Comprehensive profiling of the human fecal proteome from IBD patients with DIA-MS enables evaluation of disease-relevant proteins.

PURPOSE: Inflammatory bowel disease (IBD), which includes ulcerative colitis (UC) and Crohn's disease (CD), is characterized by chronic gastrointestinal inflammation. A high unmet need exists for noninvasive biomarkers in IBD to monitor changes in disease activity and guide treatment decisions. Stool is an easily accessed, disease proximal matrix in IBD, however the composition of the IBD fecal proteome remains poorly characterized. EXPERIMENTAL DESIGN: A data-independent acquisition LC-MS/MS approach was used to profile the human fecal proteome in two independent cohorts (Cohort 1: healthy n = 5, UC n = 5, CD n = 5, Cohort 2: healthy n = 20, UC n = 10, and CD n = 10) to identify noninvasive biomarkers reflective of disease activity. RESULTS: 688 human proteins were quantified, with 523 measured in both cohorts. In UC stool 96 proteins were differentially abundant and in CD stool 126 proteins were differentially abundant compared to healthy stool (absolute log2 fold change > 1, p-value < 0.05). Many of these fecal proteins are associated with infiltrating immune cells and ulceration/rectal bleeding, which are hallmarks of IBD pathobiology. Mapping the identified fecal proteins to a whole blood single-cell RNA sequencing data set revealed the involvement of various immune cell subsets to the IBD fecal proteome. CONCLUSIONS AND CLINICAL RELEVANCE: Findings from this study not only confirmed the presence of established fecal biomarkers for IBD, such as calprotectin and lactoferrin, but also revealed new fecal proteins from multiple pathways known to be dysregulated in IBD. These novel proteins could serve as potential noninvasive biomarkers to monitor specific aspects of IBD disease activity which could expedite clinical development of novel therapeutic targets.

Emerging Cerebrospinal Fluid Biomarkers of Disease Activity and Progression in Multiple Sclerosis.

Importance: Biomarkers distinguishing nonrelapsing progressive disease biology from relapsing biology in multiple sclerosis (MS) are lacking. Cerebrospinal fluid (CSF) is an accessible fluid that most closely reflects central nervous system biology. Objective: To identify CSF biological measures associated with progressive MS pathobiology. Design, Setting, and Participants: This cohort study assessed data from 2 prospective MS cohorts: a test cohort provided serial CSF, clinical, and imaging assessments in a multicenter study of patients with relapsing MS (RMS) or primary progressive MS (PPMS) who were initiating anti-CD20 treatment (recruitment: 2016-2018; analysis: 2020-2023). A single-site confirmation cohort was used to assess CSF at baseline and long-term (>10 year) clinical follow-up (analysis: 2022-2023). Exposures: Test-cohort participants initiated standard-of-care ocrelizumab treatment. Confirmation-cohort participants were untreated or received standard-of-care disease-modifying MS therapies. Main Outcomes and Measures: Twenty-five CSF markers, including neurofilament light chain, neurofilament heavy chain, and glial fibrillary acid protein (GFAP); 24-week confirmed disability progression (CDP24); and brain magnetic resonance imaging measures reflecting focal injury, tissue loss, and progressive biology (slowly expanding lesions [SELs]). Results: The test cohort (n = 131) included 100 patients with RMS (mean [SD] age, 36.6 [10.4] years; 68 [68%] female and 32 [32%] male; Expanded Disability Status Scale [EDSS] score, 0-5.5), and 31 patients with PPMS (mean [SD] age, 44.9 [7.4] years; 15 [48%] female and 16 [52%] male; EDSS score, 3.0-6.5). The confirmation cohort (n = 68) included 41 patients with RMS and 27 with PPMS enrolled at diagnosis (age, 40 years [range, 20-61 years]; 47 [69%] female and 21 [31%] male). In the test cohort, GFAP was correlated with SEL count (r = 0.33), greater proportion of T2 lesion volume from SELs (r = 0.24), and lower T1-weighted intensity within SELs (r = -0.33) but not with acute inflammatory measures. Neurofilament heavy chain was correlated with SEL count (r = 0.25) and lower T1-weighted intensity within SELs (r = -0.28). Immune markers correlated with measures of acute inflammation and, unlike GFAP, were impacted by anti-CD20. In the confirmation cohort, higher baseline CSF GFAP levels were associated with long-term CDP24 (hazard ratio, 2.1; 95% CI, 1.3-3.4; P = .002). Conclusions and Relevance: In this study, activated glial markers (in particular GFAP) and neurofilament heavy chain were associated specifically with nonrelapsing progressive disease outcomes (independent of acute inflammatory activity). Elevated CSF GFAP was associated with long-term MS disease progression.

