Publications by authors named "Anil Madugundu"

52 Publications

Tyrosine Phosphoproteomics of Patient-Derived Xenografts Reveals Ephrin Type-B Receptor 4 Tyrosine Kinase as a Therapeutic Target in Pancreatic Cancer.

Cancers (Basel) 2021 Jul 7;13(14). Epub 2021 Jul 7.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN 55905, USA.

Pancreatic ductal adenocarcinoma is a recalcitrant tumor with minimal response to conventional chemotherapeutic approaches. Oncogenic signaling by activated tyrosine kinases has been implicated in cancers resulting in activation of diverse effector signaling pathways. Thus, the discovery of aberrantly activated tyrosine kinases is of great interest in developing novel therapeutic strategies in the treatment and management of pancreatic cancer. Patient-derived tumor xenografts (PDXs) in mice serve as potentially valuable preclinical models as they maintain the histological and molecular heterogeneity of the original human tumor. Here, we employed high-resolution mass spectrometry combined with immunoaffinity purification using anti-phosphotyrosine antibodies to profile tyrosine phosphoproteome across 13 pancreatic ductal adenocarcinoma PDX models. This analysis resulted in the identification of 1199 tyrosine-phosphorylated sites mapping to 704 proteins. The mass spectrometric analysis revealed widespread and heterogeneous activation of both receptor and non-receptor tyrosine kinases. Preclinical studies confirmed ephrin type-B receptor 4 (EphB4) as a potential therapeutic target based on the efficacy of human serum albumin-conjugated soluble EphB4 in mice bearing orthotopic xenografts. Immunohistochemistry-based validation using tissue microarrays from 346 patients with PDAC showed significant expression of EphB4 in >70% of patients. In summary, we present a comprehensive landscape of tyrosine phosphoproteome with EphB4 as a promising therapeutic target in pancreatic ductal adenocarcinoma.
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http://dx.doi.org/10.3390/cancers13143404DOI Listing
July 2021

DIA-Based Proteome Profiling of Nasopharyngeal Swabs from COVID-19 Patients.

J Proteome Res 2021 Jul 22. Epub 2021 Jul 22.

Department of Laboratory Medicine and Pathology, Mayo Clinic, 200 First Street Southwest, Rochester, Minnesota 55905, United States.

Since the recent outbreak of COVID-19, there have been intense efforts to understand viral pathogenesis and host immune response to combat SARS-CoV-2. It has become evident that different host alterations can be identified in SARS-CoV-2 infection based on whether infected cells, animal models or clinical samples are studied. Although nasopharyngeal swabs are routinely collected for SARS-CoV-2 detection by RT-PCR testing, host alterations in the nasopharynx at the proteomic level have not been systematically investigated. Thus, we sought to characterize the host response through global proteome profiling of nasopharyngeal swab specimens. A mass spectrometer combining trapped ion mobility spectrometry (TIMS) and high-resolution QTOF mass spectrometer with parallel accumulation-serial fragmentation (PASEF) was deployed for unbiased proteome profiling. First, deep proteome profiling of pooled nasopharyngeal swab samples was performed in the PASEF enabled DDA mode, which identified 7723 proteins that were then used to generate a spectral library. This approach provided peptide level evidence of five missing proteins for which MS/MS spectrum and mobilograms were validated with synthetic peptides. Subsequently, quantitative proteomic profiling was carried out for 90 individual nasopharyngeal swab samples (45 positive and 45 negative) in DIA combined with PASEF, termed as diaPASEF mode, which resulted in a total of 5023 protein identifications. Of these, 577 proteins were found to be upregulated in SARS-CoV-2 positive samples. Functional analysis of these upregulated proteins revealed alterations in several biological processes including innate immune response, viral protein assembly, and exocytosis. To the best of our knowledge, this study is the first to deploy diaPASEF for quantitative proteomic profiling of clinical samples and shows the feasibility of adopting such an approach to understand mechanisms and pathways altered in diseases.
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http://dx.doi.org/10.1021/acs.jproteome.1c00506DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8315246PMC
July 2021

High-resolution mass spectrometric analysis of cardiolipin profiles in Barth syndrome.

Mitochondrion 2021 Jul 15;60:27-32. Epub 2021 Jul 15.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, United States; Center for Individualized Medicine, Mayo Clinic, Rochester, MN, United States. Electronic address:

Barth syndrome is an X-linked recessive disorder caused by pathogenic variants in TAZ, which leads to a reduction in cardiolipin with a concomitant elevation of monolysocardiolipins. There is a paucity of studies characterizing changes in individual species of monolysocardiolipins, dilysocardiolipins and cardiolipin in Barth syndrome using high resolution untargeted lipidomics that can accurately annotate and quantify diverse lipids. We confirmed the structural diversity monolysocardiolipins, dilysocardiolipins and cardiolipin and identified individual species that showed previously unreported alterations in BTHS. Development of mass spectrometry-based targeted assays for these lipid biomarkers should provide an important tool for clinical diagnosis of Barth syndrome.
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http://dx.doi.org/10.1016/j.mito.2021.07.003DOI Listing
July 2021

Analytical sensitivity and specificity of four point of care rapid antigen diagnostic tests for SARS-CoV-2 using real-time quantitative PCR, quantitative droplet digital PCR, and a mass spectrometric antigen assay as comparator methods.

Clin Chem 2021 Jul 8. Epub 2021 Jul 8.

Department of Laboratory Medicine and Pathology.

Background: We evaluated the analytical sensitivity and specificity of four rapid antigen diagnostic tests (Ag RDTs) for SARS-CoV-2, using reverse transcription quantitative PCR (RT-qPCR) as the reference method; and further characterizing samples using droplet digital quantitative PCR (ddPCR) and a mass spectrometric antigen test.

Methods: 350 (150 negative and 200 RT-qPCR positive) residual phosphate buffered saline (PBS) samples were tested for antigen using the BD Veritor lateral flow (LF), ACON LF, ACON fluorescence immunoassay (FIA), and LumiraDx FIA. ddPCR was performed on RT-qPCR positive samples to quantitate the viral load in copies/mL applied to each Ag RDT. Mass spectrometric antigen testing was performed on PBS samples to obtain a set of RT-qPCR positive, antigen positive samples for further analysis.

