A TRIM21-based bioPROTAC highlights the therapeutic benefit of HuR degradation.

Human antigen R (HuR) is a ubiquitously expressed RNA-binding protein, which functions as an RNA regulator. Overexpression of HuR correlates with high grade tumours and poor patient prognosis, implicating it as an attractive therapeutic target. However, an effective small molecule antagonist to HuR for clinical use remains elusive. Here, a single domain antibody (VHH) that binds HuR with low nanomolar affinity was identified and shown to inhibit HuR binding to RNA. This VHH was used to engineer a TRIM21-based biological PROTAC (bioPROTAC) that could degrade endogenous HuR. Significantly, HuR degradation reverses the tumour-promoting properties of cancer cells in vivo by altering the HuR-regulated proteome, highlighting the benefit of HuR degradation and paving the way for the development of HuR-degrading therapeutics. These observations have broader implications for degrading intractable therapeutic targets, with bioPROTACs presenting a unique opportunity to explore targeted-protein degradation through a modular approach.

Multiomics links global surfactant dysregulation with airflow obstruction and emphysema in COPD.

Rationale: Pulmonary surfactant is vital for lung homeostasis as it reduces surface tension to prevent alveolar collapse and provides essential immune-regulatory and antipathogenic functions. Previous studies demonstrated dysregulation of some individual surfactant components in COPD. We investigated relationships between COPD disease measures and dysregulation of surfactant components to gain new insights into potential disease mechanisms. Methods: Bronchoalveolar lavage proteome and lipidome were characterised in ex-smoking mild/moderate COPD subjects (n=26) and healthy ex-smoking (n=20) and never-smoking (n=16) controls using mass spectrometry. Serum surfactant protein analysis was performed. Results: Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, surfactant protein (SP)-B, SP-A and SP-D concentrations were lower in COPD versus controls (log2 fold change (log2FC) -2.0, -2.2, -1.5, -0.5, -0.7 and -0.5 (adjusted p<0.02), respectively) and correlated with lung function. Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D, napsin A and CD44 inversely correlated with computed tomography small airways disease measures (expiratory to inspiratory mean lung density) (r= -0.56, r= -0.58, r= -0.45, r= -0.36, r= -0.44, r= -0.37, r= -0.40 and r= -0.39 (adjusted p<0.05)). Total phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, SP-A, SP-B, SP-D and NAPSA inversely correlated with emphysema (% low-attenuation areas): r= -0.55, r= -0.61, r= -0.48, r= -0.51, r= -0.41, r= -0.31 and r= -0.34, respectively (adjusted p<0.05). Neutrophil elastase, known to degrade SP-A and SP-D, was elevated in COPD versus controls (log2FC 0.40, adjusted p=0.0390), and inversely correlated with SP-A and SP-D. Serum SP-D was increased in COPD versus healthy ex-smoking volunteers, and predicted COPD status (area under the curve 0.85). Conclusions: Using a multiomics approach, we demonstrate, for the first time, global surfactant dysregulation in COPD that was associated with emphysema, giving new insights into potential mechanisms underlying the cause or consequence of disease.

Comparative systeomics to elucidate physiological differences between CHO and SP2/0 cell lines.

Omics-based tools were coupled with bioinformatics for a systeomics analysis of two biopharma cell types: Chinese hamster ovary (M-CHO and CHO-K1) and SP2/0. Exponential and stationary phase samples revealed more than 10,000 transcripts and 6000 proteins across these two manufacturing cell lines. A statistical comparison of transcriptomics and proteomics data identified downregulated genes involved in protein folding, protein synthesis and protein metabolism, including PPIA-cyclophilin A, HSPD1, and EIF3K, in M-CHO compared to SP2/0 while cell cycle and actin cytoskeleton genes were reduced in SP2/0. KEGG pathway comparisons revealed glycerolipids, glycosphingolipids, ABC transporters, calcium signaling, cell adhesion, and secretion pathways depleted in M-CHO while retinol metabolism was upregulated. KEGG and IPA also indicated apoptosis, RNA degradation, and proteosomes enriched in CHO stationary phase. Alternatively, gene ontology analysis revealed an underrepresentation in ion and potassium channel activities, membrane proteins, and secretory granules including Stxbpt2, Syt1, Syt9, and Cma1 proteins in M-CHO. Additional enrichment strategies involving ultracentrifugation, biotinylation, and hydrazide chemistry identified over 4000 potential CHO membrane and secretory proteins, yet many secretory and membrane proteins were still depleted. This systeomics pipeline has revealed bottlenecks and potential opportunities for cell line engineering in CHO and SP2/0 to improve their production capabilities.