Development of an LC-MS/MS Method to Measure Sphingolipids in CSF from Patients with Multiple Sclerosis.

Multiple sclerosis is an inflammatory and degenerative disease characterized by different clinical courses including relapsing multiple sclerosis (RMS) and primary progressive multiple sclerosis (PPMS). A hallmark of patients with multiple sclerosis (pwMS) includes a putative autoimmune response, which results in demyelination and neuroaxonal damage in the central nervous system. Sphingolipids in cerebrospinal fluid (CSF) have been proposed as potential biomarkers reflective of disease activity in pwMS. Hence, sensitive methods to accurately quantify sphingolipids in CSF are needed. In this study, we report the development of a sensitive high-throughput multiplexed liquid chromatography coupled to a tandem mass spectrometry method to perform quantitation on 14 species of sphingolipids in human CSF. We applied this method to measure CSF sphingolipids in healthy controls (n = 10), PPMS (n = 27), and RMS (n = 17) patients before and after ocrelizumab treatment. The median CSF levels of the 14 sphingolipids measured herein was higher in PPMS (17.2 ng/mL) and RMS (17.6 ng/mL) when compared with the healthy controls (13.8 ng/mL). Levels of sphingolipids were decreased by 8.6% at week 52 after treatment with ocrelizumab in RMS patients but not in PPMS patients. Specifically, C16 glucosylceramide (-26%; P = 0.004) and C18 ceramides (-13%; P = 0.042) decreased from baseline in RMS patients. Additionally, in PPMS patients C16 glucosylceramide levels correlated with CSF neurofilament heavy levels at baseline (Rho =0.532; P = 0.004) and after treatment (Rho =0.424; P = 0.028). Collectively, these results indicate that CSF sphingolipid levels are altered in pwMS and treatment with ocrelizumab results in significant shifts in the sphingolipid profile that may reflect a reduction in disease activity supporting further investigation into sphingolipids as tools to monitor disease state. SIGNIFICANCE STATEMENT: This study describes the development of a new method to measure 14 sphingolipid species in CSF. These results demonstrate that sphingolipids levels are elevated in CSF from pwMS compared to healthy controls. Distinct sphingolipid signatures were observed between patients with different clinical disease courses, and these lipid signatures changed after treatment with ocrelizumab, especially in RMS patients. This method enables further investigation into the role of sphingolipids as candidate biomarkers in pwMS and other central nervous system disorders.

Randomized Phase II Study of the Safety and Efficacy of Semorinemab in Participants With Mild-to-Moderate Alzheimer Disease: Lauriet.

BACKGROUND AND OBJECTIVES: Accumulation of tau pathology in Alzheimer disease (AD) correlates with cognitive decline. Anti-tau immunotherapies were proposed as potential interventions in AD. While antibodies targeting N-terminal tau failed to demonstrate clinical efficacy in prodromal-to-mild AD, their utility at other disease stages was not evaluated in prior studies. Lauriet is a phase 2 study of an anti-tau monoclonal antibody, semorinemab, in patients with mild-to-moderate AD. METHODS: The phase 2 Lauriet study included a randomized, placebo-controlled, double-blind period, during which participants with mild-to-moderate AD received 4,500 mg of IV semorinemab or placebo every 4 weeks for 48 or 60 weeks. Participants who chose to continue in the subsequent optional open-label extension received 4,500 mg of semorinemab every 4 weeks for up to 96 weeks. Coprimary efficacy endpoints were change from baseline to week 49 or 61 on the 11-item version of the Alzheimer's Disease Assessment Scale-Cognitive Subscale (ADAS-Cog11) and the Alzheimer's Disease Cooperative Study-Activities of Daily Living (ADCS-ADL) scale. Secondary efficacy endpoints included change from baseline on the Mini-Mental State Examination (MMSE) and Clinical Dementia Rating-Sum of Boxes (CDR-SB). Safety, pharmacokinetics, and pharmacodynamic effects were also evaluated. RESULTS: Between December 3, 2018, and February 27, 2020, 624 individuals were screened, 272 participants were randomized, and 238 were included in the modified intent-to-treat population (received ≥1 dose(s) of study medication and underwent baseline and ≥1 postbaseline assessment(s)). Baseline characteristics were well balanced. At week 49, the semorinemab arm demonstrated a 42.2% reduction (-2.89 points, 95% CI -4.56 to -1.21, p = 0.0008) in decline on the ADAS-Cog11 (coprimary endpoint) relative to the placebo arm. However, no treatment effects were observed on the ADCS-ADL scale (coprimary endpoint; absolute difference between the 2 treatment arms in the ADCS-ADL score change from baseline of -0.83 points, 95% CI -3.39 to 1.72, p = 0.52) or on the MMSE or CDR-SB (secondary endpoints). Semorinemab was safe and well tolerated. DISCUSSION: Based on the results of the prespecified coprimary endpoints, this study was negative. While semorinemab had a significant effect on cognition measured by the ADAS-Cog11, this effect did not extend to improved functional or global outcomes. These results may warrant further exploration of semorinemab or other anti-tau therapies in mild-to-moderate AD. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that semorinemab does not slow functional decline in patients with mild-to-moderate AD. TRIAL REGISTRATION INFORMATION: The Lauriet study is registered on ClinicalTrials.gov, NCT03828747, and EudraCT 2018-003398-87.