Results: All Ag RDTs had nearly 100% specificity compared to RT-qPCR. Overall analytical sensitivity varied from 66.5% to 88.3%. All methods detected antigen in samples with viral load >1,500,000 copies/mL RNA, and detected ≥75% of samples with viral load of 500,000 to 1,500,000 copies/mL. The BD Veritor LF detected only 25% of samples with viral load between 50,000-500,000 copies/mL, compared to 75% for the ACON LF device and >80% for LumiraDx and ACON FIA. The ACON FIA detected significantly more samples with viral load <50,000 copies/mL compared to the BD Veritor. Among samples with detectable antigen and viral load <50,000 copies/mL, sensitivity of the Ag RDT varied between 13.0% (BD Veritor) and 78.3% (ACON FIA).

Conclusions: Ag RDTs differ significantly in analytical sensitivity, particularly at viral load <500,000 copies/mL.
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http://dx.doi.org/10.1093/clinchem/hvab138DOI Listing
July 2021

A mass spectrometry-based targeted assay for detection of SARS-CoV-2 antigen from clinical specimens.

EBioMedicine 2021 Jul 3;69:103465. Epub 2021 Jul 3.

Department of Laboratory Medicine and Pathology, Division of Clinical Biochemistry and Immunology, Mayo Clinic, MN 55905, USA; Center for Individualized Medicine, Mayo Clinic, Rochester, MN 55905, USA; Center for Molecular Medicine, National Institute of Mental Health and Neurosciences, Hosur Road, Bangalore, Karnataka 560029, India. Electronic address:

Background: The COVID-19 pandemic caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) has overwhelmed health systems worldwide and highlighted limitations of diagnostic testing. Several types of diagnostic tests including RT-PCR-based assays and antigen detection by lateral flow assays, each with their own strengths and weaknesses, have been developed and deployed in a short time.

Methods: Here, we describe an immunoaffinity purification approach followed a by high resolution mass spectrometry-based targeted qualitative assay capable of detecting SARS-CoV-2 viral antigen from nasopharyngeal swab samples. Based on our discovery experiments using purified virus, recombinant viral protein and nasopharyngeal swab samples from COVID-19 positive patients, nucleocapsid protein was selected as a target antigen. We then developed an automated antibody capture-based workflow coupled to targeted high-field asymmetric waveform ion mobility spectrometry (FAIMS) - parallel reaction monitoring (PRM) assay on an Orbitrap Exploris 480 mass spectrometer. An ensemble machine learning-based model for determining COVID-19 positive samples was developed using fragment ion intensities from the PRM data.

Findings: The optimized targeted assay, which was used to analyze 88 positive and 88 negative nasopharyngeal swab samples for validation, resulted in 98% (95% CI = 0.922-0.997) (86/88) sensitivity and 100% (95% CI = 0.958-1.000) (88/88) specificity using RT-PCR-based molecular testing as the reference method.

Interpretation: Our results demonstrate that direct detection of infectious agents from clinical samples by tandem mass spectrometry-based assays have potential to be deployed as diagnostic assays in clinical laboratories, which has hitherto been limited to analysis of pure microbial cultures.

Funding: This study was supported by DBT/Wellcome Trust India Alliance Margdarshi Fellowship grant IA/M/15/1/502023 awarded to AP and the generosity of Eric and Wendy Schmidt.
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http://dx.doi.org/10.1016/j.ebiom.2021.103465DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8253671PMC
July 2021

Cerebrospinal fluid lipidomics for biomarkers of Alzheimer's disease.

Mol Omics 2021 Jun;17(3):454-463

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN, 55902, USA.

Alzheimer's disease (AD) is the most common cause of dementia and is associated with serious neurologic sequelae resulting from neurodegenerative changes. Identification of markers of early-stage AD could be important for designing strategies to arrest the progression of the disease. The brain is rich in lipids because they are crucial for signal transduction and anchoring of membrane proteins. Cerebrospinal fluid (CSF) is an excellent specimen for studying the metabolism of lipids in AD because it can reflect changes occurring in the brain. We aimed to identify CSF lipidomic alterations associated with AD, using untargeted lipidomics, carried out in positive and negative ion modes. We found CSF lipids that were significantly altered in AD cases. In addition, comparison of CSF lipid profiles between persons with mild cognitive impairment (MCI) and AD showed a strong positive correlation between the lipidomes of the MCI and AD groups. The novel lipid biomarkers identified in this study are excellent candidates for validation in a larger set of patient samples and as predictive biomarkers of AD through future longitudinal studies. Once validated, the lipid biomarkers could lead to early detection, disease monitoring and the ability to measure the efficacy of potential therapeutic interventions in AD.
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http://dx.doi.org/10.1039/d0mo00186dDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210464PMC
June 2021

Mass Spectrometric Analysis of Urine from COVID-19 Patients for Detection of SARS-CoV-2 Viral Antigen and to Study Host Response.

J Proteome Res 2021 07 2;20(7):3404-3413. Epub 2021 Jun 2.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905, United States.

SARS-CoV-2 infection has become a major public health burden and affects many organs including lungs, kidneys, the liver, and the brain. Although the virus is readily detected and diagnosed using nasopharyngeal swabs by reverse transcriptase polymerase chain reaction (RT-PCR), detection of its presence in body fluids is fraught with difficulties. A number of published studies have failed to detect viral RNA by RT-PCR methods in urine. Although microbial identification in clinical microbiology using mass spectrometry is undertaken after culture, here we undertook a mass spectrometry-based approach that employed an enrichment step to capture and detect SARS-CoV-2 nucleocapsid protein directly from urine of COVID-19 patients without any culture. We detected SARS-CoV-2 nucleocapsid protein-derived peptides from 13 out of 39 urine samples. Further, a subset of COVID-19 positive and COVID-19 negative urine samples validated by mass spectrometry were used for the quantitative proteomics analysis. Proteins with increased abundance in urine of SARS-CoV-2 positive individuals were enriched in the acute phase response, regulation of complement system, and immune response. Notably, a number of renal proteins such as podocin (NPHS2), an amino acid transporter (SLC36A2), and sodium/glucose cotransporter 5 (SLC5A10), which are intimately involved in normal kidney function, were decreased in the urine of COVID-19 patients. Overall, the detection of viral antigens in urine using mass spectrometry and alterations of the urinary proteome could provide insights into understanding the pathogenesis of COVID-19.
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http://dx.doi.org/10.1021/acs.jproteome.1c00391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8189038PMC
July 2021

Mapping the micro-proteome of the nuclear lamina and lamina-associated domains.

