Large molecule bioanalysis using Q-TOF without predigestion and its data processing challenges

Background: Recent developments in LC-MS have turned it into a viable and valid alternative to ligand-binding assays. Large molecule bioanalysis by LC-MS is generally performed by tryptic digestion, purification and detection of one or more small signature peptides. High-resolution MS instruments offer quantification of intact small proteins or peptides and are able to increase the selectivity, while maintaining sensitivity. Results: Unlike multiple reaction monitoring assays, several factors affecting data processing were presented and the optimal parameters to consider during quantification method building were also demonstrated. MUC5AC-I3 (MW 1709.8 Da), human hepcidin/LEAP-I (MW 2797.4 Da), porcine calcitonin (MW 3604 Da) and chicken lysozyme (MW 14.3 kDa) were selected as model compounds and the possibility of intact peptide and small protein quantification, without tryptic digestion, was demonstrated. Conclusion: Selectivity and sensitivity were improved using different scan modes, such as TOF-MS and TOF-MS/MS.