SAkuraBONSAI: Protocol design of a novel, prospective study to explore clinical, imaging, and biomarker outcomes in patients with AQP4-IgG-seropositive neuromyelitis optica spectrum disorder receiving open-label satralizumab.

Background: Neuromyelitis optica spectrum disorder (NMOSD) is a rare, autoimmune disease of the central nervous system that produces acute, unpredictable relapses causing cumulative neurological disability. Satralizumab, a humanized, monoclonal recycling antibody that targets the interleukin-6 receptor, reduced NMOSD relapse risk vs. placebo in two Phase 3 trials: SAkuraSky (satralizumab ± immunosuppressive therapy; NCT02028884) and SAkuraStar (satralizumab monotherapy; NCT02073279). Satralizumab is approved to treat aquaporin-4 IgG-seropositive (AQP4-IgG+) NMOSD. SAkuraBONSAI (NCT05269667) will explore fluid and imaging biomarkers to better understand the mechanism of action of satralizumab and the neuronal and immunological changes following treatment in AQP4-IgG+ NMOSD. Objectives: SAkuraBONSAI will evaluate clinical disease activity measures, patient-reported outcomes (PROs), pharmacokinetics, and safety of satralizumab in AQP4-IgG+ NMOSD. Correlations between imaging markers (magnetic resonance imaging [MRI] and optical coherence tomography [OCT]) and blood and cerebrospinal fluid (CSF) biomarkers will be investigated. Study design: SAkuraBONSAI is a prospective, open-label, multicenter, international, Phase 4 study that will enroll approximately 100 adults (18-74 years) with AQP4-IgG+ NMOSD. This study includes two patient cohorts: newly diagnosed, treatment-naïve patients (Cohort 1; n = 60); and inadequate responders to recent (<6 months) rituximab infusion (Cohort 2; n = 40). Satralizumab monotherapy (120 mg) will be administered subcutaneously at Weeks 0, 2, 4, and Q4W thereafter for a total of 92 weeks. Endpoints: Disease activity related to relapses (proportion relapse-free, annualized relapse rate, time to relapse, and relapse severity), disability progression (Expanded Disability Status Scale), cognition (Symbol Digit Modalities Test), and ophthalmological changes (visual acuity; National Eye Institute Visual Function Questionnaire-25) will all be assessed. Peri-papillary retinal nerve fiber layer and ganglion cell complex thickness will be monitored using advanced OCT (retinal nerve fiber layer and ganglion cell plus inner plexiform layer thickness). Lesion activity and atrophy will be monitored by MRI. Pharmacokinetics, PROs, and blood and CSF mechanistic biomarkers will be assessed regularly. Safety outcomes include the incidence and severity of adverse events. Conclusions: SAkuraBONSAI will incorporate comprehensive imaging, fluid biomarker, and clinical assessments in patients with AQP4-IgG+ NMOSD. SAkuraBONSAI will provide new insights into the mechanism of action of satralizumab in NMOSD, while offering the opportunity to identify clinically relevant neurological, immunological, and imaging markers.

