Intact protein analysis is performed for a number of reasons, including determination of molecular mass (during top-down analysis) and posttranslational modification (PTM) (e.g., monoclonal antibody glycosylation). Full characterization of intact proteins is often performed on proteins that have been enriched or purified. These proteins can be introduced into the mass spectrometer (MS) by either direct infusion or through liquid chromatography (LC) coupled with electrospray ionization (ESI).
The primary advantage of direct infusion is that it allots more time for signal averaging. However, most intact protein mixtures require separation prior to introduction into the MS to reduce precursor spectral complexity and minimize ion suppression. This separation can be performed by either on- or off-line LC. High-resolution accurate mass (HRAM) analysis is also crucial for intact protein analysis.
Sample complexity must be minimized if intact protein analysis by MS is to be successful. Proteins must be well-resolved from one another and from various proteoforms to minimize the low thresholds of intact protein detection. Thermo Scientific ProSwift RP-4H LC Columns are ideal for use in intact protein separation prior to MS analysis; they are also designed to work with milliliter to microliter flow.
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Screen, identify, and characterize your proteins with higher productivity and confidence. Confirm amino acid sequences, identify post-translational modifications (PTMs), or localize modifications using the automated top-down workflow. From intact protein through to peptide mapping and multi-attribute monitoring, the BioPharma Finder software helps you understand your therapeutics like never before.
