In top-down proteomics, proteins are introduced into a mass analyzer, where they are subjected to gas-phase fragmentation.

The following figure compares bottom-up and top-down methods:

Figure Comparing top-down and bottom-up proteomics
Comparing top-down and bottom-up proteomics

Top-down proteomics is a technique for protein identification and characterization. Combining top-down proteomics searches with the shotgun annotation process provides a method for rapid and accurate protein definition.

Top-down proteomics can provide thorough sequence coverage of proteoforms, supporting characterization of complex combinations of PTMs.

Separation and ionization of intact proteins present many challenges beyond the scope of this guide. The bioinformatics of top-down proteomics, though, has some challenges that the application addresses.

  • ProSightPD automates the determination of neutral masses from both precursor and fragmentation spectra. The resultant masses are then used for protein identification and characterization.
  • Precursor masses may not originate from full-length, unmodified proteins. Therefore, multiple search strategies may be necessary to identify and characterize all proteoforms in a sample. ProSightPD provides several search modes to identify different proteoforms. Search modes such as the Subsequence search can be used to find truncated forms while Annotated Proteoform searches can be used to identify highly modified proteoforms. More in-depth descriptions are provided in the ProSightPD database search algorithms section.