Using SILAC to Develop Quantitative Data-Independent Acquisition (DIA) Proteomic Methods.

Proteins are integral to biological systems and functions. Identifying and quantifying proteins can therefore offer systems-wide insights into protein-protein interactions, cellular signaling, and biological pathway activity. The use of quantitative proteomics has become a method of choice for identifying and quantifying proteins in complex matrices. Proteomics allows researchers to survey hundreds to thousands of proteins in a less biased manner than classical antibody-based protein capture strategies. Typically, discovery approaches have used data-dependent acquisition (DDA) methods, but this approach suffers from stochasticity that compromises quantitation. Recent developments in data-independent acquisition (DIA) proteomics workflows enable proteomic profiling of thousands of samples with increased peak picking consistency making it an excellent candidate for discovering and assessing biomarkers in clinical samples. However, quantitation of peptides from DIA datasets is computationally intensive, and guidelines on how to establish DIA methods are lacking. Method development and optimization require novel tools to visualize and filter DIA datasets appropriately. Here, a protocol and novel script workflow for the optimization of quantitative DIA methods using stable isotope labeling of amino acids in culture (SILAC) are presented. This protocol includes steps for cell growth and labeling, peptide digestion and preparation, and optimization of quantitative DIA methods. In addition, important steps for (1) computational analysis to identify and quantify peptides, (2) data visualizations to identify the linear abundance ranges for all peptides in the sample, and (3) descriptions of how to find high confidence quantitation abundance thresholds are described herein.

Surfactant protein D is a biomarker of influenza-related pediatric lung injury.

BACKGROUND: Biomarkers that can risk-stratify children with influenza virus lower respiratory infection may identify patients for targeted intervention. Early elevation of alveolar-related proteins in the bloodstream in these patients could indicate more severe lung damage portending worse outcomes. METHODS: We used a mouse model of human influenza infection and evaluated relationships between lung pathophysiology and surfactant protein D (SP-D), SP-A, and Club cell protein 16 (CC16). We then measured SP-A, SP-D, and CC16 levels in plasma samples from 94 children with influenza-associated acute respiratory failure (PICFLU cohort), excluding children with underlying conditions explaining disease severity. We tested for associations between levels of circulating proteins and disease severity including the diagnosis of acute respiratory distress syndrome (ARDS), mechanical ventilator, intensive care unit and hospital days, and hospital mortality. RESULTS: Circulating SP-D showed a greater increase than SP-A and CC16 in mice with increased alveolar-vascular permeability following influenza infection. In the PICFLU cohort, SP-D was associated with moderate-severe ARDS diagnosis (p = 0.01) and with mechanical ventilator (r = 0.45, p = 0.002), ICU (r = 0.44, p = 0.002), and hospital days (r = 0.37, p = 0.001) in influenza-infected children without bacterial coinfection. Levels of SP-D were lower in children with secondary bacterial pneumonia (p = 0.01) and not associated with outcomes. CC16 and SP-A levels did not differ with bacterial coinfection and were not consistently associated with severe outcomes. CONCLUSIONS: SP-D has potential as an early circulating biomarker reflecting a degree of lung damage caused directly by influenza virus infection in children. Secondary bacterial pneumonia alters SP-D biomarker performance.

An Integrated Genomic, Proteomic, and Immunopeptidomic Approach to Discover Treatment-Induced Neoantigens.

