Proteases are in the center of many diseases, and consequently, proteases and their substrates are important drug targets as represented by an estimated 5-10% of all drugs under development. Mass spectrometry has been an indispensable tool for the discovery of novel protease substrates, particularly through the proteome-scale enrichment of so-called N-terminal peptides representing endogenous protein N termini. Methods such as combined fractional diagonal chromatography (COFRADIC) 1 and, later,

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... terminal amine isotopic labeling of substrates (TAILS) have revealed numerous insights into protease substrates and consensus motifs. We present an alternative and simple protocol for N-terminal peptide enrichment, based on charge-based fractional diagonal chromatography (ChaFRADIC) and requiring only wellestablished protein chemistry and a pipette tip. Using iTRAQ-8-plex, we quantified on average 2,073 ؎ 52 unique N-terminal peptides from only 4.3 g per sample/channel, allowing the identification of proteolytic targets and consensus motifs. This high sensitivity may even allow work-ing with clinical samples such as needle biopsies in the future. We applied our method to study the dynamics of staurosporine-induced apoptosis. Our data demonstrate an orchestrated regulation of specific pathways after 1.5 h, 3 h, and 6 h of treatment, with many important players of homeostasis targeted already after 1.5 h. We additionally observed an early multilevel modulation of the splicing machinery both by proteolysis and phosphorylation. This may reflect the known role of alternative splicing variants for a variety of apoptotic genes, which seems to be a driving force of staurosporineinduced apoptosis. Molecular & Cellular Proteomics Proteolysis plays a crucial role in maintaining cellular homeostasis by modulating protein function and activity and its dysregulation underlies many diseases such as cancer and Alzheimer (1-3). Through proteolytic cleavage, novel protein N termini are generated. The identification of these so-called neo N termini is an important step toward understanding which proteins are substrates of a specific protease and revealing regulatory proteolytic networks in health and disease. Moreover, it also allows identifying protease cleavage motifs, which is important for developing protease inhibitors or chemical proteomics tools. As protein N termini and, more importantly, neo N termini are significantly underrepresented in the proteome, specific methods have been developed for the enrichment of N-terminal peptides followed by mass spectrometry (N-terminomics) to enable the system-wide identification of protease substrates and cleavage patterns (4). Several methods, namely combined fractional diagonal chromatography (COFRADIC) (5), subtiligase N-terminal labeling and enrichment (6) and, later, terminal amine isotopic labeling of substrates (TAILS) (7) pioneered the field of N-terminomics. COFRADIC and TAILS utilize the specific labeling of protein N termini (and Lys residues) as an initial step of the enrichment procedure. Upon proteolytic cleavage as part of the common bottom-up proteomics strategy, this labeling allows quantifying N-terminal peptides but also distinguishing them from internal peptides with free N termini generated during in vitro digestion. Both methods have been used in numerous studies providing novel From the ‡Leibniz-