Life Sci Alliance 2021 05 23;4(5). Epub 2021 Mar 23.

Department of Biological Chemistry, Johns Hopkins University of Medicine, Baltimore, MD, USA

The nuclear lamina is a proteinaceous network of filaments that provide both structural and gene regulatory functions by tethering proteins and large domains of DNA, the so-called lamina-associated domains (LADs), to the periphery of the nucleus. LADs are a large fraction of the mammalian genome that are repressed, in part, by their association to the nuclear periphery. The genesis and maintenance of LADs is poorly understood as are the proteins that participate in these functions. In an effort to identify proteins that reside at the nuclear periphery and potentially interact with LADs, we have taken a two-pronged approach. First, we have undertaken an interactome analysis of the inner nuclear membrane bound LAP2β to further characterize the nuclear lamina proteome. To accomplish this, we have leveraged the BioID system, which previously has been successfully used to characterize the nuclear lamina proteome. Second, we have established a system to identify proteins that bind to LADs by developing a chromatin-directed BioID system. We combined the BioID system with the m6A-tracer system which binds to LADs in live cells to identify both LAD proximal and nuclear lamina proteins. In combining these datasets, we have further characterized the protein network at the nuclear lamina, identified putative LAD proximal proteins and found several proteins that appear to interface with both micro-proteomes. Importantly, several proteins essential for LAD function, including heterochromatin regulating proteins related to H3K9 methylation, were identified in this study.
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http://dx.doi.org/10.26508/lsa.202000774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8008952PMC
May 2021

Quantitative Proteomics Reveals that the OGT Interactome Is Remodeled in Response to Oxidative Stress.

Mol Cell Proteomics 2021 Mar 12;20:100069. Epub 2021 Mar 12.

Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States. Electronic address:

The dynamic modification of specific serine and threonine residues of intracellular proteins by O-linked N-acetyl-β-D-glucosamine (O-GlcNAc) mitigates injury and promotes cytoprotection in a variety of stress models. The O-GlcNAc transferase (OGT) and the O-GlcNAcase are the sole enzymes that add and remove O-GlcNAc, respectively, from thousands of substrates. It remains unclear how just two enzymes can be specifically controlled to affect glycosylation of target proteins and signaling pathways both basally and in response to stress. Several lines of evidence suggest that protein interactors regulate these responses by affecting OGT and O-GlcNAcase activity, localization, and substrate specificity. To provide insight into the mechanisms by which OGT function is controlled, we have used quantitative proteomics to define OGT's basal and stress-induced interactomes. OGT and its interaction partners were immunoprecipitated from OGT WT, null, and hydrogen peroxide-treated cell lysates that had been isotopically labeled with light, medium, and heavy lysine and arginine (stable isotopic labeling of amino acids in cell culture). In total, more than 130 proteins were found to interact with OGT, many of which change their association upon hydrogen peroxide stress. These proteins include the major OGT cleavage and glycosylation substrate, host cell factor 1, which demonstrated a time-dependent dissociation after stress. To validate less well-characterized interactors, such as glyceraldehyde 3-phosphate dehydrogenase and histone deacetylase 1, we turned to parallel reaction monitoring, which recapitulated our discovery-based stable isotopic labeling of amino acids in cell culture approach. Although the majority of proteins identified are novel OGT interactors, 64% of them are previously characterized glycosylation targets that contain varied domain architecture and function. Together these data demonstrate that OGT interacts with unique and specific interactors in a stress-responsive manner.
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http://dx.doi.org/10.1016/j.mcpro.2021.100069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8079276PMC
March 2021

Expanding the clinical and metabolic phenotype of DPM2 deficient congenital disorders of glycosylation.

Mol Genet Metab 2021 01 17;132(1):27-37. Epub 2020 Oct 17.

Mayo Clinic, Department of Clinical Genomics, Rochester, MN, USA; Mayo Clinic, Department of Laboratory of Medical Pathology, Rochester, MN, USA.

Pathogenic alterations in the DPM2 gene have been previously described in patients with hypotonia, progressive muscle weakness, absent psychomotor development, intractable seizures, and early death. We identified biallelic DPM2 variants in a 23-year-old male with truncal hypotonia, hypertonicity, congenital heart defects, intellectual disability, and generalized muscle wasting. His clinical presentation was much less severe than that of the three previously described patients. This is the second report on this ultra-rare disorder. Here we review the characteristics of previously reported individuals with a defect in the DPM complex while expanding the clinical phenotype of DPM2-Congenital Disorders of Glycosylation. In addition, we offer further insights into the pathomechanism of DPM2-CDG disorder by introducing glycomics and lipidomics analysis.
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http://dx.doi.org/10.1016/j.ymgme.2020.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855207PMC
January 2021

PASS-DIA: A Data-Independent Acquisition Approach for Discovery Studies.

Anal Chem 2020 11 20;92(21):14466-14475. Epub 2020 Oct 20.

Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, Minnesota 55905, United States.