All nucleated mammalian cells express major histocompatibility complex (MHC) proteins that present peptides on cell surfaces for immune surveillance. These MHC-presented peptides (pMHC) are necessary for directing T-cell responses against cells harboring non-self antigens derived from pathogens or from somatic mutations. Alterations in tumor-specific antigen repertoires - particularly novel MHC presentation of mutation-bearing peptides (neoantigens) - can be potent targets of anti-tumor immune responses. Here we employed an integrated genomic and proteomic antigen discovery strategy aimed at measuring how interferon gamma (IFN-γ) alters antigen presentation, using a human lymphoma cell line, GRANTA-519. IFN-γ treatment resulted in 126 differentially expressed proteins (2% of all quantified proteins), which included components of antigen presentation machinery and interferon signaling pathways, and MHC molecules themselves. In addition, several proteasome subunits were found to be modulated, consistent with previous reports of immunoproteasome induction by IFN-γ exposure. This finding suggests that a modest proteomic response to IFN-γ could create larger alteration to cells' antigen/epitope repertoires. Accordingly, MHC immunoprecipitation followed by mass spectrometric analysis of eluted peptide repertoires revealed exclusive signatures of IFN-γ induction, with 951 unique peptides reproducibly presented by MHC-I and 582 presented by MHC-II. Furthermore, an additional set of pMHCs including several candidate neoantigens, distinguished control and the IFN-γ samples by their altered relative abundances. Accordingly, we developed a classification system to distinguish peptides which are differentially presented due to altered expression from novel peptides resulting from changes in antigen processing. Taken together, these data demonstrate that IFN-γ can re-shape antigen repertoires by identity and by abundance. Extending this approach to models with greater clinical relevance could help develop strategies by which immunopeptide repertoires are intentionally reshaped to improve endogenous or vaccine-induced anti-tumor immune responses and potentially anti-viral immune responses.

Early Amplified Respiratory Bioactive Lipid Response Is Associated With Worse Outcomes in Pediatric Influenza-Related Respiratory Failure.

BACKGROUND: Biomarkers are needed for early identification of patients at risk of severe complications from influenza infection, including prolonged respiratory failure and death. Eicosanoids are bioactive lipid mediators with pro- and anti-inflammatory properties produced in response to infection. This study assessed the relationships between the host bioactive lipid response, influenza viral load, and clinical outcomes. METHODS: Influenza-positive, intubated children ≤18 years old were enrolled across 26 US pediatric intensive care units (PICUs). Mass spectrometry was used to measure >100 lipid metabolites in endotracheal and nasopharyngeal samples. Influenza viral load was measured by quantitative polymerase chain reaction. RESULTS: Age and bacterial co-infection were associated with multiple bioactive lipids (P < .05). Influenza viral load was lower in patients with bacterial co-infection compared with those without, and pro-inflammatory bioactive lipids positively correlated with viral load in bacterially co-infected children (P < .05). Lipids associated with disease resolution correlated with viral load in patients without bacterial co-infection (P < .01). After adjusting for age and bacterial co-infection status, elevated pro- and anti-inflammatory lipids measured early in the intensive care unit course were associated with higher mortality, whereas influenza viral load and endotracheal cytokine levels were not associated with clinical outcomes. Prostaglandin E2, arachidonic acid, docosahexaenoic acid, and 12-hydroxyeicosatetraenoic acid measured within 72 hours of PICU admission predicted death or prolonged (≥28 days) mechanical ventilator support (area under the curve, 0.72-0.79; P < .02) not explained by admission illness severity. CONCLUSIONS: Children with influenza-related complications have early bioactive lipid responses that may reflect lung disease severity. Respiratory bioactive lipids are candidate prognostic biomarkers to identify children with the most severe clinical outcomes.

Discovery and Qualification of Candidate Urinary Biomarkers of Disease Activity in Lupus Nephritis.

Lupus nephritis (LN) is a severe clinical manifestation of systemic lupus erythematosus (SLE) associated with significant morbidity and mortality. Assessment of severity and activity of renal involvement in SLE requires a kidney biopsy, an invasive procedure with limited prognostic value. Noninvasive biomarkers are needed to inform treatment decisions and to monitor disease activity. Proteinuria is associated with disease progression in LN; however, the composition of the LN urinary proteome remains incompletely characterized. To address this, we profiled LN urine samples using complementary mass spectrometry-based methods:  protein gel fractionation, chemical labeling using tandem mass tags, and data-independent acquisition. Combining results from these approaches yielded quantitative information on 2573 unique proteins in urine from LN patients. A multiple-reaction monitoring (MRM) method was established to confirm eight proteins in an independent cohort of LN patients, and seven proteins (transferrin, α-2-macroglobulin, haptoglobin, afamin, α-1-antitrypsin, vimentin, and ceruloplasmin) were confirmed to be elevated in LN urine compared to healthy controls. In this study, we demonstrate that deep mass spectrometry profiling of a small number of patient samples can identify high-quality biomarkers that replicate in an independent LN disease cohort. These biomarkers are being used to inform clinical biomarker strategies to support longitudinal and interventional studies focused on evaluating disease progression and treatment efficacy of novel LN therapeutics.

