Missed Cleavage Sites Research Articles

Discovery and validation of glucose-sensitive peptide biomarkers from human serum albumin to diagnose type 2 diabetes mellitus

Glycated albumin (GA), which represents the global glycation level of albumin, has emerged as a biomarker for diagnosing prediabetes and diabetes. In our previous study, we developed a peptide-based strategy and found three putative peptide biomarkers from the tryptic peptides of GA to diagnose type 2 diabetes mellitus (T2DM). However, the trypsin cleavage sites at the carboxyl side of lysine (K) and arginine (R) are consistent with the nonenzymatic glycation modification site residues, which considerably increases the number of missed cleavage sites and half-cleaved peptides. To solve this problem, the endoproteinase Glu-C was used to digest GA from human serum to screen putative peptides to diagnose T2DM. In the discovery phase, we found eighteen and fifteen glucose-sensitive peptides from purified albumin and human serum incubated with 13C glucose in vitro, respectively. In the validation phase, eight glucose-sensitive peptides were screened and validated in 72 clinical samples (28 healthy controls and 44 patients with diabetes) using label-free LC-ESI-MRM. Three putative sensitive peptides (VAHRFKDLGEE, FKPLVEEPQNLIKQNCE and NQDSISSKLKE) from albumin exhibited good specificity and sensitivity based on receiver operating characteristic analysis. In summary, three peptides were found as promising biomarkers for the diagnosis and assessment of T2DM based on mass spectrometry.

Maximizing Cumulative Trypsin Activity with Calcium at Elevated Temperature for Enhanced Bottom-Up Proteome Analysis.

Simple SummaryTrypsin is frequently employed to cleave proteins ahead of mass spectrometry characterization. Traditionally, enzyme digestion involves overnight incubation of proteins at 37 °C, which is time consuming though still may yield poor digestion efficiency. While raising the temperature should theoretically accelerate the digestion, it also destabilizes the enzyme and promotes trypsin de-activation. We therefore questioned whether elevated temperature is beneficial for improving tryptic digestion. Here, we quantify protein digestion kinetics at elevated temperatures for calcium-stabilized trypsin and enforce the critical importance of calcium ions to preserve the enzyme. We quantitatively demonstrate that 1 h at 47 °C provides a superior digest when compared to conventional (overnight, 37 °C) processing of the proteome. The practical impact of our enhanced digestion protocol is shown through bottom-up mass spectrometry analysis of a complex proteome mixture.Bottom-up proteomics relies on efficient trypsin digestion ahead of MS analysis. Prior studies have suggested digestion at elevated temperature to accelerate proteolysis, showing an increase in the number of MS-identified peptides. However, improved sequence coverage may be a consequence of partial digestion, as higher temperatures destabilize and degrade the enzyme, causing enhanced activity to be short-lived. Here, we use a spectroscopic (BAEE) assay to quantify calcium-stabilized trypsin activity over the complete time course of a digestion. At 47 °C, the addition of calcium contributes a 25-fold enhancement in trypsin stability. Higher temperatures show a net decrease in cumulative trypsin activity. Through bottom-up MS analysis of a yeast proteome extract, we demonstrate that a 1 h digestion at 47 °C with 10 mM Ca2+ provides a 29% increase in the total number of peptide identifications. Simultaneously, the quantitative proportion of peptides with 1 or more missed cleavage sites was diminished in the 47 °C digestion, supporting enhanced digestion efficiency with the 1 h protocol. Trypsin specificity also improves, as seen by a drop in the quantitative abundance of semi-tryptic peptides. Our enhanced digestion protocol improves throughput for bottom-up sample preparation and validates the approach as a robust, low-cost alternative to maximized protein digestion efficiency.

Development of an Online LC‐MS/MS Method for the Investigation of Enzyme Kinetics Using Trypsinization of Apomyoglobin Protein as a Model System

