**5. Conclusion**

A huge progress has been made in the field of TDMS, allowing the identification and comprehensive analysis of the composition of atoms of proteoforms, especially if they are smaller than 30 kDa. TDMS analysis of larger proteoforms still is more challenging. However, until today the most critical point is the purification of a proteoform towards near homogeneity or at least the significant reduction of complexity of the sample, which is desorbed and ionized into a tandem mass spectrometer for TDMS. A low complexity of the composition of a protein mixture entering the MS still is mandatory for getting high quality spectra. Thus, efficient separation methods are needed for obtaining fractions with low complexity. For developing strategies for separating proteoforms, therapeutic proteins are well suited, however challenging because of their heterogeneity. In depth separation of the proteoforms of a therapeutic protein requires the combination of fractionation techniques based on orthogonal mechanisms. In addition, the combination of gradient chromatography and displacement chromatography will add further opportunities for successful separations.