Mitochondrial membrane potential-enriched CHO host: a novel and powerful tool for improving biomanufacturing capability.

With the aim of increasing protein productivity of Chinese hamster ovary (CHO) cells, we sought to generate new CHO hosts with favorable biomanufacturing phenotypes and improved functionality. Here, we present an innovative approach of enriching the CHO host cells with a high mitochondrial membrane potential (MMP). Stable transfectant pools and clonal cell lines expressing difficult-to-express bispecific molecules generated from the MMP-enriched host outperformed the parental host by displaying (1) improved fed-batch productivity; (2) enhanced long-term cell viability of pools; (3) more favorable lactate metabolism; and (4) improved cell cloning efficiency during monoclonal cell line generation. Proteomic analysis together with Western blot validation were used to investigate the underlying mechanisms by which high MMP influenced production performance. The MMP-enriched host exhibited multifaceted protection against mitochondrial dysfunction and endoplasmic reticulum stress. Our findings indicate that the MMP-enriched host achieved an overall "fitter" phenotype that contributes to the significant improvement in biomanufacturing capability.

Characterization of Citrullination Sites in Neutrophils and Mast Cells Activated by Ionomycin via Integration of Mass Spectrometry and Machine Learning.

Citrullination is an important post-translational modification implicated in many diseases including rheumatoid arthritis (RA), Alzheimer's disease, and cancer. Neutrophil and mast cells have different expression profiles for protein-arginine deiminases (PADs), and ionomycin-induced activation makes them an ideal cellular model to study proteins susceptible to citrullination. We performed high-resolution mass spectrometry and stringent data filtration to identify citrullination sites in neutrophil and mast cells treated with and without ionomycin. We identified a total of 833 validated citrullination sites on 395 proteins. Several of these citrullinated proteins are important components of pathways involved in innate immune responses. Using this benchmark primary sequence data set, we developed machine learning models to predict citrullination in neutrophil and mast cell proteins. We show that our models predict citrullination likelihood with 0.735 and 0.766 AUCs (area under the receiver operating characteristic curves), respectively, on independent validation sets. In summary, this study provides the largest number of validated citrullination sites in neutrophil and mast cell proteins. The use of our novel motif analysis approach to predict citrullination sites will facilitate the discovery of novel protein substrates of protein-arginine deiminases (PADs), which may be key to understanding immunopathologies of various diseases.

Resistance to Pyrrolobenzodiazepine Dimers Is Associated with SLFN11 Downregulation and Can Be Reversed through Inhibition of ATR.

Resistance to antibody-drug conjugates (ADCs) has been observed in both preclinical models and clinical studies. However, mechanisms of resistance to pyrrolobenzodiazepine (PBD)-conjugated ADCs have not been well characterized and thus, this study was designed to investigate development of resistance to PBD dimer warheads and PBD-conjugated ADCs. We established a PBD-resistant cell line, 361-PBDr, by treating human breast cancer MDA-MB-361 cells with gradually increasing concentrations of SG3199, the PBD dimer released from the PBD drug-linker tesirine. 361-PBDr cells were over 20-fold less sensitive to SG3199 compared with parental cells and were cross-resistant to other PBD warhead and ADCs conjugated with PBDs. Proteomic profiling revealed that downregulation of Schlafen family member 11 (SLFN11), a putative DNA/RNA helicase, sensitizing cancer cells to DNA-damaging agents, was associated with PBD resistance. Confirmatory studies demonstrated that siRNA knockdown of SLFN11 in multiple tumor cell lines conferred reduced sensitivity to SG3199 and PBD-conjugated ADCs. Treatment with EPZ011989, an EZH2 inhibitor, derepressed SLFN11 expression in 361-PBDr and other SLFN11-deficient tumor cells, and increased sensitivity to PBD and PBD-conjugated ADCs, indicating that the suppression of SLFN11 expression is associated with histone methylation as reported. Moreover, we demonstrated that combining an ataxia telangiectasia and Rad3-related protein (ATR) inhibitor, AZD6738, with SG3199 or PBD-based ADCs led to synergistic cytotoxicity in either resistant 361-PBDr cells or cells that SLFN11 was knocked down via siRNA. Collectively, these data provide insights into potential development of resistance to PBDs and PBD-conjugated ADCs, and more importantly, inform strategy development to overcome such resistance.