The optimization of enzymatic protein cleavage by trypsin is often performed “in the dark” wherein researchers perform the reaction under different conditions on a laboratory bench for a single or a small selection of time points. Upon quenching of the trypsinization reaction with a strong acid, the resulting protein reaction mixture is injected onto a liquid chromatography column for profiling of the peptide mixture. The arbitrary selection of enzyme reaction time presents a major limitation to this experimental approach. Herein, we present a “real time” optimization of the enzymatic reaction enabled by performing the reaction within a vial in an autosampler with regular sampling of the mixture. Using this method, we are able to simultaneously monitor degradation of the intact protein target and the release of its tryptic peptides. Peptides with missed cleavage sites may appear and then disappear with time as fully tryptic peptides dominate the endpoint of the reaction. The method can be multiplexed in that we can sample many parallel mixtures in series. This allows us to directly compare such variables as pH, solvent, ionic strength, protein to enzyme ratio, enzyme concentration and metal salts on digestion efficiency while always including a positive control for reference. Our liquid chromatograph-tandem mass spectrometry analysis platform uses a Dionex Ultimate 3000 interfaced with a hybrid quadrupole-Orbitrap (Q-Exactive) mass spectrometer capable of mass resolution of 140,000 and a mass accuracy better than 10 ppm. We are able to monitor the efficiency and kinetics of tryptic digestion of apomyoglobin with high resolution in the time dimension. At room temperature, the enzymatic reaction can take in excess of 12 hours to reach completion compared with about 3 hours at 38oC. A low percentage of ethanol (~ 0.1%) added to the reaction mixture was shown to double the yield of most peptides while the addition of alkaline earth metals also had beneficial effects, in particular 10 mM barium chloride. Other metals were shown to either inhibit the degradation of apomyoglobin (zinc) or to prevent the detection of the resulting peptides (lead). Overall, the effects of adding barium in the presence of 0.1% ethanol improved peptide yield as much as 11-fold for the LFTGHPETLEK peptide of apomyoglobin while showing no effect on the tryptic release of the YLEFISDAIIHVLHSK peptide. This method offers a promising strategy for the optimization of targeted protein quantitation as well as a means to study the kinetics and characterize the specificity of other proteolytic enzymes.

DeepDigest: Prediction of Protein Proteolytic Digestion with Deep Learning.

Proteolytic digestion of proteins by one or more proteases is a key step in shotgun proteomics, in which the proteolytic products, i.e., peptides, are taken as the surrogates of their parent proteins for further qualitative or quantitative analysis. The proteases generally cleave proteins at specific amino acid residue sites, but digestion is hardly complete (wide existence of missed cleavage sites). Therefore, it would be of great help to improve the prior experimental design and the posterior data analysis if the digestion behaviors of proteases can be accurately modeled and predicted. At present, systematic studies about the commonly used proteases in proteomics are insufficient, and there is a lack of easy-to-use tools to predict the cleavage sites of different proteases. Here, we propose a novel sequence-based deep learning algorithm-DeepDigest, which integrates convolutional neural networks and long short-term memory networks for protein digestion prediction. DeepDigest can predict the cleavage probability of each potential cleavage site on the protein sequences for eight popular proteases including trypsin, ArgC, chymotrypsin, GluC, LysC, AspN, LysN, and LysargiNase. We compared DeepDigest with three traditional machine learning algorithms, i.e., logistic regression, random forest, and support vector machine. On the eight training data sets, the 10-fold cross-validation accuracies (AUCs) of DeepDigest were 0.956-0.982, significantly higher than those of the three traditional algorithms. On the 11 independent test data sets, DeepDigest achieved AUCs between 0.849 and 0.978, outperforming the other traditional algorithms in most cases. Transfer learning then further improved the prediction accuracy. Besides, some interesting characteristics of different proteases were revealed and discussed. Ultimately, as an application, we used DeepDigest to predict the digestibilities of peptides and demonstrated that peptide digestibility is an informative new feature to discriminate between correct and incorrect peptide identifications.

The Optimized Workflow for Sample Preparation in LC-MS/MS-Based Urine Proteomics

The sample condition is an important factor in urine proteomics with stability and accuracy. However, a general protocol of urine protein preparation in mass spectrometry analysis has not yet been established. Here, we proposed a workflow for optimized sample preparation based on methanol/chloroform (M/C) precipitation and in-solution trypsin digestion in LC-MS/MS-based urine proteomics. The urine proteins prepared by M/C precipitation showed around 80% of the protein recovery rate. The samples showed the largest number of identified proteins, which were over 1000 on average compared with other precipitation methods in LC-MS/MS-based urine proteomics. For further improvement of the workflow, the essences were arranged in protein dissolving and trypsin digestion step for the extraction of urine proteins. Addition of Ethylene diamine tetraacetic acid (EDTA) dramatically enhanced the dissolution of protein and promoted the trypsin activity in the digestion step because the treatment increased the number of identified proteins with less missed cleavage sites. Eventually, an optimized workflow was established by a well-organized strategy for daily use in the LC-MS/MS-based urine proteomics. The workflow will be of great help for several aims based on urine proteomics approaches, such as diagnosis and biomarker discovery.