A data-independent acquisition (DIA) approach is being increasingly adopted as a promising strategy for identification and quantitation of proteomes. As most DIA data sets are acquired with wide isolation windows, highly complex MS/MS spectra are generated, which negatively impacts obtaining peptide information through classical protein database searches. Therefore, the analysis of DIA data mainly relies on the evidence of the existence of peptides from prebuilt spectral libraries. Consequently, one major weakness of this method is that it does not account for peptides that are not included in the spectral library, precluding the use of DIA for discovery studies. Here, we present a strategy termed Precursor ion And Small Slice-DIA (PASS-DIA) in which MS/MS spectra are acquired with small isolation windows (slices) and MS/MS spectra are interpreted with accurately determined precursor ion masses. This method enables the direct application of conventional spectrum-centric analysis pipelines for peptide identification and precursor ion-based quantitation. The performance of PASS-DIA was observed to be superior to both data-dependent acquisition (DDA) and conventional DIA experiments with 69 and 48% additional protein identifications, respectively. Application of PASS-DIA for the analysis of post-translationally modified peptides again highlighted its superior performance in characterizing phosphopeptides (77% more), N-terminal acetylated peptides (56% more), and N-glycopeptides (83% more) as compared to DDA alone. Finally, the use of PASS-DIA to characterize a rare proteome of human fallopian tube organoids enabled 34% additional protein identifications than DDA alone and revealed biologically relevant pathways including low abundance proteins. Overall, PASS-DIA is a novel DIA approach for use as a discovery tool that outperforms both conventional DDA and DIA experiments to provide additional protein information. We believe that the PASS-DIA method is an important strategy for discovery-type studies when deeper proteome characterization is required.
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http://dx.doi.org/10.1021/acs.analchem.0c02513DOI Listing
November 2020

High-quality nuclear genome for Sarcoptes scabiei-A critical resource for a neglected parasite.

PLoS Negl Trop Dis 2020 10 1;14(10):e0008720. Epub 2020 Oct 1.

Cell and Molecular Biology Department, Infectious Diseases Program, QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia.

The parasitic mite Sarcoptes scabiei is an economically highly significant parasite of the skin of humans and animals worldwide. In humans, this mite causes a neglected tropical disease (NTD), called scabies. This disease results in major morbidity, disability, stigma and poverty globally and is often associated with secondary bacterial infections. Currently, anti-scabies treatments are not sufficiently effective, resistance to them is emerging and no vaccine is available. Here, we report the first high-quality genome and transcriptomic data for S. scabiei. The genome is 56.6 Mb in size, has a a repeat content of 10.6% and codes for 9,174 proteins. We explored key molecules involved in development, reproduction, host-parasite interactions, immunity and disease. The enhanced 'omic data sets for S. scabiei represent comprehensive and critical resources for genetic, functional genomic, metabolomic, phylogenetic, ecological and/or epidemiological investigations, and will underpin the design and development of new treatments, vaccines and/or diagnostic tests.
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http://dx.doi.org/10.1371/journal.pntd.0008720DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7591027PMC
October 2020

Mutation-Specific and Common Phosphotyrosine Signatures of G12D and G13D Alleles.

J Proteome Res 2021 01 27;20(1):670-683. Epub 2020 Oct 27.

Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, United States.

is one of the most frequently mutated genes across all cancer subtypes. Two of the most frequent oncogenic mutations observed in patients result in glycine to aspartic acid substitution at either codon 12 (G12D) or 13 (G13D). Although the biochemical differences between these two predominant mutations are not fully understood, distinct clinical features of the resulting tumors suggest involvement of disparate signaling mechanisms. When we compared the global phosphotyrosine proteomic profiles of isogenic colorectal cancer cell lines bearing either G12D or G13D mutation, we observed both shared as well as unique signaling events induced by the two mutations. Remarkably, while the G12D mutation led to an increase in membrane proximal and adherens junction signaling, the G13D mutation led to activation of signaling molecules such as nonreceptor tyrosine kinases, MAPK kinases, and regulators of metabolic processes. The importance of one of the cell surface molecules, MPZL1, which was found to be hyperphosphorylated in G12D cells, was confirmed by cellular assays as its knockdown led to a decrease in proliferation of G12D but not G13D expressing cells. Overall, our study reveals important signaling differences across two common mutations and highlights the utility of our approach to systematically dissect subtle differences between related oncogenic mutants and potentially lead to individualized treatments.
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http://dx.doi.org/10.1021/acs.jproteome.0c00587DOI Listing
January 2021

Signature Fragment Ions of Biotinylated Peptides.

J Am Soc Mass Spectrom 2020 Feb 13;31(2):394-404. Epub 2020 Jan 13.

Department of Laboratory Medicine and Pathology , Mayo Clinic , Rochester , Minnesota 55905 , United States.

The use of biotin or biotin-containing reagents is an essential component of many protein purification and labeling technologies. Owing to its small size and high affinity to the avidin family of proteins, biotin is a versatile molecular handle that permits both enrichment and purity that is not easily achieved by other reagents. Traditionally, the use of biotinylation to enrich for proteins has not required the detection of the site of biotinylation. However, newer technologies for discovery of protein-protein interactions, such as APEX and BioID, as well as some of the click chemistry-based labeling approaches have underscored the importance of determining the exact residue that is modified by biotin. Anti-biotin antibody-based enrichment of biotinylated peptides (e.g., BioSITe) coupled to LC-MS/MS permit large-scale detection and localization of sites of biotinylation. As with any chemical modification of peptides, understanding the fragmentation patterns that result from biotin modification is essential to improving its detection by LC-MS/MS. Tandem mass spectra of biotinylated peptides has not yet been studied systematically. Here, we describe the various signature fragment ions generated with collision-induced dissociation of biotinylated peptides. We focused on biotin adducts attached to peptides generated by BioID and APEX experiments, including biotin, isotopically heavy biotin, and biotin-XX-phenol, a nonpermeable variant of biotin-phenol. We also highlight how the detection of biotinylated peptides in high-throughput studies poses certain computational challenges for accurate quantitation which need to be addressed. Our findings about signature fragment ions of biotinylated peptides should be helpful in the confirmation of biotinylation sites.
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http://dx.doi.org/10.1021/jasms.9b00024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7199424PMC
February 2020

Transcriptomic Profiles of Confirmed Pediatric Tuberculosis Patients and Household Contacts Identifies Active Tuberculosis, Infection, and Treatment Response Among Indian Children.

