Pandey, Akhilesh

Abstract A quadrupole time‐of‐flight mass spectrometer coupled with a trapped ion mobility spectrometry (timsTOF) operated in parallel accumulation‐serial fragmentation (PASEF) mode has recently emerged as a platform capable of providing four‐dimensional (4D) features comprising of elution time, collision cross section (CCS), mass‐to‐charge ratio, and intensity of peptides. The PASEF mode provides ∼100% ion sampling efficiency both in data‐dependent acquisition (DDA) and data‐independent acquisition (DIA) modes without sacrificing sensitivity. In addition, targeted measurements using PASEF integrated parallel reaction monitoring (PRM) mode have also been described. However, only limited number of studies have used timsTOF for analysis of clinical samples. Although Orbitrap mass spectrometers have been used for biomarker discovery from cerebrospinal fluid (CSF) in a variety of neurological diseases, these Orbitrap‐derived datasets cannot readily be applied for driving experiments on timsTOF mass spectrometers. We generated a catalog of peptides and proteins in human CSF in DDA mode on a timsTOF mass spectrometer and used these data to build a spectral library. This strategy allowed us to use elution times and ion mobility values from the spectral library to design PRM experiments for quantifying previously discovered biomarkers from CSF samples in Alzheimer's disease. When the same samples were analyzed using a DIA approach combined with a spectral library search, a higher number of proteins were identified than in a library‐free approach. Overall, we have established a spectral library of CSF as a resource and demonstrated its utility for PRM and DIA studies, which should facilitate studies of neurological disorders.

575 Background: Cholangiocarcinoma (CCA) is a lethal hepatobiliary adenocarcinoma with increasing incidence and poor outcomes. Treatment success has been hindered by resistance, and new therapeutic strategies are necessary. The Src-family tyrosine kinase, LCK, activates Yes-associated protein (YAP), a known oncogene in CCA. While YAP has been historically difficult to target, we recently described LCK inhibition as a potential therapeutic option in CCA. We probed the LCK interactome employing biotinylation site identification technology (BioSITe) to discover molecular co-dependencies in the LCK-YAP axis. Methods: LCK-deficient HuCCT1, wild-type HuCCT1, and wild-type RBE cells were transfected with pEF-LCK-TurboID and used to reveal protein-protein interactions. Biotinylated peptides were enriched using anti-biotin antibodies followed by mass spectrometry analysis. Co-immunoprecipitation (Co-IP) was used to confirm interactions between LCK and identified interactors. Phosphoproteome profiling of human CCA tumors and patient derived xenografts was conducted to identify active kinase signatures in CCA. Tyrosine phosphorylated peptides were enriched using anti-phosphotyrosine antibody beads and analyzed by high-resolution mass spectrometery/liquid chromatography. Immunoblot analysis of HuCCT1 and LCK-deficient HuCCT1 cells was used to map the mechanistic interaction between LCK and epidermal growth factor receptor, EGFR. Inhibitors of EGFR and LCK, afatinib and NTRC 0652-0, respectively, were tested in vitro on human and murine CCA cell lines. Cell viability was determined with CellTiter-Glo; Calcusyn software was used to determine synergistic drug effects. Results: We discovered intracellular interactors of LCK in CCA using BioSITe. We identified both known and novel LCK interactors, including EGFR. Given that EGFR functions as a molecular driver of tumorigenesis and is increasingly recognized as a biomarker of resistance in a variety of tumors, we performed a phosphoproteomic evaluation of CCA tumors in which activated EGFR was identified by the presence of EGFR-Y1092, Y1197, and Y1172 phosphorylation. EGFR phosphorylation decreased under conditions of genetic LCK knock out and inhibition by NTRC 0652-0. Co-IP confirmed the interaction of EGFR with LCK. EGFR-Y1092 phosphorylation was downregulated with genetic knockout of LCK by immunoblot. Finally, EGFR inhibition was evaluated as a therapeutic strategy utilizing afatinib. Effects on cell viability were noted in both human and murine CCA cells. Afatinib and NTRC 0652-0 demonstrated a synergistic effect in vitro, with calculated combination indices of 0.007 – 0.790. Conclusions: A novel interaction between EGFR and LCK in CCA was identified using an unbiased proximity-dependent biotinylation technique. Dual blockade of LCK and EGFR is synergistic in CCA and may be a viable therapeutic approach for patients.

Protein arginine methylation is an extensive and functionally significant post-translational modification. However, little is known about its role in differentiation at the systems level. Using stable isotope labeling by amino acids in cell culture (SILAC) proteomics of whole proteome analysis in proliferating or five-day differentiated mouse C2C12 myoblasts, followed by high-resolution mass spectrometry, biochemical assays, and specific immunoprecipitation of mono- or dimethylated arginine peptides, we identified several protein families that were differentially methylated on arginine. Our study is the first to reveal global changes in the arginine mono- or dimethylation of proteins in proliferating myoblasts and differentiated myocytes and to identify enriched protein domains and novel short linear motifs (SLiMs). Our data may be crucial for dissecting the links between differentiation and cancer growth.