Generic Workflow for Mapping of Complex Disulfide Bonds Using In-Source Reduction and Extracted Ion Chromatograms from Data-Dependent Mass Spectrometry.

Disulfide bond mapping is a critical task in protein characterization as protein stability, structure, and function is dependent on correct cysteine connectivities. Mass spectrometry (MS) is the method of choice for this, providing fast and accurate characterization of simple disulfide bonds. Disulfide mapping by liquid chromatography tandem mass spectrometry (LC-MS/MS) is performed by identifying disulfide-bonded partner peptides following proteolytic digestion. With the recently introduced ability to assign complex disulfide patterns by online postcolumn partial disulfide reduction by in-source reduction (ISR) in a LC-ISR-MS/MS methodology, the main challenge is data analysis to ensure detection of both expected and unexpected disulfide species. In this study, we introduced a workflow for confident and unbiased mapping of complex disulfide bonds using the powerful combination of extracted ion chromatograms (XICs) of LC-ISR-MS/MS data. With postcolumn partial reduction, identical LC retention times of intact disulfide-bonded species, their constituting free peptides, and partially reduced variants were observed. Subsequent selective MS/MS fragmentation of all reduction products allowed confident identification of free cysteine-containing peptides using a classical shotgun proteomics database search. Matching XICs of the identified cysteine-containing peptides allowed identification of both predicted and unpredicted disulfide species, including unforeseen proteolytic specificities, missed cleavage sites, scrambled disulfide variants, and the presence of disulfide-entangled complexes. Applying this workflow, we successfully mapped the complex disulfide bonds of tertiapin and the epidermal growth factor (EGF) family members transforming growth factor α (TGFα) and EGF. In addition, we were able to characterize the disulfide patterns of the special disulfide fold of the TGFβ superfamily in an all-online methodology.

Enzymatic Reactor with Trypsin Immobilized on Graphene Oxide Modified Polymer Microspheres To Achieve Automated Proteome Quantification.

Protein digestion and isotope labeling are two critical steps in proteome quantification. However, the conventional in-solution protocol unavoidably suffers from disadvantages such as time-consuming, low labeling efficiency, and tedious off-line manual operation, which might affect the quantification accuracy, reproducibility, and throughput. To address these problems, we developed a fully automated proteome quantification platform, in which an ultraperformance immobilized microreactor (upIMER) with graphene-oxide-modified polymer microspheres as the matrix was developed, to achieve not only the simultaneous protein digestion and 18O labeling, but also the online integration with nano-high-pressure liquid chromatography-electrospray ionization-tandem mass spectrometry (nanoHPLC-ESI-MS/MS). Compared to the conventional off-line protocols, such a platform exhibits obviously improved digestion and 18O labeling efficiency (only 8% peptides with missed cleavage sites, 99% labeling efficiency, and 2.5 min reaction time), leading to the increased quantification coverage, accuracy, precision and throughput. All the results demonstrated that our developed fully automated platform should provide new opportunities to improve the accuracy, reproducibility, and throughput for proteome quantification.

Validation, Identification, and Biological Consequences of the Site-specific O-GlcNAcylation Dynamics of Carbohydrate-responsive Element-binding Protein (ChREBP).

O-GlcNAcylation of carbohydrate-responsive element-binding protein (ChREBP) is believed as an important modulator of ChREBP activities, however little direct evidence of O-GlcNAcylation on ChREBP and no exact O-GlcNAcylation sites have been reported so far. Here, we validate O-GlcNAcylation on ChREBP in cell-free coupled transcription/translation system and in cells by chemoenzymatic and metabolic labeling, respectively. Moreover, for the first time, we identify O-GlcNAcylation on Ser614 in the C-terminus of ChREBP by mass spectrometry and validate two important sites, Thr517 and Ser839 for O-GlcNAcylation and their function via molecular and chemical biological method. Under high glucose conditions, Ser514 phosphorylation enhances ChREBP O-GlcNAcylation, maintaining the transcriptional activity of ChREBP; Ser839 O-GlcNAcylation is essential for Mlx-heterodimerization and DNA-binding activity enhancement, consequently inducing transcriptional activity. Ser839 O-GlcNAcylation is also crucial for ChREBP nuclear export partially by strengthening interactions with CRM1 and 14-3-3. This work is a detailed study of ChREBP O-GlcNAcylation and highlights the biological consequences of the site-specific O-GlcNAcylation dynamics of ChREBP.

