Application of middle-down approach in quantitation and catabolite identification of protein by LC-high-resolution mass spectrometry.

To further enhance the detection sensitivity and increase resolving power of top-down intact protein bioanalysis, middle-down approach was explored. An monoclonal antibody (mAb) was used as a model protein to evaluate quantitative bioanalytical assay performance and a disulfide linked dimer protein was investigated for its pharmacokinetics properties and catabolism by middle-down approach. For quantitation of the mAb, different subunits generated by middle-down approach provided different level of signal improvement in biological samples with Lc and half Fc giving five-times better sensitivity than intact mAb. For the dimer protein, middle-down analysis by reduction enabled effective differentiation of the unchanged protein and its oxidized form, and clearly elucidated their respective proteolytic catabolites.