In the Top Down Analysis workflow, the BioPharma Finder application identifies and characterizes the molecular composition of an unknown intact protein using LC/MS data acquired on Thermo Scientific instruments.

In a Top Down Analysis experiment, intact precursor proteins are first ionized and analyzed by mass spectrometry and then isolated component peaks are further subjected to fragmentation. Depending on the type of MS data (full-scan or MS/MS) acquired during the experiment, the BioPharma Finder application deconvolves the spectra using either the Xtract or ReSpect deconvolution algorithm or the ProSight Lite application.

Intact Deconvolution: Full-scan MS data processing

For full-scan MS data, the application uses a deconvolution algorithm (Xtract or ReSpect) to transform a charge state series into a molecular mass, similar to the process used in the Intact Mass Analysis workflow. To do this, the application identifies multiple peaks in the mass spectrum associated with different charge states of the same component and displays information about the mass and abundance of that component.

TIP

For more information on the Xtract and ReSpect deconvolution algorithms, refer to the "Deconvolution algorithms" topic in the Intact Mass Analysis User Guide.

Intact Fragmentation: MS/MS data processing

For MS/MS data, the application uses features of the ProSight Lite application to process the deconvolved MS/MS data. To do this, the application searches for fragmentation matches using the ProSight Lite application, a database of known proteoforms for a specific organism, based on its sequenced genome. The application then compares the MS mass measurements and MS/MS fragment ion data of the unknown proteins to those of the identified proteoforms (with known or predicted PTMs) to identify and characterize the unknown protein.

Identifications are not always exact, but may be matched within a fragmentation tolerance. To determine the best match, the application scores the matches by using various fragment-based scoring functions.