Evaluation of Endoproteinase Lys-C/Trypsin Sequential Digestion Used in Proteomics Sample Preparation

Abstract Endoproteinase lysine-C (Lys-C)/trypsin sequential digestion and trypsin digestion were used in 293T cell proteomics sample preparation and the differences of Lys-C/trypsin sequential digestion and trypsin digestion in proteomics sample preparation were systematically evaluated. It was found that the number of identified peptides and proteins increased significantly and missed cleavage sites, especially K sites decreased dramatically through Lys-C/trypsin sequential digestion. The average sequence coverage of identified proteins in Lys-C/trypsin sequential digestion sample was higher than that in trypsin digestion sample. Besides, different amounts of enzymes were tested to select the optimal usage of enzymes in Lys-C/trypsin sequential digestion. This study provided the references for proteomics sample preparation.

Studying the effect of microwave heating on the digestion process and identification of proteins.

The impact of microwave irradiation on the in-solution digestion processes and the detection limit of proteins are systematically studied. Kinetic processes of many peptides produced through the trypsin digestion of various proteins under microwave heating at 50°C were investigated with MALDI-MS. This study also examines the detection limits and digestion completeness of individual proteins under microwave heating at 50°C and at different time intervals (1, 5 and 30 min) using LC-MS. We conclude that if the peptides without missed cleavage dictate the detection limit, conventional digestion will lead to a better detection limit. The detection limit may not differ between the microwave and conventional heating if the peptides with missed cleavage sites and strong intensity are formed at the very early stage (i.e., less than 1min) and are not further digested throughout the entire digestion process. The digestion of Escherichia coli lysate was compared under conventional and short time (microwave) conditions. The number of proteins identified under conventional heating exceeded that obtained from microwave heating over heating periods less than 5 min. The overall results show that the microwave-assisted digestion is not complete. Although the sequence coverage might be better, the detection limit might be worse than that under conventional heating.

Quantitative Evaluation of Filter Aided Sample Preparation (FASP) and Multienzyme Digestion FASP Protocols.

Filter aided sample preparation (FASP) and related methods gain increasing popularity for proteomic sample preparation. Nevertheless, the originally published FASP method has been criticized by several authors, who reported low digestion performance. In this work, we re-evaluate FASP and the related multienzyme digestion (MED) FASP method. We use different types of animal tissues and cultured cells and test the performance of the method under various conditions. We analyze the protein to peptide conversion by assessing the yield of peptides, frequency of peptides with missed cleavage sites, and the reproducibility of FASP. We identify conditions allowing efficient protein processing with high peptide yields and demonstrate advantages of the two step digestion strategy over single step digestion with trypsin. In addition, we show that FASP outperforms in-solution cleavage strategies. Our results clearly demonstrate that the performance of digestion varies between different types of samples. We show that MED FASP in combination with the total protein approach provides highly reproducible protein abundance values. The presented data can be used as a guide for optimization of sample processing.

Processed Meat Protein and Heat-Stable Peptide Marker Identification Using Microwave-Assisted Tryptic Digestion.

New approaches to rapid examination of proteins and peptides in complex food matrices are of great interest to the community of food scientists. The aim of the study is to examine the influence of microwave irradiation on the acceleration of enzymatic cleavage and enzymatic digestion of denatured proteins in cooked meat of five species (cattle, horse, pig, chicken and turkey) and processed meat products (coarsely minced, smoked, cooked and semi-dried sausages). Severe protein aggregation occurred not only in heated meat under harsh treatment at 190 °C but also in processed meat products. All the protein aggregates were thoroughly hydrolyzed after 1 h of trypsin treatment with short exposure times of 40 and 20 s to microwave irradiation at 138 and 303 W. There were much more missed cleavage sites observed in all microwave-assisted digestions. Despite the incompleteness of microwave-assisted digestion, six unique peptide markers were detected, which allowed unambiguous identification of processed meat derived from the examined species. Although the microwave-assisted tryptic digestion can serve as a tool for rapid and high-throughput protein identification, great caution and pre-evaluation of individual samples is recommended in protein quantitation.

Insight into Trypsin Miscleavage: Comparison of Kinetic Constants of Problematic Peptide Sequences.

