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Mass Spectrometry at the Protein Level

Proteins are typically measured in positive ion mode, with the charge produced on the protein originating from protonation of basic residues; arginine, lysine, histidine, and the N-terminus.

With protein level analysis, it is the intact protein, or large fragments thereof, being analyzed.  In the case of monoclonal antibodies, the light and heavy chains are large fragments, generated by disulfide bond reduction, the Fab and Fc fragments are generated by papain cleavage, and the F(ab’)2 fragment by pepsin or immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS) cleavage.  These slightly smaller fragments are more amenable to chromatographic and mass spectrometric analysis, making the identification of particular modifications at specific domains easier.  Several techniques are used for protein level analysis and each provides different information (Table 1). 

Level Technique Separation principle Detection principle Characteristics
Protein RPLC Hydrophobicity UV, MS Purity, impurities,
post-translational modifications
IEX Charge UV, MS
HIC Hydrophobicity UV, MS
SEC Size UV, MS Purity, impurities, aggregation, fragmentation
MS (MS/MS) m/z - MW, structural integrity, modifications, partial sequence, higher-order structures

Table 1: Techniques used and characteristics revealed at the protein level.

MS analysis of the intact mAb trastuzumab with heavy (Hc) and light chains (Lc) generated following interchain disulfide bond reduction using dithiothreitol (DTT), and the crystallizable fragment (Fc) generated from papain cleavage (Figure 1), provides initial confirmation of the gene-derived protein sequence, reveals the structural integrity, and any post-translational modifications.  At each level the measured molecular weight can be accurately calculated (with typical mass error of less than 0.005% of the theoretical value).  The presence of several glycoforms is revealed in the spectra of the intact, Hc, and Lc fragments - these spectra all exhibit characteristic 146 and 162 Da spacings indicative of fucose and galactose and consequently N-glycosylation.  The Hc spectrum reveals the presence of four main glycoforms; G0, G0F, G1F, and G2F.  The peak intensities are indicative of the relative amounts of these glycoforms.  mAbs contain two heavy chains linked by disulfide bridges, hence, the measurement of the intact species and Fc fragment allows simultaneous interrogation of the two N-glycosylation sites which provides further structural information.  

 
 

Figure 1: Deconvoluted spectra of trastuzumab (Hereceptin) acquired using Q-ToF mass spectrometry. 
Samples introduced following on-line RP desalting.

 

 

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