Development of a Parallel Reaction Monitoring Mass Spectrometry Assay for the Detection of SARS-CoV-2 Spike Glycoprotein and Nucleoprotein.

There is an urgent need for robust and high-throughput methods for SARS-CoV-2 detection in suspected patient samples to facilitate disease management, surveillance, and control. Although nucleic acid detection methods such as reverse transcription polymerase chain reaction (RT-PCR) are the gold standard, during the current pandemic, the deployment of RT-PCR tests has been extremely slow, and key reagents such as PCR primers and RNA extraction kits are at critical shortages. Rapid point-of-care viral antigen detection methods have been previously employed for the diagnosis of respiratory viruses such as influenza and respiratory syncytial viruses. Therefore, the direct detection of SARS-CoV-2 viral antigens in patient samples could also be used for diagnosis of active infection, and alternative methodologies for specific and sensitive viral protein detection should be explored. Targeted mass spectrometry techniques have enabled the identification and quantitation of a defined subset of proteins/peptides at single amino acid resolution with attomole level sensitivity and high reproducibility. Herein, we report a targeted mass spectrometry assay for the detection of SARS-CoV-2 spike protein and nucleoprotein in a relevant biological matrix. Recombinant full-length spike protein and nucleoprotein were digested and proteotypic peptides were selected for parallel reaction monitoring (PRM) quantitation using a high-resolution Orbitrap instrument. A spectral library, which contained seven proteotypic peptides (four from spike protein and three from nucleoprotein) and the top three to four transitions, was generated and evaluated. From the original spectral library, we selected two best performing peptides for the final PRM assay. The assay was evaluated using mock test samples containing inactivated SARS-CoV-2 virions, added to in vitro derived mucus. The PRM assay provided a limit of detection of ∼200 attomoles and a limit of quantitation of ∼ 390 attomoles. Extrapolating from the test samples, the projected titer of virus particles necessary for the detection of SARS-CoV-2 spike and nucleoprotein detection was approximately 2 × 105 viral particles/mL, making it an attractive alternative to RT-PCR assays. Potentially, mass spectrometry-based methods for viral antigen detection may deliver higher throughput and could serve as a complementary diagnostic tool to RT-PCR. Furthermore, this assay could be used to evaluate the presence of SARS-CoV-2 in archived or recently collected biological fluids, in vitro-derived research materials, and wastewater samples.

Actomyosin contraction during cellularization is regulated in part by Src64 control of Actin 5C protein levels.

Src64 is required for actomyosin contraction during cellularization of the Drosophila embryonic blastoderm. The mechanism of actomyosin ring constriction is poorly understood even though a number of cytoskeletal regulators have been implicated in the assembly, organization, and contraction of these microfilament rings. How these cytoskeletal processes are regulated during development is even less well understood. To investigate the role of Src64 as an upstream regulator of actomyosin contraction, we conducted a proteomics screen to identify proteins whose expression levels are controlled by src64. Global levels of actin are reduced in src64 mutant embryos. Furthermore, we show that reduction of the actin isoform Actin 5C causes defects in actomyosin contraction during cellularization similar to those caused by src64 mutation, indicating that a relatively high level of Actin 5C is required for normal actomyosin contraction and furrow canal structure. However, reduction of Actin 5C levels only slows down actomyosin ring constriction rather than preventing it, suggesting that src64 acts not only to modulate actin levels, but also to regulate the actomyosin cytoskeleton by other means.

Staphylococcus aureus drives expansion of low-density neutrophils in diabetic mice.

