| 41 |
IPD9367 |
Contryphan sequence diversity:Conus amadis |
Prof. P. Balaram |
Mass Spectrometric Characterisation of contryphans.
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Molecular Biophysics Unit, Indian Institute of Science, Bangalore, Karnataka, India |
Top-down |
2025-05-25 |
36587728
|
| 42 |
IPD2486 |
Mass spectral Analysis of Synthetic Peptides: Implications in Proteomics |
Dr. Medicharla Venkata Jagannadham |
Tryptic synthetic peptides generated in silico from human proteome are used as standards to evaluate the performance of MS and algorithm used to identify the proteins.
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Centre for Cellular and Molecular Biology, Hyderabad, Telangana, India |
Shotgun proteomics |
2025-06-18 |
33953644
|
| 43 |
IPD5120 |
Characterization of the novel broad-spectrum kinase inhibitor CTx-0294885 as an affinity reagent for mass spectrometry-based kinome profiling |
Dr. Medicharla Venkata Jagannadham |
Kinome profiling of human basal breast cancer cell line MDA-MB-231 using CTx-0294885 or mixture of 4 broad-spectrum kinase inhibitor (Purvalanol B, SU6668, VI16832 and CTx-0294885). Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still...
Kinome profiling of human basal breast cancer cell line MDA-MB-231 using CTx-0294885 or mixture of 4 broad-spectrum kinase inhibitor (Purvalanol B, SU6668, VI16832 and CTx-0294885). Kinase enrichment utilizing broad-spectrum kinase inhibitors enables the identification of large proportions of the expressed kinome by mass spectrometry. However, the existing inhibitors are still inadequate in covering the entire kinome. Here, we identified a novel bis-anilino pyrimidine, CTx-0294885, exhibiting inhibitory activity against a broad range of kinases in vitro, and further developed it into a Sepharose supported kinase capture reagent. Use of a quantitative proteomics approach confirmed the selectivity of CTx-0294885-bound beads for kinase enrichment. Large-scale CTx-0294885-based affinity purification followed by LC-MS/MS led to the identification of 235 protein kinases from MDA-MB-231 cells, including all members of the AKT family that had not been previously detected by other broad spectrum kinase inhibitors. Addition of CTx-0294885 to a mixture of three kinase inhibitors commonly used for kinase-enrichment increased the number of kinase identifications to 261, representing the largest kinome coverage from a single cell line reported to date. Coupling phosphopeptide enrichment with affinity purification using the four inhibitors enabled the identification of 799 high confidence phosphosites on 183 kinases, approximately 10 % of which were localized to the activation loop, and included previously unreported phosphosites on BMP2K, MELK, HIPK2 and PRKDC. Therefore, CTx 0294885 represents a powerful new reagent for analysis of kinome signalling networks that may facilitate development of targeted therapeutic strategies. Data processing and bioinformatics: Raw files were processed with MaxQuant (version 1.1.1.25) for feature detection, protein identification and quantification, using the Andromeda search engine integrated into the MaxQuant environment for database searching. Extracted peak lists were searched against the UniProtKB/Swiss-Prot Homo sapiens database (Uniprot_human_2010_10) containing 35052 entries and a separate reverse decoy database for controlling the false discovery rate (FDR). The following search parameters were selected; fixed cysteine carbamidomethylation modification; variable methionine oxidation modification, variable protein N-acetylation, variable phosphorylation of serine, threonine and tyrosine; minimum peptide length of 6 amino acids and up to 2 missed cleavages were allowed. In addition, for SILAC experiments, the SILAC labels Arg10 and Lys8 were selected as modifications, and minimum peptide count for protein quantification was set to 1. The initial first search mass tolerance was 20 ppm for precursor ions and 0.5 Da for fragment ions, with individualized peptide mass tolerances used for the subsequent searches. The ‘match between runs’ option in MaxQuant was used to transfer identifications between runs based on matching of precursors with high mass accuracy. The FDR was limited to 1 % for both protein and peptide identifications. Peptides with posterior error probability greater than 10 % were removed and protein identification required a minimum of 1 unique peptide. For phosphopeptides, those exhibiting a phosphosite localization probability (LP) > 0.75 were included in further analyses.
