Nowadays, mass spectrometry-based proteomics is carried out primarily in a 
bottom-up fashion, with peptides obtained after proteolytic digest of a whole 
proteome lysate as the primary analytes instead of the proteins themselves. This 
experimental setup crucially relies on a protease to digest an abundant and 
complex protein mixture into a far more complex peptide mixture. Full knowledge 
of the working mechanism and specificity of the used proteases is therefore 
crucial, both for the digestion step itself as well as for the downstream 
identification and quantification of the (fragmentation) mass spectra acquired 
for the peptides in the mixture. Targeted protein analysis through selected 
reaction monitoring, a relative newcomer in the specific field of mass 
spectrometry-based proteomics, even requires a priori understanding of protease 
behavior for the proteins of interest. Because of the rapidly increasing 
popularity of proteomics as an analytical tool in the life sciences, there is 
now a renewed demand for detailed knowledge on trypsin, the workhorse protease 
in proteomics. This review addresses this need and provides an overview on the 
structure and working mechanism of trypsin, followed by a critical analysis of 
its cleavage behavior, typically simply accepted to occur exclusively yet 
consistently after Arg and Lys, unless they are followed by a Pro. In this 
context, shortcomings in our ability to understand and predict the behavior of 
trypsin will be highlighted, along with the downstream implications. 
Furthermore, an analysis is carried out on the inherent shortcomings of trypsin 
with regard to whole proteome analysis, and alternative approaches will be 
presented that can alleviate these issues. Finally, some reflections on the 
future of trypsin as the workhorse protease in mass spectrometry-based 
proteomics will be provided.