Native and Intact Protein
Characterization
Biotherapeutic compounds, such as proteins and monoclonal antibodies (mAbs), as well as conjugates,
complexes and assemblies based on them, make up the vast majority of the rapidly growing biologics
drug market. Full characterization of these compounds by mass spectrometry includes determination of
protein sequences, and identification and relative quantitation of protein isoforms, including identification
and localization of one or multiple post-translational modifications (PTMs). Traditional workflows for such
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.
These same research endeavors would, in many cases, also benefit from intact and native mass
spectrometric analyses.
Intact and native protein characterization by mass spectrometry (MS) has emerged as a valuable
technology that has numerous advantages over bottom up sequencing. Intact and native MS with
high-resolution, accurate mass (HRAM) technologies provide accurate information on various protein
properties, such as intact molecular mass, glycosylation forms, amino acid sequence, post-translational
modifications, and minor impurities due to sample processing and storage, as well as higher-order
structural information, such as protein conformational changes upon modifications, noncovalent
interactions between protein drugs and receptor proteins, and protein aggregation caused by misfolding.
Mass analysis at the intact protein level is usually the first step of the structural characterization.
High-resolution mass spectrometry is essential for resolving co-eluting intact proteins as well as isotopic
peaks of highly charged proteins for charge state determination and accurate mass determination.
Additionally, due to the number of product ions generated during fragmentation of intact proteins, high-
resolution mass analysis is required for accurate detection and assignment of product ions in the resultant
complex MS/MS spectra.
The high-resolution accurate mass of the Thermo Scientific
™
Orbitrap
™
family of mass spectrometers,
including the extended m/z range capability of selected models, enables novel native and intact
workflows. Orbitrap-based mass spectrometers have the potential to become essential tools for routine
intact and native biopharmaceutical analysis as demonstrated by a variety of examples below.