Diabetic individuals are at considerable risk for invasive infection by Staphylococcus aureus, however, the mechanisms underlying this enhanced susceptibility to infection are unclear. We observed increased mortality following i.v. S. aureus infection in diabetic mice compared with nondiabetic controls, correlating with increased numbers of low-density neutrophils (LDNs) and neutrophil extracellular traps (NETs). LDNs have been implicated in the inflammatory pathology of diseases such as lupus, given their release of large amounts of NETs. Our goal was to describe what drives LDN increases during S. aureus infection in the diabetic host and mechanisms that promote increased NET production by LDNs. LDN development is dependent on TGF-ß, which we found to be more activated in the diabetic host. Neutralization of TGF-ß, or the TGF-ß-activating integrin αvß8, reduced LDN numbers and improved survival during S. aureus infection. Targeting S. aureus directly with MEDI4893*, an α toxin-neutralizing monoclonal antibody, blocked TGF-ß activation, reduced LDNs and NETs, and significantly improved survival. A comparison of gene and protein expression in high-density neutrophils and LDNs identified increased GPCRs and elevated phosphatase and tensin homolog (PTEN) in the LDN subset. Inhibition of PTEN improved the survival of infected diabetic mice. Our data identify a population of neutrophils in infected diabetic mice that correlated with decreased survival and increased NET production and describe 3 therapeutic targets, a bacterial target and 2 host proteins, that prevented NET production and improved survival.

Proteogenomic Annotation of Chinese Hamsters Reveals Extensive Novel Translation Events and Endogenous Retroviral Elements.

A high-quality genome annotation greatly facilitates successful cell line engineering. Standard draft genome annotation pipelines are based largely on de novo gene prediction, homology, and RNA-Seq data. However, draft annotations can suffer from incorrect predictions of translated sequence, inaccurate splice isoforms, and missing genes. Here, we generated a draft annotation for the newly assembled Chinese hamster genome and used RNA-Seq, proteomics, and Ribo-Seq to experimentally annotate the genome. We identified 3529 new proteins compared to the hamster RefSeq protein annotation and 2256 novel translational events (e.g., alternative splices, mutations, and novel splices). Finally, we used this pipeline to identify the source of translated retroviruses contaminating recombinant products from Chinese hamster ovary (CHO) cell lines, including 119 type-C retroviruses, thus enabling future efforts to eliminate retroviruses to reduce the costs incurred with retroviral particle clearance. In summary, the improved annotation provides a more accurate resource for CHO cell line engineering, by facilitating the interpretation of omics data, defining of cellular pathways, and engineering of complex phenotypes.

Correction to: Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

[This corrects the article DOI: 10.1186/s12014-018-9197-x.].

Evidence for a direct link between PAD4-mediated citrullination and the oxidative burst in human neutrophils.

Neutrophils are critical for the defense against pathogens, in part through the extrusion of extracellular DNA traps, phagocytosis, and the production of reactive oxygen species. Neutrophils may also play an important role in the pathogenesis of rheumatoid arthritis (RA) through the activation of protein arginine deiminases (PADs) that citrullinate proteins that subsequently act as autoantigens. We report that PAD4 is physically associated with the cytosolic subunits of the oxidative burst machinery, p47phox (also known as neutrophil cytosol factor 1, NCF1) and p67phox (NCF2). Activation of PAD4 by membranolytic insults that result in high levels of intracellular calcium (higher than physiological neutrophil activation) leads to rapid citrullination of p47phox/NCF1 and p67phox/NCF2, as well as their dissociation from PAD4. This dissociation prevents the assembly of an active NADPH oxidase complex and an oxidative burst in neutrophils stimulated by phorbol-ester or immune complexes. In further support of a substrate-to-inactive enzyme interaction, small-molecule PAD inhibitors also disrupt the PAD4-NCF complex and reduce oxidase activation and phagocytic killing of Staphylococcus aureus. This novel role of PAD4 in the regulation of neutrophil physiology suggests that targeting PAD4 with active site inhibitors for the treatment of RA may have a broader impact on neutrophil biology than just inhibition of citrullination.

Quantitative phosphoproteomic analysis reveals reciprocal activation of receptor tyrosine kinases between cancer epithelial cells and stromal fibroblasts.

