by DNA sequences, disease-related amino acid mutations and insoluble membrane proteins remain problematic and necessitate the development of new proteomic tools to complement genomic analysis.; This work focuses on the development of new mass spectrometric methods for comprehensive analysis of protein sequences and modifications for both soluble and insoluble proteins. A new de novo peptide sequencing algorithm has been developed to identify unknown proteins isolated from shark cartilage that has no completely sequenced DNA database. A novel concept of two-dimensional mass spectrometry using metabolic stable isotope labeling technique was developed to facilitate de novo peptide sequencing and to enhance the identification of low abundance proteins with incomplete MS/MS spectrum. Alternatively, chemical derivatization in vitro has also been investigated in order to achieve unambiguous sequence assignment.; In particular, a technique of microwave assisted ladder generation from intact proteins was successfully developed to detect modifications and amino acid mutations. The presented fragmentation method is specific to polypeptide bonds and results in two series of ladders from N- and C-terminus of intact proteins. In addition to the experimental observations, theoretical simulation was also studied to investigate the kinetic process of microwave assisted protein hydrolysis. Amino acid mutations and many kinds of modifications such as acetylation, methylation, oxidization, phosphorylation and so on can be easily located from the resultant MALDI-TOF spectrum.; The well-established mass spectrometric methods are not compatible with membrane proteins due to the poor solubility, low abundance and extensive modifications. In this work a detergent-free mass spectrometric method was developed. All posttranslational modifications and all trans-membrane domains were detected in a model protein bacteriorhodopsin.; Applications of the developed methods to human cancer cell lines demonstrate that these techniques are fast and feasible for large-scale analysis of functional proteomics.