Biological mass spectrometry-based proteomics analysis methods mainly include Bottom-up and Top-down methods.
Ÿ Bottom-up is a traditional method that (enzymatically) digests a mixture of large fragments of proteins into small fragments of peptides and then analyzes them. It is widely used in proteomics research.
Ÿ Top-down technology can directly analyze intact proteins, including post-translationally modified proteins and other large protein fragments rather than just peptides.
Although a variety of protein separation technologies have been developed, most of them are not capable of separating proteins from complex mixtures except the top-down proteomics strategy, which can characterize multiple intact proteins in a mixture.
Procedures of top-down mass spectrometry method
In top-down proteomics, intact proteins are first separated from complex biological samples by reversed-phase liquid chromatography, and then directly ionized by electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) techniques. The generated ions are fragmented by collision-induced dissociation (CID), high energy collision-induced dissociation (HCD), electron capture dissociation (ECD), or electron transfer dissociation (ETD), and are analyzed in tandem mass spectrometry.
Advantages of top-down technology
The top-down proteomics method has great potential in protein identification, analysis, sequence analysis, and characterization of post-translational modifications.
The traditional bottom-up method is to first digest a mixture of large protein fragments into small fragments of peptides before analyzing them. Obtaining information on the relationship between different PTMs is impossible to be achieved in this way. Because many PTMs cannot be kept in a stable state when analyzed and the integrity of the detected peptides cannot be guaranteed, a few PTMs often cannot be detected. The top-down technology can detect the complete protein, not the peptide, which allows PTM-related information to be preserved to the greatest extent.
Since top-down mass spectrometry can analyze intact proteins/peptides without proteolysis, it has a strong ability to comprehensively analyze protein modifications. When analyzing protein therapeutics such as monoclonal antibodies and recombinant proteins, this strategy can retain most of the unstable modification motifs lost in the shotgun method and CID lysis method.
Flaws of the top-down approach
Compared with bottom-up technology, top-down technology has less sensitivity and throughput. In addition, top-down technology requires a large number of samples, for which the sample preparation can be technically challenging with a variety of proteins in organisms, with complex properties, large quantities, and wide dynamic range. To add to the challenge, protein samples must be pre-separated before entering the mass spectrometer. Conventional pre-separation techniques include two-dimensional gel electrophoresis, multi-dimensional liquid chromatography, and capillary electrophoresis. There are other separation methods for special objects, such as the removal of high-abundance proteins and the enrichment and separation of low-abundance proteins.
The analysis of fragment ion spectra generated by the top-down method is also more complicated.
Top-down mass spectrometry based on ECD and ETD is suitable for:
Ÿ Complete protein/peptide quality determination
Ÿ Quantification of multiple modified protein forms
Ÿ High coverage modification mapping
Ÿ Unpredicted PTMs identification
Ÿ The order of various modifications, etc.