BACKGROUND: Cancer-associated fibroblasts (CAFs) are one of the most important components of tumor stroma and play a key role in modulating tumor growth. However, a mechanistic understanding of how CAFs communicate with tumor cells to promote their proliferation and invasion is far from complete. A major reason for this is that most current techniques and model systems do not capture the complexity of signal transduction that occurs between CAFs and tumor cells. METHODS: In this study, we employed a stable isotope labeling with amino acids in cell culture (SILAC) strategy to label invasive breast cancer cells, MDA-MB-231, and breast cancer patient-derived CAF this has already been defined above cells. We used an antibody-based phosphotyrosine peptide enrichment method coupled to LC-MS/MS to catalog and quantify tyrosine phosphorylation-mediated signal transduction events induced by the bidirectional communication between patient-derived CAFs and tumor cells. RESULTS: We discovered that distinct signaling events were activated in CAFs and in tumor epithelial cells during the crosstalk between these two cell types. We identified reciprocal activation of a number of receptor tyrosine kinases including EGFR, FGFR1 and EPHA2 induced by this bidirectional communication. CONCLUSIONS: Our study not only provides insights into the mechanisms of the interaction between CAFs and tumor cells, but the model system described here could be used as a prototype for analysis of intercellular communication in many different tumor microenvironments.

A novel bicistronic gene design couples stable cell line selection with a fucose switch in a designer CHO host to produce native and afucosylated glycoform antibodies.

The conserved glycosylation site Asn297 of a monoclonal antibody (mAb) can be decorated with a variety of sugars that can alter mAb pharmacokinetics and recruitment of effector proteins. Antibodies lacking the core fucose at Asn297 (afucosylated mAbs) show enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and increased efficacy. Here, we describe the development of a robust platform for the manufacture of afucosylated therapeutic mAbs by engineering a Chinese hamster ovary (CHO) host cell line to co-express a mAb with GDP-6-deoxy-D-lyxo-4-hexulose reductase (RMD), a prokaryotic enzyme that deflects an intermediate in the de novo synthesis of fucose to a dead-end product, resulting in the production of afucosylated mAb (GlymaxX™ Technology, ProBioGen). Expression of the mAb and RMD genes was coordinated by co-transfection of separate mAb and RMD vectors or use of an internal ribosome entry site (IRES) element to link the translation of RMD with either the glutamine synthase selection marker or the mAb light chain. The GS-IRES-RMD vector format was more suitable for the rapid generation of high yielding cell lines, secreting afucosylated mAb with titers exceeding 6.0 g/L. These cell lines maintained production of afucosylated mAb over 60 generations, ensuring their suitability for use in large-scale manufacturing. The afucosylated mAbs purified from these RMD-engineered cell lines showed increased binding in a CD16 cellular assay, demonstrating enhancement of ADCC compared to fucosylated control mAb. Furthermore, the afucosylation in these mAbs could be controlled by simple addition of L-fucose in the culture medium, thereby allowing the use of a single cell line for production of the same mAb in fucosylated and afucosylated formats for multiple therapeutic indications.

Reverse and Random Decoy Methods for False Discovery Rate Estimation in High Mass Accuracy Peptide Spectral Library Searches.

Spectral library searching (SLS) is an attractive alternative to sequence database searching (SDS) for peptide identification due to its speed, sensitivity, and ability to include any selected mass spectra. While decoy methods for SLS have been developed for low mass accuracy peptide spectral libraries, it is not clear that they are optimal or directly applicable to high mass accuracy spectra. Therefore, we report the development and validation of methods for high mass accuracy decoy libraries. Two types of decoy libraries were found to be suitable for this purpose. The first, referred to as Reverse, constructs spectra by reversing a library's peptide sequences except for the C-terminal residue. The second, termed Random, randomly replaces all non-C-terminal residues and either retains the original C-terminal residue or replaces it based on the amino-acid frequency of the library's C-terminus. In both cases the m/z values of fragment ions are shifted accordingly. Determination of FDR is performed in a manner equivalent to SDS, concatenating a library with its decoy prior to a search. The utility of Reverse and Random libraries for target-decoy SLS in estimating false-positives and FDRs was demonstrated using spectra derived from a recently published synthetic human proteome project (Zolg, D. P.; et al. Nat. Methods 2017, 14, 259-262). For data sets from two large-scale label-free and iTRAQ experiments, these decoy building methods yielded highly similar score thresholds and spectral identifications at 1% FDR. The results were also found to be equivalent to those of using the decoy-free PeptideProphet algorithm. Using these new methods for FDR estimation, MSPepSearch, which is freely available search software, led to 18% more identifications at 1% FDR and 23% more at 0.1% FDR when compared with other widely used SDS engines coupled to postprocessing approaches such as Percolator. An application of these methods for FDR estimation for the recently reported "hybrid" library search (Burke, M. C.; et al. J. Proteome Res. 2017, 16, 1924-1935) method is also made. The application of decoy methods for high mass accuracy SLS permits the merging of these results with those of SDS, thereby increasing the assignment of more peptides, leading to deeper proteome coverage.