J Infect Dis 2020 04;221(10):1647-1658

Center for Clinical Global Health Education, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

Background: Gene expression profiling is emerging as a tool for tuberculosis diagnosis and treatment response monitoring, but limited data specific to Indian children and incident tuberculosis infection (TBI) exist.

Methods: Sixteen pediatric Indian tuberculosis cases were age- and sex-matched to 32 tuberculosis-exposed controls (13 developed incident TBI without subsequent active tuberculosis). Longitudinal samples were collected for ribonucleic acid sequencing. Differential expression analysis generated gene lists that identify tuberculosis diagnosis and tuberculosis treatment response. Data were compared with published gene lists. Population-specific risk score thresholds were calculated.

Results: Seventy-one genes identified tuberculosis diagnosis and 25 treatment response. Within-group expression was partially explained by age, sex, and incident TBI. Transient changes in gene expression were identified after both infection and treatment. Application of 27 published gene lists to our data found variable performance for tuberculosis diagnosis (sensitivity 0.38-1.00, specificity 0.48-0.93) and treatment response (sensitivity 0.70-0.80, specificity 0.40-0.80). Our gene lists found similarly variable performance when applied to published datasets for diagnosis (sensitivity 0.56-0.85, specificity 0.50-0.85) and treatment response (sensitivity 0.49- 0.86, specificity 0.50-0.84).

Conclusions: Gene expression profiles among Indian children with confirmed tuberculosis were distinct from adult-derived gene lists, highlighting the importance of including distinct populations in differential gene expression models.
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http://dx.doi.org/10.1093/infdis/jiz639DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184902PMC
April 2020

Integrative phosphoproteome and interactome analysis of the role of Ubash3b in BCR-ABL signaling.

Leukemia 2020 01 9;34(1):301-305. Epub 2019 Aug 9.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.

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http://dx.doi.org/10.1038/s41375-019-0535-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934410PMC
January 2020

Hotspot SF3B1 mutations induce metabolic reprogramming and vulnerability to serine deprivation.

J Clin Invest 2019 08 8;129(11):4708-4723. Epub 2019 Aug 8.

Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, and.

Cancer-associated mutations in the spliceosome gene SF3B1 create a neomorphic protein that produces aberrant mRNA splicing in hundreds of genes, but the ensuing biologic and therapeutic consequences of this missplicing are not well understood. Here we have provided evidence that aberrant splicing by mutant SF3B1 altered the transcriptome, proteome, and metabolome of human cells, leading to missplicing-associated downregulation of metabolic genes, decreased mitochondrial respiration, and suppression of the serine synthesis pathway. We also found that mutant SF3B1 induces vulnerability to deprivation of the nonessential amino acid serine, which was mediated by missplicing-associated downregulation of the serine synthesis pathway enzyme PHGDH. This vulnerability was manifest both in vitro and in vivo, as dietary restriction of serine and glycine in mice was able to inhibit the growth of SF3B1MUT xenografts. These findings describe a role for SF3B1 mutations in altered energy metabolism, and they offer a new therapeutic strategy against SF3B1MUT cancers.
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http://dx.doi.org/10.1172/JCI125022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6819102PMC
August 2019

Surgery, Octreotide, Temozolomide, Bevacizumab, Radiotherapy, and Pegvisomant Treatment of an AIP Mutation‒Positive Child.

J Clin Endocrinol Metab 2019 08;104(8):3539-3544

Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine, Queen Mary University of London, London, United Kingdom.

Context: Inactivating germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene are linked to pituitary adenoma predisposition. Here, we present the youngest known patient with AIP-related pituitary adenoma.

Case Description: The patient presented at the age of 4 years with pituitary apoplexy and left ptosis with severe visual loss following a 1-year history of abdominal pain, headaches, and rapid growth. His IGF-1 level was 5× the upper limit of normal, and his random GH level was 1200 ng/mL. MRI showed a 43 × 24 × 35‒mm adenoma with suprasellar extension invading the left cavernous sinus (Knosp grade 4). After transsphenoidal surgery, histology showed a grade 2A sparsely granulated somatotropinoma with negative O6-methylguanine-DNA methyltransferase and positive vascular endothelial growth factor staining. Genetic testing identified a heterozygous germline nonsense AIP mutation (p.Arg81Ter). Exome sequencing of the tumor revealed that it had lost the entire maternal chromosome-11, rendering it hemizygous for chromosome-11 and therefore lacking functional copies of AIP in the tumor. He was started on octreotide, but because the tumor rapidly regrew and IGF-1 levels were unchanged, temozolomide was initiated, and intensity-modulated radiotherapy was administered 5 months after surgery. Two months later, bevacizumab was added, resulting in excellent tumor response. Although these treatments stabilized tumor growth over 4 years, IGF-1 was normalized only after pegvisomant treatment, although access to this medication was intermittent. At 3.5 years of follow-up, gamma knife treatment was administered, and pegvisomant dose increase was indicated.

Conclusion: Multimodal treatment with surgery, long-acting octreotide, radiotherapy, temozolomide, bevacizumab, and pegvisomant can control genetically driven, aggressive, childhood-onset somatotropinomas.
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http://dx.doi.org/10.1210/jc.2019-00432DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6619489PMC
August 2019

Family-Based Next-Generation Sequencing Study Identifies an Variant in an Infant with Primary Immunodeficiency.

OMICS 2019 05;23(5):285-290

3 Center for Molecular Medicine, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India.

Primary immunodeficiencies (PIDs) are a rare and heterogeneous group of inherited genetic disorders that are characterized by an absent or impaired immune system. In this report, we describe the use of next-generation sequencing to investigate a male infant with clinical and immunological manifestations suggestive of a PID. Whole-exome sequencing of the infant along with his parents revealed a novel nucleotide variant (cytosine to adenine substitution at nucleotide position 252) in the coding region of the interleukin 2 receptor subunit gamma () gene. The mother was found to be a carrier. These findings are consistent with a diagnosis of X-linked severe combined immunodeficiency and represent the first such reported mutation in an Indian family. This mutation leads to an asparagine to lysine substitution ( p.Asn84Lys ) located in the extracellular domain of IL2RG, which is predicted to be pathogenic. Our study demonstrates the power of next-generation sequencing in identifying potential causative mutations to enable accurate clinical diagnosis, prenatal screening, and carrier female detection in PID patients. We believe that this approach, which is not a current routine in clinical practice, will become a mainstream component of individualized medicine in the near future.
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http://dx.doi.org/10.1089/omi.2018.0196DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534087PMC
May 2019

Integrated Transcriptomic and Proteomic Analysis of Primary Human Umbilical Vein Endothelial Cells.