Uncovering a Dual Regulatory Role for Caspases During Endoplasmic Reticulum Stress-induced Cell Death.

Many diseases are associated with endoplasmic reticulum (ER) stress, which results from an accumulation of misfolded proteins. This triggers an adaptive response called the "unfolded protein response" (UPR), and prolonged exposure to ER stress leads to cell death. Caspases are reported to play a critical role in ER stress-induced cell death but the underlying mechanisms by which they exert their effect continue to remain elusive. To understand the role caspases play during ER stress, a systems level approach integrating analysis of the transcriptome, proteome, and proteolytic substrate profile was employed. This quantitative analysis revealed transcriptional profiles for most human genes, provided information on protein abundance for 4476 proteins, and identified 445 caspase substrates. Based on these data sets many caspase substrates were shown to be downregulated at the protein level during ER stress suggesting caspase activity inhibits their cellular function. Additionally, RNA sequencing revealed a role for caspases in regulation of ER stress-induced transcriptional pathways and gene set enrichment analysis showed expression of multiple gene targets of essential transcription factors to be upregulated during ER stress upon inhibition of caspases. Furthermore, these transcription factors were degraded in a caspase-dependent manner during ER stress. These results indicate that caspases play a dual role in regulating the cellular response to ER stress through both post-translational and transcriptional regulatory mechanisms. Moreover, this study provides unique insight into progression of the unfolded protein response into cell death, which may help identify therapeutic strategies to treat ER stress-related diseases.

SUMO deconjugation is required for arsenic-triggered ubiquitylation of PML.

Acute promyelocytic leukemia is characterized by a chromosomal translocation that produces an oncogenic fusion protein of the retinoic acid receptor α (RARα) and promyelocytic leukemia protein (PML). Arsenic trioxide chemotherapy of this cancer induces the PML moiety to organize nuclear bodies, where the oncoprotein is degraded. This process requires the participation of two SUMO paralogs (SUMO1 and SUMO2) to promote PML ubiquitylation mediated by the ubiquitin E3 ligase RNF4 and reorganization of PML nuclear bodies. We demonstrated that the ubiquitylation of PML required the SUMO deconjugation machinery, primarily the deconjugating enzyme SENP1, and was suppressed by expression of non-deconjugatable SUMO2. We hypothesized that constitutive SUMO2 conjugation and deconjugation occurred basally and that arsenic trioxide treatment caused the exchange of SUMO2 for SUMO1 on a fraction of Lys(65) in PML. On the basis of data obtained with mutational analysis and quantitative proteomics, we propose that the SUMO switch at Lys(65) of PML enhanced nuclear body formation, subsequent SUMO2 conjugation to Lys(160), and consequent RNF4-dependent ubiquitylation of PML. Our work provides insights into how the SUMO system achieves selective SUMO paralog modification and highlights the crucial role of SENPs in defining the specificity of SUMO signaling.

Transcription Factor ATF4 Induces NLRP1 Inflammasome Expression during Endoplasmic Reticulum Stress.

Perturbation of endoplasmic reticulum (ER) homeostasis triggers the ER stress response (also known as Unfolded Protein Response), a hallmark of many pathological disorders. However the connection between ER stress and inflammation remains largely unexplored. Recent data suggest that ER stress controls the activity of inflammasomes, key signaling platforms that mediate innate immune responses. Here we report that expression of NLRP1, a core inflammasome component, is specifically up-regulated during severe ER stress conditions in human cell lines. Both IRE1α and PERK, but not the ATF6 pathway, modulate NLRP1 gene expression. Furthermore, using mutagenesis, chromatin immunoprecipitation and CRISPR-Cas9-mediated genome editing technology, we demonstrate that ATF4 transcription factor directly binds to NLRP1 promoter during ER stress. Although involved in different types of inflammatory responses, XBP-1 splicing was not required for NLRP1 induction. This study provides further evidence that links ER stress with innate.

Proteomic tools for the characterization of cell death mechanisms in drug discovery.