Spontaneous Secretion of the Citrullination Enzyme PAD2 and Cell Surface Exposure of PAD4 by Neutrophils.

Autoantibodies directed against citrullinated epitopes of proteins are highly diagnostic of rheumatoid arthritis (RA), and elevated levels of protein citrullination can be found in the joints of patients with RA. Calcium-dependent peptidyl-arginine deiminases (PAD) are the enzymes responsible for citrullination. PAD2 and PAD4 are enriched in neutrophils and likely drive citrullination under inflammatory conditions. PADs may be released during NETosis or cell death, but the mechanisms responsible for PAD activity under physiological conditions have not been fully elucidated. To understand how PADs citrullinate extracellular proteins, we investigated the cellular localization and activity of PAD2 and PAD4, and we report that viable neutrophils from healthy donors have active PAD4 exposed on their surface and spontaneously secrete PAD2. Neutrophil activation by some stimulatory agents increased the levels of immunoreactive PAD4 on the cell surface, and some stimuli reduced PAD2 secretion. Our data indicate that live neutrophils have the inherent capacity to express active extracellular PADs. These novel pathways are distinguished from intracellular PAD activation during NETosis and calcium influx-mediated hypercitrullination. Our study implies that extracellular PADs may have a physiological role under non-pathogenic conditions as well as a pathological role in RA.

Lessons from the Hamster: Cricetulus griseus Tissue and CHO Cell Line Proteome Comparison.

Chinese hamster ovary cells represent the dominant host for therapeutic recombinant protein production. However, few large-scale data sets have been generated to characterize this host organism and derived CHO cell lines at the proteomics level. Consequently, an extensive label-free quantitative proteomics analysis of two cell lines (CHO-S and CHO DG44) and two Chinese hamster tissues (liver and ovary) was used to identify a total of 11 801 unique proteins containing at least two unique peptides. 9359 unique proteins were identified specifically in the cell lines, representing a 56% increase over previous work. Additionally, 6663 unique proteins were identified across liver and ovary tissues, providing the first Chinese hamster tissue proteome. Protein expression was more conserved within cell lines during both growth phases than across cell lines, suggesting large genetic differences across cell lines. Overall, both gene ontology and KEGG pathway analysis revealed enrichment of cell-cycle activity in cells. In contrast, upregulated molecular functions in tissue include glycosylation and lipid transporter activity. Furthermore, cellular components including Golgi apparatus are upregulated in both tissues. In conclusion, this large-scale proteomics analysis enables us to delineate specific changes between tissues and cells derived from these tissues, which can help explain specific tissue function and the adaptations cells incur for applications in biopharmaceutical productions.

Statistical Models for the Analysis of Isobaric Tags Multiplexed Quantitative Proteomics.

Mass spectrometry is being used to identify protein biomarkers that can facilitate development of drug treatment. Mass spectrometry-based labeling proteomic experiments result in complex proteomic data that is hierarchical in nature often with small sample size studies. The generalized linear model (GLM) is the most popular approach in proteomics to compare protein abundances between groups. However, GLM does not address all the complexities of proteomics data such as repeated measures and variance heterogeneity. Linear models for microarray data (LIMMA) and mixed models are two approaches that can address some of these data complexities to provide better statistical estimates. We compared these three statistical models (GLM, LIMMA, and mixed models) under two different normalization approaches (quantile normalization and median sweeping) to demonstrate when each approach is the best for tagged proteins. We evaluated these methods using a spiked-in data set of known protein abundances, a systemic lupus erythematosus (SLE) data set, and simulated data from multiplexed labeling experiments that use tandem mass tags (TMT). Data are available via ProteomeXchange with identifier PXD005486. We found median sweeping to be a preferred approach of data normalization, and with this normalization approach there was overlap with findings across all methods with GLM being a subset of mixed models. The conclusion is that the mixed model had the best type I error with median sweeping, whereas LIMMA had the better overall statistical properties regardless of normalization approaches.

