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Stress and injury to the retinal pigment epithelium (RPE) often lead to dedifferentiation and epithelial-to-mesenchymal transition (EMT). These processes have been implicated in several retinal diseases, including proliferative vitreoretinopathy, diabetic retinopathy, and age-related macular degeneration. Despite the importance of RPE-EMT and the large body of data characterizing malignancy-related EMT, comprehensive proteomic studies to define the protein changes and pathways underlying RPE-EMT have not been reported. This study sought to investigate the temporal protein expression changes that occur in a human-induced pluripotent stem cell–based RPE-EMT model. We utilized multiplexed isobaric tandem mass tag labeling followed by high-resolution tandem MS for precise and in-depth quantification of the RPE-EMT proteome. We have identified and quantified 7937 protein groups in our tandem mass tag–based MS analysis. We observed a total of 532 proteins that are differentially regulated during RPE-EMT. Furthermore, we integrated our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into RPE-EMT mechanisms. To validate these results, we have performed a label-free single-shot data-independent acquisition MS study. Our integrated analysis indicates both the commonality and uniqueness of RPE-EMT compared with malignancy-associated EMT. Our comparative analysis also revealed that multiple age-related macular degeneration–associated risk factors are differentially regulated during RPE-EMT. Together, our integrated dataset provides a comprehensive RPE-EMT atlas and resource for understanding the molecular signaling events and associated biological pathways that underlie RPE-EMT onset. This resource has already facilitated the identification of chemical modulators that could inhibit RPE-EMT, and it will hopefully aid in ongoing efforts to develop EMT inhibition as an approach for the treatment of retinal disease., Graphical Abstract, Highlights • Proteomics data were integrated with prior transcriptomic (RNA-Seq) data on RPE-EMT. • Dysregulated RPE-EMT proteome shares commonality with malignancy-associated EMT. • Altered RPE-EMT proteome signatures correlated with known AMD-associated risk factors. • Protein kinases and phosphatases crosstalk modulate RPE-EMT., In Brief EMT can play a role in retinal diseases. Here, we present a comprehensive proteomic analysis aimed at defining the temporal protein expression changes associated with EMT of stem cell–derived retinal pigment epithelial cells. Tandem mass tag and direct data-independent acquisition MS approaches were performed after inducing RPE-EMT by enzymatic dissociation. We present integration of our proteomic data with prior transcriptomic (RNA-Seq) data to provide additional insights into the RPE-EMT progression.

The mitogen-activated protein kinase pathway is one of the most frequently altered pathways in cancer. It is involved in the control of cell proliferation, invasion, and metabolism, and can cause resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer, and glioma, are driven by a constitutively activating missense mutation (V600E) in the v-Raf murine sarcoma viral oncogene homolog B (BRAF) component of the pathway. Mitogen-activated protein kinase kinase (MEK) inhibition is initially effective in targeting these cancers, but reflexive activation of mammalian target of rapamycin (mTOR) signaling contributes to frequent therapy resistance. We have previously demonstrated that combination treatment with the MEK inhibitor trametinib and the dual mammalian target of rapamycin complex 1/2 inhibitor TAK228 improves survival and decreases vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of this combination therapy and understand the ensuing tumor response, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228. We identified 13,313 proteins and 30,928 localized phosphosites, of which 12,526 proteins and 17,444 phosphosites were quantified across all samples (data available via ProteomeXchange; identifier PXD022329). We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the mitogen-activated protein kinase and mTOR pathways. Combination therapy also increased apoptotic signaling, suppressed angiogenesis signaling, and broadly suppressed the activity of the cyclin-dependent kinases. In response to combination therapy, both epidermal growth factor receptor and class 1 histone deacetylase proteins were activated. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies new targets for the development of rational combination therapies for BRAF-driven tumors., Graphical Abstract, Highlights • Large-scale proteomic and phosphoproteomic analysis of an in vivo BRAFV600E glioma model. • In-depth analysis of tumor and stromal cellular response to protein kinase inhibitors. • Insights into acquired resistance and alternative therapeutic approaches., In Brief BRAFV600E is a key oncogenic driver in glioma, melanoma, and colon cancer. These tumors escape mitogen-activated protein kinase pathway inhibition by upregulating mammalian target of rapamycin signaling. Using comprehensive unbiased proteomic and phosphoproteomic analysis of an in vivo BRAFV600E mutant glioma model treated with inhibitors of both these key pathways, we characterize the tumor and stromal response and suggest additional therapeutic targets for BRAF-driven cancers, including epidermal growth factor receptor and class 1 histone deacetylases.

The extracellular matrix is a key component of tissues, yet it is underrepresented in proteomic datasets. Identification and evaluation of proteins in the extracellular matrix (ECM) has proved challenging due to the insolubility of many ECM proteins in traditional protein extraction buffers. Here we separate the decellularization and ECM extraction steps of several prominent methods for evaluation under real-world conditions. The results are used to optimize a two-fraction ECM extraction method. Approximately one dozen additional parameters are tested, and recommendations for analysis based on overall ECM coverage or specific ECM classes are given. Compared with a standard in-solution digest, the optimized method yielded a fourfold improvement in unique ECM peptide identifications., Graphical abstract, Highlights • Decellularization drastically improves ECM coverage. • ECM coverage is the highest with a dual chemical/enzymatic digestion method. • SPEED is the best tested single-shot method for ECM identification. • A fourfold improvement in ECM peptide IDs was achieved compared with standard methods., Due to their insolubility, many extracellular matrix (ECM) proteins are resistant to typical extraction and require optimized extraction methods for proteomic analysis. A wide variety of decellularization and ECM extraction methods have been published, but it remains unclear how these methods perform compared with one another on a complex, ECM-rich sample. A direct comparison of both cell and ECM extraction methods on a whole organism sample and four additional organs serves as a reference for future ECM proteomics.

The choice for adjuvant chemotherapy in stage II colorectal cancer is controversial as many patients are cured by surgery alone and it is difficult to identify patients with high risk of recurrence of the disease. There is a need for better stratification of this group of patients. Mass spectrometry imaging could identify patients at risk. We report here the N-glycosylation signatures of the different cell populations in a group of stage II colorectal cancer tissue samples. The cancer cells, compared with normal epithelial cells, have increased levels of sialylation and high-mannose glycans, as well as decreased levels of fucosylation and highly branched N-glycans. When looking at the interface between cancer and its microenvironment, it seems that the cancer N-glycosylation signature spreads into the surrounding stroma at the invasive front of the tumor. This finding was more outspoken in patients with a worse outcome within this sample group., Graphical Abstract, Highlights • N-glycan MS imaging as tool for better cancer stratification. • Increased levels of sialylated and high-mannose glycans in CRC stage II cancer. • Characterization of the interaction between cancer and its direct microenvironment. • Insight into N-glycosylation signature of high-risk patients with short survival., In Brief There is a need for better stratification of patients with stage II colorectal cancer as the treatment option remains controversial. Using MS imaging, we have characterized the N-glycosylation signatures of the different cell populations in stage II CRC tissues. We found, looking at the tumor microenvironment, that the cancer N-glycosylation signature spreads into the surrounding stroma at the invasive front of the tumor. This finding was more outspoken in patients with a worse outcome.