Proteomics 2019 08 26;19(15):e1800315. Epub 2019 Jun 26.

Center for Molecular Medicine, National Institute of Mental Health and Neurosciences, Hosur Road, Bangalore, 560029, Karnataka, India.

Understanding the molecular profile of every human cell type is essential for understanding its role in normal physiology and disease. Technological advancements in DNA sequencing, mass spectrometry, and computational methods allow us to carry out multiomics analyses although such approaches are not routine yet. Human umbilical vein endothelial cells (HUVECs) are a widely used model system to study pathological and physiological processes associated with the cardiovascular system. In this study, next-generation sequencing and high-resolution mass spectrometry to profile the transcriptome and proteome of primary HUVECs is employed. Analysis of 145 million paired-end reads from next-generation sequencing confirmed expression of 12 186 protein-coding genes (FPKM ≥0.1), 439 novel long non-coding RNAs, and revealed 6089 novel isoforms that were not annotated in GENCODE. Proteomics analysis identifies 6477 proteins including confirmation of N-termini for 1091 proteins, isoforms for 149 proteins, and 1034 phosphosites. A database search to specifically identify other post-translational modifications provide evidence for a number of modification sites on 117 proteins which include ubiquitylation, lysine acetylation, and mono-, di- and tri-methylation events. Evidence for 11 "missing proteins," which are proteins for which there was insufficient or no protein level evidence, is provided. Peptides supporting missing protein and novel events are validated by comparison of MS/MS fragmentation patterns with synthetic peptides. Finally, 245 variant peptides derived from 207 expressed proteins in addition to alternate translational start sites for seven proteins and evidence for novel proteoforms for five proteins resulting from alternative splicing are identified. Overall, it is believed that the integrated approach employed in this study is widely applicable to study any primary cell type for deeper molecular characterization.
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http://dx.doi.org/10.1002/pmic.201800315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6812510PMC
August 2019

Integrated Transcriptomic and Proteomic Analysis of Human Eccrine Sweat Glands Identifies Missing and Novel Proteins.

Mol Cell Proteomics 2019 07 12;18(7):1382-1395. Epub 2019 Apr 12.

From the ‡McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland;; §Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland;; Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland;; ‡‡Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland;. Electronic address:

The eccrine sweat gland is an exocrine gland that is involved in the secretion of sweat for control of temperature. Malfunction of the sweat glands can result in disorders such as miliaria, hyperhidrosis and bromhidrosis. Understanding the transcriptome and proteome of sweat glands is important for understanding their physiology and role in diseases. However, no systematic transcriptome or proteome analysis of sweat glands has yet been reported. Here, we isolated eccrine sweat glands from human skin by microdissection and performed RNA-seq and proteome analysis. In total, ∼138,000 transcripts and ∼6,100 proteins were identified. Comparison of the RNA-seq data of eccrine sweat glands to other human tissues revealed the closest resemblance to the cortex region of kidneys. The proteome data showed enrichment of proteins involved in secretion, reabsorption, and wound healing. Importantly, protein level identification of the calcium ion channel TRPV4 suggests the importance of eccrine sweat glands in re-epithelialization of wounds and prevention of dehydration. We also identified 2 previously missing proteins from our analysis. Using a proteogenomic approach, we identified 7 peptides from 5 novel genes, which we validated using synthetic peptides. Most of the novel proteins were from short open reading frames (sORFs) suggesting that many sORFs still remain to be annotated in the human genome. This study presents the first integrated analysis of the transcriptome and proteome of the human eccrine sweat gland and would become a valuable resource for studying sweat glands in physiology and disease.
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http://dx.doi.org/10.1074/mcp.RA118.001101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6601213PMC
July 2019

Quantitative Proteomic Profiling of Cerebrospinal Fluid to Identify Candidate Biomarkers for Alzheimer's Disease.

Proteomics Clin Appl 2019 07 25;13(4):e1800105. Epub 2019 Jan 25.

Center for Molecular Medicine, National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, 560029, India.

Purpose: The aim of this study is to identify the potential cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease and to evaluate these markers on independent CSF samples using parallel reaction monitoring (PRM) assays.

Experimental Design: High-Resolution mass spectrometry and tandem mass tag (TMT) multiplexing technology are employed to identify potential biomarkers for Alzheimer's disease. Some of the identified potential biomarkers are validated using PRM assays.

Results: A total of 2327 proteins are identified in the CSF of which 139 are observed to be significantly altered in the CSF of AD patients. The proteins altered in AD includes a number of known AD marker such as MAPT, NPTX2, VGF, GFAP, and NCAM1 as well as novel biomarkers such as PKM and YWHAG. These findings are validated in a separate set of CSF specimens from AD dementia patients and controls. NPTX2, in combination with PKM or YWHAG, leads to the best results with AUCs of 0.935 and 0.933, respectively.

Conclusions And Clinical Relevance: The proteins that are found to be altered in the CSF of patients with AD could be used for monitoring disease progression and therapeutic response and perhaps also for early detection once they are validated in larger studies.
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http://dx.doi.org/10.1002/prca.201800105DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6639119PMC
July 2019

Proteomic Analysis of the Human Anterior Pituitary Gland.

