The potential of fractional diagonal chromatography strategies for the enrichment of post-translational modifications

More than 450 post-translational modifications (PTMs) are known, however, currently only some of those can be enriched and analyzed from complex samples such as cell lysates. Therefore, we need additional methods and concepts to improve our understanding about the dynamic crosstalk of PTMs and the highly context-dependent regulation of protein function by so-called ‘PTM codes’. The mere focus on affinity-based enrichment techniques may not be sufficient to achieve this ambitious goal. However, the complementary use of two-dimensional chromatography-based strategies such as COFRADIC and ChaFRADIC might open new avenues for enriching a variety of so far inaccessible PTMs for large-scale proteome studies

Identification of cleavage sites and substrate proteins for two mitochondrial intermediate peptidases in Arabidopsis thaliana

Most mitochondrial proteins contain an N-terminal targeting signal that is removed by specific proteases following import. In plant mitochondria, only mitochondrial processing peptidase (MPP) has been characterized to date. Therefore, we sought to determine the substrates and cleavage sites of the Arabidopsis thaliana homologues to the yeast Icp55 and Oct1 proteins, using the newly developed ChaFRADIC method for N-terminal protein sequencing. We identified 88 and seven putative substrates for Arabidopsis ICP55 and OCT1, respectively. It was determined that the Arabidopsis ICP55 contains an almost identical cleavage site to that of Icp55 from yeast. However, it can also remove a far greater range of amino acids. The OCT1 substrates from Arabidopsis displayed no consensus cleavage motif, and do not contain the classical –10R motif identified in other eukaryotes. Arabidopsis OCT1 can also cleave presequences independently, without the prior cleavage of MPP. It was concluded that while both OCT1 and ICP55 were probably acquired early on in the evolution of mitochondria, their substrate profiles and cleavage sites have either remained very similar or diverged completely

Shedding light on black boxes in protein identification

Performing a well thought-out proteomics data analysis can be a daunting task, especially for newcomers to the field. Even researchers experienced in the proteomics field can find it challenging to follow existing publication guidelines for MS-based protein identification and characterization in detail. One of the primary goals of bioinformatics is to enable any researcher to interpret the vast amounts of data generated in modern biology, by providing user-friendly and robust end-user applications, clear documentation, and corresponding teaching materials. In that spirit, we here present an extensive tutorial for peptide and protein identification, available at http://compomics.com/bioinformatics-for-proteomics. The material is completely based on freely available and open-source tools, and has already been used and refined at numerous international courses over the past 3 years. During this time, it has demonstrated its ability to allow even complete beginners to intuitively conduct advanced bioinformatics workflows, interpret the results, and understand their context. This tutorial is thus aimed at fully empowering users, by removing black boxes in the proteomics informatics pipeline.acceptedVersio

The potential of fractional diagonal chromatography strategies for the enrichment of post-translational modifications

More than 450 post-translational modifications (PTMs) are known, however, currently only some of those can be enriched and analyzed from complex samples such as cell lysates. Therefore, we need additional methods and concepts to improve our understanding about the dynamic crosstalk of PTMs and the highly context-dependent regulation of protein function by so-called ‘PTM codes’. The mere focus on affinity-based enrichment techniques may not be sufficient to achieve this ambitious goal. However, the complementary use of two-dimensional chromatography-based strategies such as COFRADIC and ChaFRADIC might open new avenues for enriching a variety of so far inaccessible PTMs for large-scale proteome studies

Enrichment of Cross-Linked Peptides Using Charge-Based Fractional Diagonal Chromatography (ChaFRADIC)

Chemical cross-linking of proteins is an emerging field with huge potential for the structural investigation of proteins and protein complexes. Owing to the often relatively low yield of cross-linking products, their identification in complex samples benefits from enrichment procedures prior to mass spectrometry analysis. So far, this is mainly accomplished by using biotin moieties in specific cross-linkers or by applying strong cation exchange chromatography (SCX) for a relatively crude enrichment. We present a novel workflow to enrich cross-linked peptides by utilizing charge-based fractional diagonal chromatography (ChaFRADIC). On the basis of two-dimensional diagonal SCX separation, we could increase the number of identified cross-linked peptides for samples of different complexity: pure cross-linked BSA, cross-linked BSA spiked into a simple protein mixture, and cross-linked BSA spiked into a HeLa lysate. We also compared XL-ChaFRADIC with size exclusion chromatography-based enrichment of cross-linked peptides. The XL-ChaFRADIC approach is straightforward, reproducible, and independent of the cross-linking chemistry and cross-linker properties

Novel Highly Sensitive, Specific, and Straightforward Strategy for Comprehensive N‑Terminal Proteomics Reveals Unknown Substrates of the Mitochondrial Peptidase Icp55

We present a novel straightforward method for enrichment of N-terminal peptides, utilizing charge-based fractional diagonal chromatography (ChaFRADIC). Our method is robust, easy to operate, fast, specific, and more sensitive than existing methods, enabling the differential quantitation of 1459 nonredundant N-terminal peptides between two <i>S. cerevisiae</i> samples within 10 h of LC–MS, starting from only 50 μg of protein per condition and analyzing only 40% of the obtained fractions. Using ChaFRADIC we compared mitochondrial proteins from wild-type and icp55Δ yeast (30 μg each). Icp55 is an intermediate cleaving peptidase, which, following mitochondrial processing peptidase (MPP)-dependent cleavage of signal sequences, removes a single amino acid from a specific set of proteins according to the N-end rule. Using ChaFRADIC we identified 36 icp55 substrates, 14 of which were previously unknown, expanding the set of known icp55 substrates to a total of 52 proteins. Interestingly, a novel substrate, Isa2, is likely processed by Icp55 in two consecutive steps and thus might represent the first example of a triple processing event in a mitochondrial precursor protein. Thus, ChaFRADIC is a powerful and practicable tool for protease and peptidase research, providing the sensitivity to characterize even samples that can be obtained only in small quantities

Simple, scalable and ultra-sensitive tip-based identification of protease substrates

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) and later 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 well-established protein chemistry and a pipette tip. Using iTRAQ-8plex we quantified on average 2073±52 unique N-terminal peptides from only 4.3 μg per sample/channel, allowing the identification of proteolytic targets and consensus motifs. This high sensitivity may even allow working 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 multi-level 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 staurosporine-induced apoptosis.status: publishe

PARL mediates Smac proteolytic maturation in mitochondria to promote apoptosis

Mitochondria drive apoptosis by releasing pro-apoptotic proteins that promote caspase activation in the cytosol. The rhomboid protease PARL, an intramembrane cleaving peptidase in the inner membrane, regulates mitophagy and plays an ill-defined role in apoptosis. Here, we employed PARL-based proteomics to define its substrate spectrum. Our data identified the mitochondrial pro-apoptotic protein Smac (also known as DIABLO) as a PARL substrate. In apoptotic cells, Smac is released into the cytosol and promotes caspase activity by inhibiting inhibitors of apoptosis (IAPs). Intramembrane cleavage of Smac by PARL generates an amino-terminal IAP-binding motif, which is required for its apoptotic activity. Loss of PARL impairs proteolytic maturation of Smac, which fails to bind XIAP. Smac peptidomimetics, downregulation of XIAP or cytosolic expression of cleaved Smac restores apoptosis in PARL-deficient cells. Our results reveal a pro-apoptotic function of PARL and identify PARL-mediated Smac processing and cytochrome c release facilitated by OPA1-dependent cristae remodelling as two independent pro-apoptotic pathways in mitochondria