Trypsin, a high fidelity protease, is the most widely used enzyme for protein digestion in proteomic research. Optimal digestion conditions are well-known and so are the expected cleavage products. However, missed cleavage sites are frequently observed when acidic amino acids, aspartic and glutamic acids, are present near the cleavage site. Also, the sequence motifs with successive lysine and/or arginine residues represent a source of missed cleaved sites. In spite of an adverse role of missed cleaved peptides on proteomic research, the digestion kinetics of these problematic sequences is not well-known. In this work, synthetic peptides with various sequence motifs were used as trypsin substrates. Cleavage products were analyzed with reversed-phase high performance liquid chromatography, and the kinetic constants for selected missed cleavage sites were calculated. Relative digestion speed for lysine and arginine sites is compared, including the digestion motifs flanked with aspartic and glutamic acid. Our findings show that DK and DTR motifs are cleaved by trypsin with 3 orders of magnitude lower speed than the arginine site. These motifs are likely to produce missed cleavage peptides in protein tryptic digests even at prolonged digestion times.

Cleaved and Missed Sites for Trypsin, Lys-C, and Lys-N Can Be Predicted with High Confidence on the Basis of Sequence Context

Trypsin, Lys-C, and Lys-N are the most broadly used enzymes in proteomics. Here, on the basis of large-scale peptide mass spectrometry (MS) data sets, an approach is described to confidently identify missed cleavage sites in either phosphorylated or unmodified substrates for these three proteases, or any protease, on the basis of side chain species present within 15 residues of the cleavage-specificity residue. Previously known effects of proline, negatively charged side chains, and phospho-modified residues have been quantified, and additional side chain effects were noted. By applying a set of quantitative side chain rules established for each of the three proteases, scissile and nonscissile sites could be established, on the basis of protein sequence alone, with near certainty for Lys-C, and with a high degree of confidence for trypsin or Lys-N. These rules were applicable to orthogonal peptide data sets, including the two largest in the PeptideAtlas database. The approach described here facilitates the comprehensive modeling of substrate recognition in proteolysis.

Comprehensive Cell-specific Protein Analysis in Early and Late Pollen Development from Diploid Microsporocytes to Pollen Tube Growth

Pollen development in angiosperms is one of the most important processes controlling plant reproduction and thus productivity. At the same time, pollen development is highly sensitive to environmental fluctuations, including temperature, drought, and nutrition. Therefore, pollen biology is a major focus in applied studies and breeding approaches for improving plant productivity in a globally changing climate. The most accessible developmental stages of pollen are the mature pollen and the pollen tubes, and these are thus most frequently analyzed. To reveal a complete quantitative proteome map, we additionally addressed the very early stages, analyzing eight stages of tobacco pollen development: diploid microsporocytes, meiosis, tetrads, microspores, polarized microspores, bipolar pollen, desiccated pollen, and pollen tubes. A protocol for the isolation of the early stages was established. Proteins were extracted and analyzed by means of a new gel LC-MS fractionation protocol. In total, 3817 protein groups were identified. Quantitative analysis was performed based on peptide count. Exceedingly stage-specific differential protein regulation was observed during the conversion from the sporophytic to the gametophytic proteome. A map of highly specialized functionality for the different stages could be revealed from the metabolic activity and pronounced differentiation of proteasomal and ribosomal protein complex composition up to protective mechanisms such as high levels of heat shock proteins in the very early stages of development.

Protein Sets Define Disease States and Predict In Vivo Effects of Drug Treatment

Gaining understanding of common complex diseases and their treatments are the main drivers for life sciences. As we show here, comprehensive protein set analyses offer new opportunities to decipher functional molecular networks of diseases and assess the efficacy and side-effects of treatments in vivo. Using mass spectrometry, we quantitatively detected several thousands of proteins and observed significant changes in protein pathways that were (dys-) regulated in diet-induced obesity mice. Analysis of the expression and post-translational modifications of proteins in various peripheral metabolic target tissues including adipose, heart, and liver tissue generated functional insights in the regulation of cell and tissue homeostasis during high-fat diet feeding and medication with two antidiabetic compounds. Protein set analyses singled out pathways for functional characterization, and indicated, for example, early-on potential cardiovascular complication of the diabetes drug rosiglitazone. In vivo protein set detection can provide new avenues for monitoring complex disease processes, and for evaluating preclinical drug candidates.