OMICS 2018 12;22(12):759-769

24 Microbiology Laboratory, National Institute of Pathology, New Delhi, India.

The pituitary function is regulated by a complex system involving the hypothalamus and biological networks within the pituitary. Although the hormones secreted from the pituitary have been well studied, comprehensive analyses of the pituitary proteome are limited. Pituitary proteomics is a field of postgenomic research that is crucial to understand human health and pituitary diseases. In this context, we report here a systematic proteomic profiling of human anterior pituitary gland (adenohypophysis) using high-resolution Fourier transform mass spectrometry. A total of 2164 proteins were identified in this study, of which 105 proteins were identified for the first time compared with high-throughput proteomic-based studies from human pituitary glands. In addition, we identified 480 proteins with secretory potential and 187 N-terminally acetylated proteins. These are the first region-specific data that could serve as a vital resource for further investigations on the physiological role of the human anterior pituitary glands and the proteins secreted by them. We anticipate that the identification of previously unknown proteins in the present study will accelerate biomedical research to decipher their role in functioning of the human anterior pituitary gland and associated human diseases.
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http://dx.doi.org/10.1089/omi.2018.0160DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6425986PMC
December 2018

CHESS: a new human gene catalog curated from thousands of large-scale RNA sequencing experiments reveals extensive transcriptional noise.

Genome Biol 2018 11 28;19(1):208. Epub 2018 Nov 28.

Center for Computational Biology, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, USA.

We assembled the sequences from deep RNA sequencing experiments by the Genotype-Tissue Expression (GTEx) project, to create a new catalog of human genes and transcripts, called CHESS. The new database contains 42,611 genes, of which 20,352 are potentially protein-coding and 22,259 are noncoding, and a total of 323,258 transcripts. These include 224 novel protein-coding genes and 116,156 novel transcripts. We detected over 30 million additional transcripts at more than 650,000 genomic loci, nearly all of which are likely nonfunctional, revealing a heretofore unappreciated amount of transcriptional noise in human cells. The CHESS database is available at http://ccb.jhu.edu/chess .
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http://dx.doi.org/10.1186/s13059-018-1590-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6260756PMC
November 2018

Phosphotyrosine profiling of human cerebrospinal fluid.

Clin Proteomics 2018 12;15:29. Epub 2018 Sep 12.

1Center for Molecular Medicine, National Institute of Mental Health and Neurosciences (NIMHANS), Hosur Road, Bangalore, 560029 India.

Background: Cerebrospinal fluid (CSF) is an important source of potential biomarkers that affect the brain. Biomarkers for neurodegenerative disorders are needed to assist in diagnosis, monitoring disease progression and evaluating efficacy of therapies. Recent studies have demonstrated the involvement of tyrosine kinases in neuronal cell death. Thus, neurodegeneration in the brain is related to altered tyrosine phosphorylation of proteins in the brain and identification of abnormally phosphorylated tyrosine peptides in CSF has the potential to ascertain candidate biomarkers for neurodegenerative disorders.

Methods: In this study, we used an antibody-based tyrosine phosphopeptide enrichment method coupled with high resolution Orbitrap Fusion Tribrid Lumos Fourier transform mass spectrometer to catalog tyrosine phosphorylated peptides from cerebrospinal fluid. The subset of identified tyrosine phosphorylated peptides was also validated using parallel reaction monitoring (PRM)-based targeted approach.

Results: To date, there are no published studies on global profiling of phosphotyrosine modifications of CSF proteins. We carried out phosphotyrosine profiling of CSF using an anti-phosphotyrosine antibody-based enrichment and analysis using high resolution Orbitrap Fusion Lumos mass spectrometer. We identified 111 phosphotyrosine peptides mapping to 66 proteins, which included 24 proteins which have not been identified in CSF previously. We then validated a set of 5 tyrosine phosphorylated peptides in an independent set of CSF samples from cognitively normal subjects, using a PRM-based targeted approach.

Conclusions: The findings from this deep phosphotyrosine profiling of CSF samples have the potential to identify novel disease-related phosphotyrosine-containing peptides in CSF.
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http://dx.doi.org/10.1186/s12014-018-9205-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6136184PMC
September 2018

A Next-Generation Sequencing-Based Molecular Approach to Characterize a Tick Vector in Lyme Disease.

OMICS 2018 08;22(8):565-574

3 McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland.

Next-generation sequencing approaches have revolutionized genomic medicine and enabled rapid diagnosis for several diseases. These approaches are widely used for pathogen detection in several infectious diseases. Lyme disease is a tick-borne infectious disease, which affects multiple organs. The causative organism is a spirochete, Borrelia burgdorferi, which is transmitted by ticks. Lyme disease can be treated easily if detected early, but its diagnosis is often delayed or is incorrect leading to a chronic debilitating condition. Current confirmatory diagnostic tests for Lyme disease rely on detection of antigens derived from B. burgdorferi, which are prone to both false positives and false negatives. Instead of focusing only on the human host for the diagnosis of Lyme disease, one could also attempt to identify the vector (tick) and the causative organism carried by the tick. Since all ticks do not transmit Lyme disease, it can be informative to accurately identify the tick from the site of bite, which is often observed by the patient and discarded. However, identifying ticks based on morphology alone requires a trained operator and can still be incorrect. Thus, we decided to take a molecular approach by sequencing DNA and RNA from a tick collected from an individual bitten by the tick. Using next-generation sequencing, we confirmed the identity of the tick as a dog tick, Dermacentor variabilis, and did not identify any pathogenic bacterial sequences, including Borrelia species. Despite the limited availability of nucleotide sequences for many types of ticks, our approach correctly identified the tick species. This proof-of-principle study demonstrates the potential of next-generation sequencing in the diagnosis of tick-borne infections, which can also be extended to other zoonotic diseases.
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http://dx.doi.org/10.1089/omi.2018.0089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112187PMC
August 2018

Phosphoproteomics of Retinoblastoma: A Pilot Study Identifies Aberrant Kinases.

Molecules 2018 Jun 15;23(6). Epub 2018 Jun 15.

Department of Nanotechnology, Vision Research Foundation, Sankara Nethralaya, Chennai, Tamil Nadu 600 006, India.