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Centre for Cellular and Molecular Biology, Hyderabad, Telangana, India |
Bottom-up |
2025-06-18 |
23692254
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| 44 |
IPD7455 |
Phosphoproteomic Analysis of Lethal Castration Resistant Prostate Cancer Reveals Patient but not Metastatic Site Heterogeneity of Tyrosine Kinase Activation |
Dr. Medicharla Venkata Jagannadham |
Tissue lysis was performed as previously described (Drake, J.M., et al. Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648 (2012)) Briefly, greater than 300 mg of frozen tumor mass was homogenized and sonicated in urea lysis buffer (20 mM...
Tissue lysis was performed as previously described (Drake, J.M., et al. Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648 (2012)) Briefly, greater than 300 mg of frozen tumor mass was homogenized and sonicated in urea lysis buffer (20 mM HEPES pH 8.0, 9 M urea, 2.5 mM sodium pyrophosphate, 1.0 mM beta-glycerophosphate, 1% N-octyl glycoside, 2 mM sodium orthovanadate). Total protein was measured using the BCA Protein Assay Kit (Thermo Scientific/Pierce) and 25 mg of total protein was used for phospho-proteomic analysis. Phospho-tyrosine peptide enrichment and liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was performed as previously described (Drake, J.M., et al. Oncogene-specific activation of tyrosine kinase networks during prostate cancer progression. Proc Natl Acad Sci U S A 109, 1643-1648 (2012); Rubbi, L., et al. Global phosphoproteomics reveals crosstalk between Bcr-Abl and negative feedback mechanisms controlling Src signaling. Sci Signal 4, ra18 (2011); Graham, N.A., et al. Glucose deprivation activates a metabolic and signaling amplification loop leading to cell death. Molecular systems biology 8, 589 (2012)) Phospho-peptides were identified using the Proteome Discoverer software (version 1.3.0.339, Thermo Fisher Scientific). MS/MS fragmentation spectra were searched using SEQUEST against the Uniprot human reference proteome database with canonical and isoform sequences (downloaded January 2012 from uniprot.org). Search parameters included carbamidomethyl cysteine (*C) as a static modification. Dynamic modifications included phosphorylated tyrosine, serine, or threonine (pY, pS, pT, respectively) and oxidized methionine (*M). The Percolator node of Protein Discoverer was used to calculate false-discovery rate (FDR) thresholds and the FDR for the datasets was adjusted to 1% (version 1.17, Thermo Scientific). The Percolator algorithm uses a target-decoy database search strategy and discriminates true and false identifications with a support vector machine. The PhosphoRS 2.0 node was used to more accurately localize the phosphate on the peptide44. Only phospho-peptides with at least one phospho-tyrosine assignment with a reported probability above 20% were considered. MS2 spectra for all reported phosphopeptides are available under the PRIDE accession numbers.
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Centre for Cellular and Molecular Biology, Hyderabad, Telangana, India |
Bottom-up |
2025-06-18 |
24248375
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| 45 |
IPD8488 |
LC-MS analysis of human tumour cell lines |
Dr. Medicharla Venkata Jagannadham |
Main aim of this study was to identify and characterize human hypothetical proteins. These are the protein sequences for which there is no experimental evidence at translation level and are functionally unknown. First part of the project deals with identification and characterization of hypothetical proteins using label-free lc-ms/ms approaches. Second...
Main aim of this study was to identify and characterize human hypothetical proteins. These are the protein sequences for which there is no experimental evidence at translation level and are functionally unknown. First part of the project deals with identification and characterization of hypothetical proteins using label-free lc-ms/ms approaches. Second part deals with providing functional clues to those identified proteins thus connecting the missing links in biological mechanisms.