This review focuses on the use of proteomic tools for the characterization of cell death mechanisms that have contributed to drug discovery efforts. Resistance to cell death plays a major role in the development of many diseases, including numerous types of malignancies. Using a multitude of proteomic approaches, including protein-protein interaction studies, phosphorylation site mapping, ubiquitination site identification, and differential quantitative approaches, various cellular death pathways such as apoptosis and necroptosis have been investigated. These studies have aided in the development of therapeutic strategies or allowed dissection of clinical results to evaluate the success of clinical trials in addition to contributing to our understanding of these biological pathways. Here, we address the new wave of discoveries enabled by advancements in mass spectrometric technology and bioinformatic infrastructure that will hopefully lead to clinically efficacious strategies to overcome resistance to apoptosis and therefore offer improved treatment options for patients.

Complementary methods for the identification of substrates of proteolysis.

Proteolysis describes the cleavage of proteins into smaller components, which in vivo occurs typically to either activate or impair the functionality of cellular proteins. Proteolysis can occur during cellular homeostasis or can be induced due to external stress stimuli such as heat, biological or chemical insult, and is mediated by the activity of cellular enzymes, namely, proteases. Proteolytic cleavage of proteins can influence protein activation by exposing an active site or disrupting inhibitor binding. Conversely, proteolytic cleavage of many proteins has also been shown to lead to protein degradation resulting in inactivation of the substrate. Thousands of proteolytic events are known to take place in regulated cellular processes such as apoptosis and pyroptosis, however, their individual contribution to these processes remains poorly understood. Additionally, many cellular homeostatic processes are regulated by proteolytic events, however, in some cases, few proteolytic substrates have been identified. To gain further insight into the mechanism of action of these cellular processes, and to characterize biomarkers of cell death and other pathological indications, it is imperative to utilize a complete arsenal of tools for studying proteolysis events in vivo and in vitro. In this chapter, we focus on alternative methodologies to N-terminomics for profiling substrates of proteolysis and describe an additional suite of tools including orthogonal biophysical separation techniques such as COFRADIC or GASSP, and affinity capture tools that can enrich for newly formed C-termini (C-terminomics) generated as a result of caspase-mediated proteolysis.

Combinatorial approach for large-scale identification of linked peptides from tandem mass spectrometry spectra.

The combination of chemical cross-linking and mass spectrometry has recently been shown to constitute a powerful tool for studying protein-protein interactions and elucidating the structure of large protein complexes. However, computational methods for interpreting the complex MS/MS spectra from linked peptides are still in their infancy, making the high-throughput application of this approach largely impractical. Because of the lack of large annotated datasets, most current approaches do not capture the specific fragmentation patterns of linked peptides and therefore are not optimal for the identification of cross-linked peptides. Here we propose a generic approach to address this problem and demonstrate it using disulfide-bridged peptide libraries to (i) efficiently generate large mass spectral reference data for linked peptides at a low cost and (ii) automatically train an algorithm that can efficiently and accurately identify linked peptides from MS/MS spectra. We show that using this approach we were able to identify thousands of MS/MS spectra from disulfide-bridged peptides through comparison with proteome-scale sequence databases and significantly improve the sensitivity of cross-linked peptide identification. This allowed us to identify 60% more direct pairwise interactions between the protein subunits in the 20S proteasome complex than existing tools on cross-linking studies of the proteasome complexes. The basic framework of this approach and the MS/MS reference dataset generated should be valuable resources for the future development of new tools for the identification of linked peptides.

A turn-key approach for large-scale identification of complex posttranslational modifications.

The conjugation of complex post-translational modifications (PTMs) such as glycosylation and Small Ubiquitin-like Modification (SUMOylation) to a substrate protein can substantially change the resulting peptide fragmentation pattern compared to its unmodified counterpart, making current database search methods inappropriate for the identification of tandem mass (MS/MS) spectra from such modified peptides. Traditionally it has been difficult to develop new algorithms to identify these atypical peptides because of the lack of a large set of annotated spectra from which to learn the altered fragmentation pattern. Using SUMOylation as an example, we propose a novel approach to generate large MS/MS training data from modified peptides and derive an algorithm that learns properties of PTM-specific fragmentation from such training data. Benchmark tests on data sets of varying complexity show that our method is 80-300% more sensitive than current state-of-the-art approaches. The core concepts of our method are readily applicable to developing algorithms for the identifications of peptides with other complex PTMs.

Peptide level immunoaffinity enrichment enhances ubiquitination site identification on individual proteins.