Infantile neuroaxonal dystrophy (INAD) is caused by recessive variants in PLA2G6 and is a lethal pediatric neurodegenerative disorder. Loss of the Drosophila homolog of PLA2G6 , leads to ceramide accumulation, lysosome expansion, and mitochondrial defects. Here, we report that retromer function, ceramide metabolism, the endolysosomal pathway, and mitochondrial morphology are affected in INAD patient-derived neurons. We show that in INAD mouse models, the same features are affected in Purkinje cells, arguing that the neuropathological mechanisms are evolutionary conserved and that these features can be used as biomarkers. We tested 20 drugs that target these pathways and found that Ambroxol, Desipramine, Azoramide, and Genistein alleviate neurodegenerative phenotypes in INAD flies and INAD patient-derived neural progenitor cells. We also develop an AAV-based gene therapy approach that delays neurodegeneration and prolongs lifespan in an INAD mouse model.

Abstract Glycans decorate almost all proteins and lipids on the cell surface, and they play a critical role in cell-cell interactions. Terminal sialic acid addition to glycans helps the immune system distinguish between “self” and “non-self.” The enzyme ST8sia6 adds alpha-2,8 disialic acids to cell surface receptors, and this sialic acid addition promotes binding to the inhibitory receptor Siglec-E. We have previously shown ST8sia6 overexpression in tumors accelerates tumor growth in a Siglec-E dependent manner. Proteomic analysis in murine tumor lines identified CD44 as a target for ST8sia6. CD44 expression on tumors is associated with metastasis and chemoresistance. Since humans and mice have different sialic acid structures, we examined whether ST8sia6 makes CD44 a ligand to the ortholog of Siglec-E in human cells, Siglec-7. When HEK293T cells overexpress ST8sia6 and CD44 together, Siglec-7 binding was significantly increased compared to ST8sia6 or CD44 overexpression alone. Thus, ST8sia6 has a conserved role from mouse to human in decorating CD44 with sialic acid. Our study proposes that ST8sia6 sialylation of CD44 could suppress immune responses and enables cancer progression in human. Supported by grant: 2T32AI007425-23 to DJF and R01 CA243545-01S1 to V.S.S.

Abstract Background Despite extensive studies of human immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and coronavirus disease 2019 (COVID-19) vaccination, research examining protective correlates of vertical transmission following maternal exposure in pregnancy remain limited. Here, we characterized antibody and cytokine responses in maternal and cord blood following infection or vaccination at various timepoints during gestation. Methods Spike S1 protein-specific binding antibodies and antibodies capable of blocking the interaction between the receptor binding domain (RBD) and the angiotensin converting enzyme 2 (ACE2) were measured in maternal and cord blood by ELISA. Serum concentrations of 74 cytokines/chemokines were measured by multiplex assay. Humoral responses and cytokine levels from matched maternal and fetal cord sera were compared and examined for potential correlations. Results We observed a highly significant correlation between Spike S1-specific antibody titer and RBD-ACE2 blocking antibody activity between maternal and fetal cord serum (p < 2.2e-16, R > 0.90). Blocking antibody activity was significantly higher for mothers infected during the 3rd trimester compared to earlier trimesters; however, vaccinated mothers developed and transferred higher antibody titers with greater RBD-ACE2 blocking antibody activity to their neonates than infected mothers. Furthermore, vaccine-induced Spike S1 IgG transfer ratios (fetal cord/maternal) were significantly higher than those induced by infection (p = 0.002). Multiplex assay showed significantly elevated levels of 33 cytokines/chemokines, mainly pro-inflammatory in infected maternal serum samples, while the paired fetal cord samples exhibited an anti-inflammatory cytokine predominance. Conclusion Our data support selective vertical transmission of potentially protective humoral responses against SARS-CoV-2, especially following vaccination in the 3rd trimester. The anti-inflammatory cytokine predominance in cord blood that persists despite maternal SARS-CoV-2 infection may offset the adverse outcomes of inflammation in pregnancy for the neonate. Disclosures All Authors: No reported disclosures.

AbstractCongenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N‐glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N‐glycans from misfolded, retro‐translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated. We undertook a multiplexed tandem mass tags‐labeling‐based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1‐deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell‐derived factor 1, collagen alpha‐1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin‐1. We further detected a total of 3255 N‐glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N‐glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N‐glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient‐derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system‐level insights for understanding NGLY1‐CDDG.

AbstractTRIT1 defect is a rare, autosomal‐recessive disorder of transcription, initially described as a condition with developmental delay, myoclonic seizures, and abnormal mitochondrial function. Currently, only 13 patients have been reported. We reviewed the genetic, clinical, and metabolic aspects of the disease in all known patients, including two novel, unrelated TRIT1 cases with abnormalities in oxidative phosphorylation complexes I and IV in fibroblasts. Taken together the features of all 15 patients, TRIT1 defect could be identified as a potentially recognizable syndrome including myoclonic epilepsy, speech delay, strabismus, progressive spasticity, and variable microcephaly, with normal lactate levels. Half of the patients had oxidative phosphorylation complex measurements and had multiple complex abnormalities.