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Intact Protein Characterization

Full characterization of a protein by mass spectrometry includes determination of the protein sequence, and identification and relative quantitation of protein isoforms, including identification and localization of one or multiple post-translational modifications (PTMs). Traditional analyses use a “bottom-up” approach, where proteins are digested into their peptide counterparts. However, complete sequence coverage is rarely attainable, and qualitative and quantitative information about protein isoforms, including those resulting from post-translational modifications, is usually lost.
 
Top-down protein characterization by mass spectrometry is an emerging technology that has several advantages over bottom up sequencing. Top-down analysis initially involves accurate measurement of molecular weight of intact protein followed by the fragmentation of the molecular ion in the gas phase. Top-down analysis facilitates direct observation of C- and N-termini for identification of truncations, preserves the relationship between modifications in any given isoform and allows quantitative differentiation between isoforms. High mass accuracy and high mass resolution are absolute requirements for this approach due to the complexity of MS/MS data.

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Workflow Overview for Intact Protein Characterization


Full characterization of intact proteins by top-down mass spectrometry is most commonly performed on proteins that have been enriched or purified. The enriched protein mixture is introduced into the mass spectrometer using either direct infusion or liquid chromatography coupled to an ESI source. Direct infusion provides more time for signal averaging and facilitates use of multiple fragmentation techniques. For more complex intact-protein mixtures, however, on- or off-line LC separation may be required to reduce precursor spectral complexity and minimize ion suppression. Users can choose a variety of fragmentation techniques for primary sequence determination. These include collisional activations and electron-capture based dissociations. Subsequent data analysis identifies and characterizes the protein sequence based both on accurate precursor mass and fragment masses from various dissociation experiments.