Post-Translational Modifications (PTMs), Identified on Endogenous Huntingtin, Cluster within Proteolytic Domains between HEAT Repeats.

Post-translational modifications (PTMs) of proteins regulate various cellular processes. PTMs of polyglutamine-expanded huntingtin (Htt) protein, which causes Huntington's disease (HD), are likely modulators of HD pathogenesis. Previous studies have identified and characterized several PTMs on exogenously expressed Htt fragments, but none of them were designed to systematically characterize PTMs on the endogenous full-length Htt protein. We found that full-length endogenous Htt, which was immunoprecipitated from HD knock-in mouse and human post-mortem brain, is suitable for detection of PTMs by mass spectrometry. Using label-free and mass tag labeling-based approaches, we identified near 40 PTMs, of which half are novel (data are available via ProteomeXchange with identifier PXD005753). Most PTMs were located in clusters within predicted unstructured domains rather than within the predicted α-helical structured HEAT repeats. Using quantitative mass spectrometry, we detected significant differences in the stoichiometry of several PTMs between HD and WT mouse brain. The mass-spectrometry identification and quantitation were verified using phospho-specific antibodies for selected PTMs. To further validate our findings, we introduced individual PTM alterations within full-length Htt and identified several PTMs that can modulate its subcellular localization in striatal cells. These findings will be instrumental in further assembling the Htt PTM framework and highlight several PTMs as potential therapeutic targets for HD.

Quantitative Tyrosine Phosphoproteomics of Epidermal Growth Factor Receptor (EGFR) Tyrosine Kinase Inhibitor-treated Lung Adenocarcinoma Cells Reveals Potential Novel Biomarkers of Therapeutic Response.

Mutations in the Epidermal growth factor receptor (EGFR) kinase domain, such as the L858R missense mutation and deletions spanning the conserved sequence 747LREA750, are sensitive to tyrosine kinase inhibitors (TKIs). The gatekeeper site residue mutation, T790M accounts for around 60% of acquired resistance to EGFR TKIs. The first generation EGFR TKIs, erlotinib and gefitinib, and the second generation inhibitor, afatinib are FDA approved for initial treatment of EGFR mutated lung adenocarcinoma. The predominant biomarker of EGFR TKI responsiveness is the presence of EGFR TKI-sensitizing mutations. However, 30-40% of patients with EGFR mutations exhibit primary resistance to these TKIs, underscoring the unmet need of identifying additional biomarkers of treatment response. Here, we sought to characterize the dynamics of tyrosine phosphorylation upon EGFR TKI treatment of mutant EGFR-driven human lung adenocarcinoma cell lines with varying sensitivity to EGFR TKIs, erlotinib and afatinib. We employed stable isotope labeling with amino acids in cell culture (SILAC)-based quantitative mass spectrometry to identify and quantify tyrosine phosphorylated peptides. The proportion of tyrosine phosphorylated sites that had reduced phosphorylation upon erlotinib or afatinib treatment correlated with the degree of TKI-sensitivity. Afatinib, an irreversible EGFR TKI, more effectively inhibited tyrosine phosphorylation of a majority of the substrates. The phosphosites with phosphorylation SILAC ratios that correlated with the TKI-sensitivity of the cell lines include sites on kinases, such as EGFR-Y1197 and MAPK7-Y221, and adaptor proteins, such as SHC1-Y349/350, ERRFI1-Y394, GAB1-Y689, STAT5A-Y694, DLG3-Y705, and DAPP1-Y139, suggesting these are potential biomarkers of TKI sensitivity. DAPP1, is a novel target of mutant EGFR signaling and Y-139 is the major site of DAPP1 tyrosine phosphorylation. We also uncovered several off-target effects of these TKIs, such as MST1R-Y1238/Y1239 and MET-Y1252/1253. This study provides unique insight into the TKI-mediated modulation of mutant EGFR signaling, which can be applied to the development of biomarkers of EGFR TKI response.