Retinoblastoma is a malignant tumour of the retina which most often occurs in children. Earlier studies on retinoblastoma have concentrated on the identification of key players in the disease and have not provided information on activated/inhibited signalling pathways. The dysregulation of protein phosphorylation in cancer provides clues about the affected signalling cascades in cancer. Phosphoproteomics is an ideal tool for the study of phosphorylation changes in proteins. Hence, global phosphoproteomics of retinoblastoma (RB) was carried out to identify signalling events associated with this cancer. Over 350 proteins showed differential phosphorylation in RB compared to control retina. Our study identified stress response proteins to be hyperphosphorylated in RB which included H2A histone family member X (H2AFX) and sirtuin 1. In particular, Ser140 of H2AFX also known as gamma-H2AX was found to be hyperphosphorylated in retinoblastoma, which indicated the activation of DNA damage response pathways. We also observed the activation of anti-apoptosis in retinoblastoma compared to control. These observations showed the activation of survival pathways in retinoblastoma. The identification of hyperphosphorylated protein kinases including Bromodomain containing 4 (BRD4), Lysine deficient protein kinase 1 (WNK1), and Cyclin-dependent kinase 1 (CDK1) in RB opens new avenues for the treatment of RB. These kinases can be considered as probable therapeutic targets for RB, as small-molecule inhibitors for some of these kinases are already in clinical trials for the treatment other cancers.
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http://dx.doi.org/10.3390/molecules23061454DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6100359PMC
June 2018

Membrane Proteome of Invasive Retinoblastoma: Differential Proteins and Biomarkers.

Proteomics Clin Appl 2018 09 13;12(5):e1700101. Epub 2018 Jun 13.

Department of Nano-Biotechnology, Vision Research Foundation, Sankara Nethralya, Chennai, 600006, Tamil Nadu, India.

Purpose: Retinoblastoma (RB) is a pediatric ocular cancer which is caused due to the aberrations in the RB1 gene. The changes in the membrane proteomics would help in understanding the development of the retinoblastoma and could identify candidates for biomarkers and therapy.

Experimental Design: Quantitative proteomics is performed on the enriched membrane fractions from pooled normal retina (n = 5) and pooled retinoblastoma tissues (n = 5). The proteins are tryptic-digested and tagged with iTRAQ labels. Orbitrap mass spectrometry is used to analyze and quantify the deregulated membrane proteins involved in the RB tumor progression. Immunohistochemistry (IHC) is used to further validate few of the differentially expressed proteins.

Results: A total of 3122 proteins are identified of which, 663 proteins are found to be deregulated with ≥two fold change in the RB tumor compared to the retina. 282 proteins are upregulated and 381 are downregulated with ≥2 peptide identifications. Bioinformatic analysis revealed that, most of the proteins are involved in the transport, cellular communication, and growth. Overexpression of lamin B1 (LMNB1) and transferrin receptor (TFRC) are observed in RB tumors using IHC.

Conclusion And Clinical Relevance: The present study, is the first comprehensive quantitative membrane proteomic atlas of the differentially regulated proteins in RB compared to the retina. LMNB1 and TFRC could be potential biomarkers for this childhood cancer.
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http://dx.doi.org/10.1002/prca.201700101DOI Listing
September 2018

Proteomics of the Human Olfactory Tract.

OMICS 2018 01;22(1):77-87

1 Institute of Bioinformatics , International Technology Park, Bangalore, India .

Human olfactory tract plays a fundamental role in health and disease. Proteomic analysis of the olfactory tract therefore bears fundamental importance for integrative biology and clinical medicine. For example, olfactory dysfunction is one of the earliest findings in neurodegenerative disorders. The objective of the present study was to build the proteome data from human olfactory tract using a mass spectrometry-based approach. We performed a shotgun proteomic analysis of the human olfactory tract obtained from three healthy adult male subjects. The proteomics workflow consisted of fractionation based on high pH reverse phase liquid chromatography and SDS-PAGE, followed by liquid chromatography tandem-mass spectrometry (LC-MS/MS) analysis on high-resolution mass spectrometer. In total, 6055 proteins were identified, which were further subjected to bioinformatics analysis and contextualization to identify the associated biological processes and molecular functions. We found the identified proteins involved in processes and functions related to olfactory perception, cell to cell adhesion, cellular and G-coupled receptor activity, axonal growth, and transportation. Importantly, we report the identification of 83 olfactory tract-restricted proteins, 4 seven-transmembrane proteins, and 14 protein kinases. Pathway analysis of the restricted proteins revealed the enrichment of olfactory transduction, adherens junction, taste transduction, and neurotropic signaling pathways. To the best of our knowledge, this is the first study to report the human olfactory tract proteome. The study contributes to the knowledge of the human brain proteome and forms a crucial knowledge base for future applications in basic and clinical research, especially in olfactory sensation and neurodegenerative human disorders.
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http://dx.doi.org/10.1089/omi.2017.0155DOI Listing
January 2018

BioSITe: A Method for Direct Detection and Quantitation of Site-Specific Biotinylation.

J Proteome Res 2018 02 28;17(2):759-769. Epub 2017 Dec 28.

McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.

Biotin-based labeling strategies are widely employed to study protein-protein interactions, subcellular proteomes and post-translational modifications, as well as, used in drug discovery. While the high affinity of streptavidin for biotin greatly facilitates the capture of biotinylated proteins, it still presents a challenge, as currently employed, for the recovery of biotinylated peptides. Here we describe a strategy designated Biotinylation Site Identification Technology (BioSITe) for the capture of biotinylated peptides for LC-MS/MS analyses. We demonstrate the utility of BioSITe when applied to proximity-dependent labeling methods, APEX and BioID, as well as biotin-based click chemistry strategies for identifying O-GlcNAc-modified sites. We demonstrate the use of isotopically labeled biotin for quantitative BioSITe experiments that simplify differential interactome analysis and obviate the need for metabolic labeling strategies such as SILAC. Our data also highlight the potential value of site-specific biotinylation in providing spatial and topological information about proteins and protein complexes. Overall, we anticipate that BioSITe will replace the conventional methods in studies where detection of biotinylation sites is important.
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http://dx.doi.org/10.1021/acs.jproteome.7b00775DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092923PMC
February 2018
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