In-Depth Protein Characterization by Mass Spectrometry

Within this chapter, various techniques and instructions for characterizing primary structure of proteins are presented, whereas the focus lies on obtaining as much complete sequence information of single proteins as possible. Especially, in the area of protein production, mass spectrometry-based detailed protein characterization plays an increasing important role for quality control. In comparison to typical proteomics applications, wherein it is mostly sufficient to identify proteins by few peptides, several complementary techniques have to be applied to maximize primary structure information and analysis steps have to be specifically adopted. Starting from sample preparation down to mass spectrometry analysis and finally to data analysis, some of the techniques typically applied are outlined here in a summarizing and introductory manner. The field of Proteomics has been very successful in identifying the quantification of large sets of proteins (protein mixtures), for example, from whole organelles or cell lysates. Nowadays, hundreds of proteins within a complex sample can be easily identified by mass spectrometry, whereas only few peptides per protein are usually detected (1). This allows elucidating the name of the protein via searching protein sequence databases. In addition to analyzing complex protein mixtures, at least equally challenging is the art of in-depth characterization of individual proteins, or in other words, gaining as much primary structure information (including posttranslational modifications) as possible from a protein of interest. In-depth protein characterization is of great importance, as it increases the chance to detect posttranslational modification (PTM), which modulates the activity of most eukaryote proteins. Also validating and distinguishing protein isoforms within a sample