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Centre for Cellular and Molecular Biology, Hyderabad, Telangana, India |
Gel-based experiment |
2025-06-18 |
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| 46 |
IPD9282 |
Proteins from Outer Membrane Vesicules of Pseudomonas syringae Lz4W |
Dr. Medicharla Venkata Jagannadham |
The proteins from Outer Membrane Vesicles (OMVs) were extracted by chilled acetone method. The proteins were separated on 1D SDS-PAGE (12%). From the gel, total 10 fractions were made and all of them were subjected to in gel digestion using trypsin. The MS/MS spectra of the resulting tryptic peptides were...
The proteins from Outer Membrane Vesicles (OMVs) were extracted by chilled acetone method. The proteins were separated on 1D SDS-PAGE (12%). From the gel, total 10 fractions were made and all of them were subjected to in gel digestion using trypsin. The MS/MS spectra of the resulting tryptic peptides were recordedby using LC coupled ESI-MS/MS (Thermo Orbitrap Velos). The mass spectral data thus obtained was analysed by using Proteome Discoverer 1.3. Since the genome sequence of P. syringae Lz4W is not available, the data was searched against a database prepared from 20 related Pseudomonas species whose genome sequence is available on Uniprot (Updated upto Jan 2013). The search was observed by using nodes Sequest and Mascot both, the enzyme selected was trypsin with maximum 2 missed cleavages, the precursor tolerance set at 10 ppm, fragment tolerance at 0.8 Da, carbamidomethylated cystein (57.02 Da) as fixed modification, oxidised methionine (15.99 Da) as variable modification. After the search is over, the results were refined applying result filters as Peptide confidence (High) and Differentiable Proteins (including distinct proteins), which makes sure that each protein entry in the list is identified with at least one unique peptide.
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Centre for Cellular and Molecular Biology, Hyderabad, Telangana, India |
Bottom-up |
2025-06-18 |
24437924
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| 47 |
IPD2550 |
Mycobacterial strain-specific temporal modulations of newly synthesized macrophage secretome |
Dr. Kanury V.S. Rao |
THP-1 macrophages were infected with four strains of Mycobacterium tuberculosis to study the temporal dynamics of newly synthesized proteins in the secretome. Temporal snapshots of secretome reflect the macrophage response to pathogenicity which in combination with intracellular events, completes the disease picture. However, such studies are compromised by limitations...
THP-1 macrophages were infected with four strains of Mycobacterium tuberculosis to study the temporal dynamics of newly synthesized proteins in the secretome. Temporal snapshots of secretome reflect the macrophage response to pathogenicity which in combination with intracellular events, completes the disease picture. However, such studies are compromised by limitations of quantitative proteomics. Metabolic labeling by SILAC allows a 3-plex experiment while isobaric chemical labeling by iTRAQ/TMT allows up to 8 to 10-plex respectively. This makes studying temporal proteome dynamics an intangible and elusive proposition. We have developed a new variant of hyperplexing method, combining triplex SILAC with 6-plex iTRAQ to achieve 18-plex quantitation in a single MS run. THP-1 macrophages were infected with H37Ra, H37Rv, BND433 and JAL2287 and the newly synthesized secreted host proteins were studied over six temporal frame still 30 hours post infection, at a difference of 4 hours each. For quantitation, the strains were encoded with two sets of triple SILAC- H37Ra & H37Rv in one and BND433 & JAL2287 in another with a control in each. These sets were then iTRAQ labeled to encode for temporal profiles across six time points in 6-plex iTRAQ. Effectively a 36-plex design with 4 replicates of each set, these experiments were completed within few days on the mass spectrometer. Using MaxQuant and in house developed tools and pipelines, we have analysed the data to map the temporal and strain specific dynamics of newly synthesized proteins in host. Hyperplexing enables large scale spatio-temporal systems biology studies where large number of samples can be processed simultaneously and in quantitative manner.
