Motivation for DevelopmentThe development of MassMap®was inspired by studies of pharmaceutical proteins. It was the purpose of the studies to characterize the proteins in detail and/or to compare proteins produced through different processes. If the samples compared are produced through slightly differing variants of a well-established process, such studies are called comparability studies. In case an active pharmaceutical ingredient resulting from a generic process is compared against material from the original process, such studies are performed to demonstrate biosimilarity.Due to their complexity and size, proteins are usually digested by means of specific proteases before further analysis. The resulting mixture of peptides is separated chromatographically in order to produce fractions with less complexity. If the peptides are detected online with an electrospray mass spectrometer, the procedure is termed LC/MS peptide mapping.In principle, the data sets resulting from LC/MS peptide mapping contain all the information on the covalent structure of the protein. Thus, the procedure may be employed for a number of essential purposes:• Testing/verification of the primary structure• Identification and quantitative assessment of post-translational modifications• Detection of unknown or unexpected modifications as well as of unknown or unexpected other components• Assessment of higher order structure differences by means of H/D exchange (HDX) experiments.However, due to some of the properties of the data sets, the practical investigation of LC/MS data sets obtained by peptide mapping becomes extremely complicated:• In addition to the molecular ions of the peptides, fragment and adduct ions are created in the source of the mass spectrometer.• The gases employed for ionization and the solvents of the chromatographic system lead to additional signals whose intensities are often orders of magnitudes larger than the intensities of low-abundance signals.• Mass spectrometers exhibiting very high resolution lead to data sets as large as several gigabytes.• The number of molecules to be investigated for a complete or close to complete sequence coverage amounts to between 50 for smaller molecules or up to 150 for IgG antibodies. For the exhausting analysis of data sets, this number can be as large as several hundred and can even exceed 1000.MassMap®was developed to overcome these difficulties and to allow for a fast and comprehensive analysis of even very complex data sets, which was not possible with the programs supplied by the instrument manufacturers.