Global Relative Quantification with Liquid Chromatography–Matrix-assisted Laser Desorption Ionization Time-of-flight (LC-MALDI-TOF)—Cross–validation with LTQ-Orbitrap Proves Reliability and Reveals Complementary Ionization Preferences

Quantitative LC-MALDI is an underrepresented method, especially in large-scale experiments. The additional fractionation step that is needed for most MALDI-TOF-TOF instruments, the comparatively long analysis time, and the very limited number of established software tools for the data analysis render LC-MALDI a niche application for large quantitative analyses beside the widespread LC-electrospray ionization workflows. Here, we used LC-MALDI in a relative quantification analysis of Staphylococcus aureus for the first time on a proteome-wide scale. Samples were analyzed in parallel with an LTQ-Orbitrap, which allowed cross-validation with a well-established workflow. With nearly 850 proteins identified in the cytosolic fraction and quantitative data for more than 550 proteins obtained with the MASCOT Distiller software, we were able to prove that LC-MALDI is able to process highly complex samples. The good correlation of quantities determined via this method and the LTQ-Orbitrap workflow confirmed the high reliability of our LC-MALDI approach for global quantification analysis. Because the existing literature reports differences for MALDI and electrospray ionization preferences and the respective experimental work was limited by technical or methodological constraints, we systematically compared biochemical attributes of peptides identified with either instrument. This genome-wide, comprehensive study revealed biases toward certain peptide properties for both MALDI-TOF-TOF- and LTQ-Orbitrap-based approaches. These biases are based on almost 13,000 peptides and result in a general complementarity of the two approaches that should be exploited in future experiments.

Global Impact of Salmonella Pathogenicity Island 2-secreted Effectors on the Host Phosphoproteome

During the late stages of infection, Salmonella secretes numerous effectors through a type III secretion system that is encoded within Salmonella pathogenicity island 2 (SPI2). Despite the importance of SPI2 as a major virulence factor leading to the systemic spread of the bacteria and diseases, a global view of its effects on host responses is still lacking. Here, we measured global impacts of SPI2 effectors on the host phosphorylation and protein expression levels in RAW264.7 and in HeLa cells, as macrophage and nonphagocytic models of infection. We observe that SPI2 effectors differentially modulate the host phosphoproteome and cellular processes (e.g. protein trafficking, cytoskeletal regulation, and immune signaling) in a host cell-dependent manner. Our unbiased approach reveals the involvement of many previously unrecognized proteins, including E3 ligases (HERC4, RanBP2, and RAD18), kinases (CDK, SIK3, and WNK1), and histones (H2B1F, H4, and H15), in late stages of Salmonella infection. Furthermore, from this phosphoproteome analysis and other quantitative screens, we identified HSP27 as a direct in vitro and in vivo molecular target of the only type III secreted kinase, SteC. Using biochemical and cell biological assays, we demonstrate that SteC phosphorylates multiple sites in HSP27 and induces actin rearrangement through this protein. Together, these results provide a broader landscape of host players contributing to specific processes/pathways mediated by SPI2 effectors than was previously appreciated.

A SILAC-based Approach Identifies Substrates of Caspase-dependent Cleavage upon TRAIL-induced Apoptosis

The extracellular ligand-induced extrinsic pathway of apoptosis is executed via caspase protease cascades that activate downstream effectors by means of site-directed proteolysis. Here we identify proteome changes upon the induction of apoptosis by the cytokine tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in a Jurkat T cell line. We detected caspase-dependent cleavage substrates by quantifying protein intensities before and after TRAIL induction in SDS gel slices. Apoptotic protein cleavage events are identified by a characteristic stable isotope labeling with amino acids in cell culture (SILAC) ratio pattern across gel slices that results from differential migration of the cleaved and uncleaved proteins. We applied a statistical test to define apoptotic substrates in the proteome. Our approach identified more than 650 of these cleaved proteins in response to TRAIL-induced apoptosis, including many previously unknown substrates and cleavage sites. Inhibitor treatment combined with triple SILAC demonstrated that the detected cleavage events were caspase dependent. Proteins located in the lumina of organelles such as mitochondria and endoplasmic reticulum were significantly underrepresented in the substrate population. Interestingly, caspase cleavage is generally observed in not only one but several members of stable complexes, but often with lower stoichiometry. For instance, all five proteins of the condensin I complex were cleaved upon TRAIL treatment. The apoptotic substrate proteome data can be accessed and visualized in the MaxQB database and might prove useful for basic and clinical research into TRAIL-induced apoptosis. The technology described here is extensible to a wide range of other proteolytic cleavage events.

Deletion of Cysteine Cathepsins B or L Yields Differential Impacts on Murine Skin Proteome and Degradome

Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb(-/-), and Ctsl(-/-) mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl(-/-) skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl(-/-) skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl(-/-) or Ctsb(-/-) samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.