Top-down vs Bottom-up Proteomics

Protein research is one of the most challenging topics in analytical chemistry. Initial protein research focused on developing techniques to separate and identify protein sequences. The Edman degradation method was a revolutionary approach for protein amino acid sequence analysis. By combining enzymatic digestion and high-performance liquid chromatography (HPLC) separation of peptide fragments, Edman degradation could determine the complete sequence of a protein. In the 1990s, mass spectrometry replaced Edman degradation for protein identification due to its simplicity and reliability, and mass spectrometry also allowed for protein collection from samples. Over the years, more protein research techniques have been developed, defining the field as 'proteomics.' While proteomics initially referred to large-scale studies of protein sets, it now encompasses studies ranging from small to large scales.

Bottom-up and top-down are two proteomics approaches based on mass spectrometry analysis of biological materials. The bottom-up approach, also known as the shotgun method, is a widely used mass spectrometry technique in proteomics research. It involves hydrolyzing/digesting large protein fragments into peptides for analysis. In contrast, the top-down approach directly analyzes complete proteins, including post-translational modifications and other large protein fragments, rather than just peptides.

Bottom-up Proteomics

The basic process of the bottom-up approach is to digest a protein mixture into a peptide mixture; chromatographically separate and ionize the peptides; and then perform tandem mass spectrometry to generate peptide fingerprints for identification. Finally, potential proteins are inferred from the identified peptides. This method can yield a large number of identification results in a short time.

In the computational steps, existing spectrum analysis methods include sequence database searching, library searching (most commonly used), de novo sequencing, and de novo sequencing combined with error-tolerant searching. Some classic database search software includes MASCOT, SEQUEST, and X! Tandem.

Taking the shotgun technical route as an example, the basic process of database searching is as follows:

1. Cut candidate protein sequences from the database into peptides and simulate spectra of the cleaved peptide fragments.
2. Match and score experimental spectra based on their similarity to simulated spectra. Obtain highly reliable peptide identification results through specific peptide quality control methods.
3. Infer proteins based on the correspondence between peptide and protein amino acid sequences.

The bottom-up strategy is currently the most mature and widely applied strategy for protein identification, characterization of post-translational modifications, as well as relative and absolute quantification. However, limitations of the bottom-up analysis strategy also restrict its potential applications.

Top-down Proteomics

Top-down Proteomics

Despite the development of various protein separation techniques, most lack the capability to separate proteins from complex mixtures. The top-down proteomics strategy is used to characterize multiple intact proteins in mixtures. In top-down proteomics, intact proteins are first separated from complex biological samples using reverse-phase liquid chromatography, then directly ionized using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI). The generated ions are fragmented through collision-induced dissociation (CID), high-energy collision-induced dissociation (HCD), electron capture dissociation (ECD), or electron transfer dissociation (ETD) and analyzed by tandem mass spectrometry. This method has significant potential for protein identification, analysis, sequence analysis, and characterization of post-translational modifications.

Top-down vs. Bottom-up Proteomics

Workflow of Top-down and Bottom-up Proteomics (Ashley et al., 2016)

Both top-down and bottom-up proteomics strategies have their advantages and limitations. Considering the complementary information provided by both strategies, a 'middle-down' proteomics strategy has gradually emerged. In this strategy, proteins undergo limited proteolysis by enzymes such as LysC, producing products of 5-20 kDa in size. These peptides are then sequenced using the top-down approach, which has the advantages of high sequence coverage and retention of post-translational modification information. We can choose a suitable protein research strategy based on equipment and specific analytical requirements.

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