Biology subjects

White, F.

Publications and source records attributed to White, F..

24 records · Page 2Linked to original sources

bioRxiv2021

Deciphering the tumor-specific immunopeptidome in vivo with genetically engineered mouse models

Effective immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex Class I (MHC-I). Recent developments in proteomics have improved the identification of peptides that are naturally presented by MHC-I, collectively known as the "immunopeptidome". Current approaches to profile tumor immunopeptidomes have been limited to in vitro investigation, which fails to capture the in vivo repertoire of MHC-I peptides, or bulk tumor lysates, which are obscured by the lack of tumor-specific MHC-I isolation. To overcome these limitations, we report here the engineering of a Cre recombinase-inducible affinity tag into the endogenous mouse MHC-I gene and targeting of this allele to the KrasLSL-G12D/+; p53fl/fl (KP) mouse model (KP; KbStrep). This novel approach has allowed us to isolate tumor-specific MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD) in vivo. With this powerful analytical tool, we were able to profile the evolution of the LUAD immunopeptidome through tumor progression and show that in vivo MHC-I presentation is shaped by post-translational mechanisms. We also uncovered novel, putative LUAD tumor associated antigens (TAAs). Many peptides that were recurrently presented in vivo exhibited very low expression of the cognate mRNA, provoking reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance. Beyond cancer, the KbStrep allele is compatible with a broad range of Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease, and autoimmunity.

cancer biology↗

bioRxiv2021

Quantitative consequences of protein carriers in immunopeptidomics and tyrosine phosphorylation MS2 analyses

Utilizing a protein carrier in combination with isobaric labeling to "boost" the signal of other low-level samples in multiplexed analyses has emerged as an attractive strategy to enhance data quantity while minimizing protein input in mass spectrometry analyses. Recent applications of this approach include pMHC profiling and tyrosine phosphoproteomics, two applications that are often limited by large sample requirements. While including a protein carrier has been shown to increase the number of identifiable peptides in both applications, the impact of a protein carrier on quantitative accuracy remains to be thoroughly explored, particularly in relevant biological contexts where samples exhibit dynamic changes in abundance across peptides. Here, we describe two sets of analyses comparing MS2-based quantitation using a 20x protein carrier in pMHC analyses and a high ([~]100x) and low ([~]9x) protein carrier in pTyr analyses, using CDK4/6 inhibitors and EGF stimulation to drive dynamic changes in the immunopeptidome and phosphoproteome, respectively. In both applications, inclusion of a protein carrier resulted in an increased number of MHC peptide or phosphopeptide identifications, as expected. At the same time, quantitative accuracy was adversely affected by the presence of the protein carrier, altering interpretation of the underlying biological response to perturbation. Moreover, for tyrosine phosphoproteomics, the presence of high levels of protein carrier led to a large number of missing values for endogenous phosphopeptides, leading to fewer quantifiable peptides relative to the "no-boost" condition. These data highlight the unique limitations and future experimental considerations for both analysis types and provide a framework for assessing quantitative accuracy in protein carrier experiments moving forward.

bioengineering↗

bioRxiv2020

Quantitative analysis of tyrosine phosphorylation from FFPE tissues reveals patient specific signaling networks

Formalin fixed paraffin embedded (FFPE) tissues are an invaluable source of clinical specimens. Tyrosine phosphorylation (pTyr) plays a fundamental role in cellular processes and is commonly dysregulated in cancer but has not been studied to date in FFPE samples. We describe a method for quantitative analysis of pTyr signaling networks at an unprecedented sensitivity, with hundreds of sites quantified from 1-2 10-m sections of FFPE tissue specimens. Phosphotyrosine profiles of flash frozen and FFPE tissues derived from the same tumors suggest that FFPE tissues preserve pTyr signaling characteristics in PDX tumors and archived clinical specimens. Differential activation of oncogenic proteins was observed in triple negative breast cancer tumors as well as lung cancer tumors, highlighting patient specific oncogenic driving kinases and indicating potential targeted therapies for each patient. These data highlight the capability for direct translational insight from pTyr analysis of small amounts of FFPE tumor tissue specimens.

