The process for analysis of intact proteins is straightforward and requires minimal sample preparation. The sample may be deglycosylated and/or reduced prior to analysis, or may be analysed as-is, depending on the intent of the analysis. Samples are analysed through liquid chromatography (LC) coupled to high resolution mass spectrometry (MS). The LC-MS analysis is enabled by electrospray ionization (ESI). Intact mass may also be referred to as ‘top down’ analysis.

Deglycosylation prior to Intact Mass Analysis

The MS analysis of an intact antibody is often complicated by glycosylation. Since several different glycoforms (and therefore different masses) may be present in the sample, the antibody will not show as one distinct peak on the deconvoluted mass spectrum. Unless the intention is to study glycosylation profile, it is common practice to deglycosylate using PNGase prior to intact mass analysis of antibodies. Note that other post translational modifications (PTMs) will remain, and can complicate the analysis.

Studying subunits using Intact Mass Analysis

Antibody researchers may be interested in studying the regions of their molecule separately – e.g. the Fab or Fc, or the Heavy Chain or Light Chain. An enzyme (IdeS) can be used to separate the Fab and Fc, DDT can be used to separate the chains, or both can be done in succession. The resulting subunits are then separated by liquid chromatography before analysis by mass spectrometry. While the molecule is no longer whole, this type of analysis is still often referred to as intact mass analysis (or middle-down analysis) to distinguish it from peptide-level (or bottom up) analysis. See figure 1.