The global analysis of proteins (i.e. proteomics) provides, arguably, the principal and ultimate level of information required to understand how cells function. Proteomic analyses, however, are challenging due to the highly dynamic range and diversity of protein modifications. A further challenge is the interconnectivity of proteins into complexes and signalling networks that are highly divergent in time and space. Nowadays, large-scale proteome analysis has become within reach and heavily relies on mass spectrometry (MS)-based protein sequencing. MS-based proteomics is starting to mature and provide answers to important biological questions thanks to a combination of developments regarding instrumentation, sample preparation, and computational analysis.