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DBT-Translational Health Science and Technology Institute, Faridabad, Haryana, India |
Shotgun proteomics |
2025-08-06 |
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| 48 |
IPD6123 |
Defining the Akt1 interactome and delineating alterations in its composition as a function of cell cycle progression. |
Dr. Kanury V.S. Rao |
Akt1 expressing Hek 293 cells were SILAC labeled to capture dynamic changes in Akt1 interactome as the cell cycle progresses from G0 to G1S and then G2 phase. This will help in understanding how Akt1 extends its regulatory effect upon cell cycle progression.
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DBT-Translational Health Science and Technology Institute, Faridabad, Haryana, India |
Affinity purification coupled with mass spectrometry proteomics |
2025-08-06 |
28243621
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| 49 |
IPD6714 |
Delineating the Rb1 interactome data and its modulations during cell cycle progression. |
Dr. Kanury V.S. Rao |
The retinoblastoma (Rb) protein is a potent tumor suppressor which is known to negatively regulate the cell cycle as well as tumor progression. Phosphorylated Rb protein (pRb) has been demonstrated to be in-charge for the key G1 checkpoint, blocking entry into S-phase and thereby the cell growth. This study was...
The retinoblastoma (Rb) protein is a potent tumor suppressor which is known to negatively regulate the cell cycle as well as tumor progression. Phosphorylated Rb protein (pRb) has been demonstrated to be in-charge for the key G1 checkpoint, blocking entry into S-phase and thereby the cell growth. This study was designed to capture interacting protein partners of Rb1 as the cell cycle progresses. Rb1 expressing HEK-293 cells were cultured in light, medium and heavy SILAC labels to capture the changes in Rb1 interactome as the cell cycle progressed from G0 to G1S and then to G2 phase, respectively. This data might help in understanding the cell cycle regulatory effect of Rb1 protein and complement the available information on its interacting partners.
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DBT-Translational Health Science and Technology Institute, Faridabad, Haryana, India |
Affinity purification coupled with mass spectrometry proteomics |
2025-08-07 |
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| 50 |
IPD1891 |
Deep proteome analysis of more than 12000 proteins in buffalo mammary epithelial cells identifies protein signatures for active proliferation and lactation |
Dr. Ashok Kumar Mohanty |
Extensive branching morphogenesis takes place during pregnancy in the mammary gland. It is accompanied by the rapid proliferation of the Mammary Epithelial Cells (MECs). To gain insights into the proteomic changes that occur during the proliferation of MECs from buffalo (Bubalus bubalis) origin, we explored the deep proteome profile of...
Extensive branching morphogenesis takes place during pregnancy in the mammary gland. It is accompanied by the rapid proliferation of the Mammary Epithelial Cells (MECs). To gain insights into the proteomic changes that occur during the proliferation of MECs from buffalo (Bubalus bubalis) origin, we explored the deep proteome profile of buffalo mammary epithelial cells (BuMECs) using mass spectrometry (MS). To achieve this, we employed the sub-cellular fractionation approach and secretome analysis. Proteins were isolated separately from four subcellular fractions (SCFs) containing cytosolic (SCF-I), membranous and membranous organelle’s (SCF-II), nuclear (SCF-III) and cytoskeletal (SCF-IV). These sub-cellular specific protein fractions were processed using in-solution digestion and analyzed with nano-LCMS/MS. The MS analysis identified 8330, 5970, 5288 and 4818 non-redundant proteins in the fractions SCF I, II, III and IV respectively. To evaluate the secretory proteins in these cells, gel-based proteome approach was used which revealed a total of 792 non-redundant proteins. Altogether, combined analysis of all the five fractions including four sub-cellular fractions and secretome resulted in the identification of 12,609 non-redundant proteins. A total of 325 molecular pathways were identified after extensive analysis. The most enriched pathways associated with these proteins were metabolic, PI3-AKT, MAPK, mTOR, Insulin, estrogen and Oxytocin signaling. Our study demonstrated for the first time highest number of proteins identified by MS in any cell types including MECs.
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ICAR-National Dairy Research Institute, Karnal, Haryana, India |
Shotgun proteomics |
2025-08-15 |
32179766
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