systems biology↗

bioRxiv2020

Quantitative phosphoproteomics uncovers dysregulated kinase networks in Alzheimer's disease

Alzheimers disease (AD) is a form of dementia characterized by amyloid-{beta} plaques and Tau neurofibrillary tangles that progressively disrupt neural circuits in the brain. The signaling networks underlying the pathological changes in AD are poorly characterized at the level phosphoproteome. Using mass spectrometry, we performed a combined analysis of the tyrosine, serine, and threonine phosphoproteome, and proteome of temporal cortex tissue from AD patients and aged matched controls. We identified several co-correlated peptide modules that were associated with varying levels of phospho-Tau, oligodendrocyte, astrocyte, microglia, and neuronal pathologies in AD patients. We observed phosphorylation sites on kinases targeting Tau as well as other novel signaling factors that were correlated with these peptide modules. Finally, we used a data-driven statistical modeling approach to identify individual peptides and co-correlated signaling networks that were predictive of AD histopathologies. Together, these results build a map of pathology-associated phosphorylation signaling events occurring in AD.

neuroscience↗

bioRxiv2020

High-density, targeted monitoring of tyrosine phosphorylation reveals activated signaling networks in human tumors

Tyrosine phosphorylation (pTyr) plays a pivotal role in signal transduction and is commonly dysregulated in cancer. As a result, profiling tumor pTyr levels may reveal therapeutic insights critical to combating disease. Existing discovery and targeted mass spectrometry-based methods used to monitor pTyr networks involve a tradeoff between broad coverage of the pTyr network, reproducibility in target identification across analyses, and accurate quantification. To address these limitations, we developed a targeted approach, termed "SureQuant pTyr," coupling low input pTyr enrichment with a panel of isotopically labeled, tyrosine phosphorylated internal standard (IS) peptides. Using internal standard guided acquisition, the real-time detection of IS peptides during the analysis initiates the sensitive and selective quantitation of endogenous pTyr targets. This framework allows for reliable quantification of several hundred commonly dysregulated pTyr targets with high quantitative accuracy, enhances target detection success rates, and improves the robustness and usability of targeted acquisition. We establish the clinical applicability of SureQuant pTyr by profiling pTyr signaling levels in human colorectal tumors using minimal sample input, characterizing patient specific oncogenic driving mechanisms. While in some cases pTyr profiles align with previously reported proteomic, genomic, and transcriptomic molecular characterizations, we highlight instances of new insights gained using pTyr characterization and emphasize the complementary nature of pTyr measurements with traditional biomarkers for improving patient stratification and identifying therapeutic targets. The turn-key nature of this approach opens the door to rapid and reproducible pTyr profiling in research and clinical settings alike and enable pTyr-based measurements for applications in precision medicine. SummaryA targeted, mass spectrometry-based method, termed "SureQuant pTyr," enables highly sensitive and reproducible profiling of tyrosine phosphorylation levels in human colorectal tumors and reveals dysregulated signaling networks for enhanced tumor characterization and biomarker identification.

bioengineering↗

bioRxiv2020

Multiplexed relative and absolute quantitative immunopeptidomics reveals MHC I repertoire alterations induced by CDK4/6 inhibition

Peptides bound to class I major histocompatibility complexes (MHC) play a critical role in immune cell recognition and can trigger an antitumor immune response in cancer. Surface MHC levels can be modulated by anticancer agents, altering immunity. However, understanding the peptide repertoires response to treatment remains challenging and is limited by quantitative mass spectrometry-based strategies lacking robust normalization controls. We describe a novel approach that leverages recombinant heavy isotope-coded peptide MHCs (hipMHCs) and multiplex isotope tagging to quantify peptide repertoire alterations using low sample input. HipMHCs improve quantitative accuracy of peptide repertoire changes by normalizing for variation across analyses and enable absolute quantification using internal calibrants to determine copies per cell of MHC antigens, which can inform immunotherapy design. Applying this platform in melanoma to profile the immunopeptidome response to CDK4/6 inhibition and interferon gamma, known modulators of antigen presentation, we uncovered treatment-specific alterations, connecting the intracellular response to extracellular immune presentation.

immunology↗