Identifying and quantifying proteins in complex biological samples is a central challenge in research in the life sciences. Over the past two decades, mass spectrometry (MS)–based proteomics has become the central technology to achieve this on a large-scale. While the MS methods are standard and robust, the typical experimental design can be modified to obtain additional and complementary information from the biological samples. In this presentation, I will present alternative MS-based proteomics methodologies developed in our unit to address diverse biological challenges, including genome annotation, antibody sequencing, redox state determination and microbiome analysis.