The Pro in Proteomics

In the work described in this thesis, the central focus has been on the influence of proline amino acids on protein structure, protein regulation and its place in proteomics experiments. In Chapter 2, we will evaluate how different PTMs can influence the placement or removal of other PTMs. We discuss the different mechanisms possible for both positive and negative interplay and look at some well-known examples when available. Most of this chapter is aimed at the interplay between O-GlcNac and phosphorylation, an interesting situation since both modifications occur on serine and threonine residues. Due to its enrichment near serine and threonine phosphorylation sites, proline is also involved and its role is briefly discussed. In Chapter 3, we focus on the potential benefit of using proline residues as a cleavage site in proteomics. Since trypsin does not cleave when arginine or lysine are followed by a proline, the peptides resulting from cleavage at proline are expected to be highly complementary to the much more commonly analyzed tryptic peptides. In this chapter we touch upon some of the limitations of trypsin, the benefits of using alternative proteases and we compare the peptides and proteins found with either trypsin or EndoPro, the proline specific protease we explored. We focus more on the characteristics of proline in Chapter 4. Since it can stably exist in both cis and trans isomers, proline adds an interesting, dynamic dimension to protein structure and the resulting protein biology. In this chapter we employ a bioinformatics approach to evaluate the abundance of cis/trans isomers of proline in protein structures and we investigate the proline-specific protease EndoPro for its isomer specificity. 24 In Chapter 5, we studied the full-length protein Pin1, the enzyme catalyzing the cis/trans isomerization of proline residues when they are directly adjacent to a phosphorylated serine or threonine residues. Following its expression and purification, we employed affinity based pull-down methods aimed to distinguish between different interactors of Pin1. We were able to purify and detect many known and novel Pin1 interactors, but still need further validation of the interactions and their functional relevance. Finally, in Chapter 6, I close with a personal view on typical choices in proteomics workflows and how these may be suboptimal when interested in certain aspects of the proteome. My main point here is that we should always be aware of the choices we make and how these might affect the outcome of our experiments.