Ubiquitination is a process that involves the covalent attachment of the 76-residue ubiquitin protein through its C-terminal di-glycine (GG) to lysine (K) residues on substrate proteins. This post-translational modification elicits a wide range of functional consequences including targeting proteins for proteasomal degradation, altering subcellular trafficking events, and facilitating protein-protein interactions. A number of methods exist for identifying the sites of ubiquitination on proteins of interest, including site-directed mutagenesis and affinity-purification mass spectrometry (AP-MS). Recent publications have also highlighted the use of peptide-level immunoaffinity enrichment of K-GG modified peptides from whole cell lysates for global characterization of ubiquitination sites. Here we investigated the utility of this technique for focused mapping of ubiquitination sites on individual proteins. For a series of membrane-associated and cytoplasmic substrates including erbB-2 (HER2), Dishevelled-2 (DVL2), and T cell receptor α (TCRα), we observed that K-GG peptide immunoaffinity enrichment consistently yielded additional ubiquitination sites beyond those identified in protein level AP-MS experiments. To assess this quantitatively, SILAC-labeled lysates were prepared and used to compare the abundances of individual K-GG peptides from samples prepared in parallel. Consistently, K-GG peptide immunoaffinity enrichment yielded greater than fourfold higher levels of modified peptides than AP-MS approaches. Using this approach, we went on to characterize inducible ubiquitination on multiple members of the T-cell receptor complex that are functionally affected by endoplasmic reticulum (ER) stress. Together, these data demonstrate the utility of immunoaffinity peptide enrichment for single protein ubiquitination site analysis and provide insights into the ubiquitination of HER2, DVL2, and proteins in the T-cell receptor complex.

A Novel Peptide-Based SILAC Method to Identify the Posttranslational Modifications Provides Evidence for Unconventional Ubiquitination in the ER-Associated Degradation Pathway.

The endoplasmic reticulum-associated degradation (ERAD) pathway is responsible for disposing misfolded proteins from the endoplasmic reticulum by inducing their ubiquitination and degradation. Ubiquitination is conventionally observed on lysine residues and has been demonstrated on cysteine residues and protein N-termini. Ubiquitination is fundamental to the ERAD process; however, a mutant T-cell receptor α (TCRα) lacking lysine residues is targeted for the degradation by the ERAD pathway. We have shown that ubiquitination of lysine-less TCRα occurs on internal, non-lysine residues and that the same E3 ligase conjugates ubiquitin to TCRα in the presence or absence of lysine residues. Mass-spectrometry indicates that WT-TCRα is ubiquitinated on multiple lysine residues. Recent publications have provided indirect evidence that serine and threonine residues may be modified by ubiquitin. Using a novel peptide-based stable isotope labeling in cell culture (SILAC) approach, we show that specific lysine-less TCRα peptides become modified. In this study, we demonstrate that it is possible to detect both ester and thioester based ubiquitination events, although the exact linkage on lysine-less TCRα remains elusive. These findings demonstrate that SILAC can be used as a tool to identify modified peptides, even those with novel modifications that may not be detected using conventional proteomic work flows or informatics algorithms.

Complementary proteomic tools for the dissection of apoptotic proteolysis events.

Proteolysis is a key regulatory event that controls intracellular and extracellular signaling through irreversible changes in a protein's structure that greatly alters its function. Here we describe a platform for profiling caspase substrates which encompasses two highly complementary proteomic techniques--the first is a differential gel based approach termed Global Analyzer of SILAC-derived Substrates of Proteolysis (GASSP) and the second involves affinity enrichment of peptides containing a C-terminal aspartic acid residue. In combination, these techniques have enabled the profiling of a large cellular pool of apoptotic-mediated proteolytic events across a wide dynamic range. By applying this integrated proteomic work flow to analyze proteolytic events resulting from the induction of intrinsic apoptosis in Jurkat cells via etoposide treatment, 3346 proteins were quantified, of which 360 proteins were identified as etoposide-induced proteolytic substrates, including 160 previously assigned caspase substrates. In addition to global profiling, a targeted approach using BAX HCT116 isogenic cell lines was utilized to dissect pre- and post-mitochondrial extrinsic apoptotic cleavage events. By employing apoptotic activation with a pro-apoptotic receptor agonist (PARA), a limited set of apoptotic substrates including known caspase substrates such as BH3 interacting-domain death agonist (BID) and Poly (ADP-ribose) polymerase (PARP)-1, and novel substrates such as Basic Transcription Factor 3, TRK-fused gene protein (TFG), and p62/Sequestosome were also identified.