| 121 |
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.
Read more
...Read less
|
CSIR-Centre for Cellular and Molecular Biology, Hyderabad, India |
Bottom-up |
2025-06-18 |
23692254
|
| 122 |
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.
|
THSTI - National Chair and Head, Drug Discovery Research Centre Translational Health Science and Technology Institute, Faridabad, INDIA |
Affinity purification coupled with mass spectrometry proteomics |
2025-08-06 |
28243621
|
| 123 |
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.
Read more
...Read less
|
THSTI - National Chair and Head, Drug Discovery Research Centre Translational Health Science and Technology Institute, Faridabad, INDIA |
Shotgun proteomics |
2025-08-06 |
|
| 124 |
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.
Read more
...Read less
|
THSTI - National Chair and Head, Drug Discovery Research Centre Translational Health Science and Technology Institute, Faridabad, INDIA |
Affinity purification coupled with mass spectrometry proteomics |
2025-08-07 |
|
| 125 |
IPD4918 |
Identification of Potential Protein Biomarkers for Early Detection of Pregnancy in Cow Urine uses 2D DIGE and Label Free quantitation |
Dr. Ashok Kumar Mohanty |
Background: An early, reliable and noninvasive method of pregnancy diagnosis is a prerequisite for efficient reproductive management in dairy industry. The early detection of pregnancy also helps to reduce the calving interval and rebreeding time which is beneficial for farmers and dairy industries. The aim of this work to identify...
Background: An early, reliable and noninvasive method of pregnancy diagnosis is a prerequisite for efficient reproductive management in dairy industry. The early detection of pregnancy also helps to reduce the calving interval and rebreeding time which is beneficial for farmers and dairy industries. The aim of this work to identify potential biomarker for pregnancy detection at earlier stages (16-25 days). To achieve this goal, we performed differential in gel electrophoresis (DIGE) and label free quantitation (LFQ) for identification of protein which have significant differential expression during pregnancy. Results: DIGE experiment revealed eleven differentially expressed proteins out of which nine proteins were up regulated having fold change ≥1.5. The LFQ data analysis gave 202 differentially expressed protein out of 30 proteins were up-regulated and 40 down regulated having significant fold change ≥1.5 and ≤0.6 respectively. Further bioinformatic analysis showed that majority of proteins was involved in regulation of leukocyte immunity, endopeptidase inhibitor activity, regulation of peptidase activity and polysaccharide binding. Conclusion: To the best of our knowledge, this is first report on identification of differentially expressed proteins in urine of cows during various time points of pregnancy using DIGE and LFQ. In our investigation, we have discussed functional significance of few selected proteins such as A2HS, MBP, GRP, IGFBP-II, SERPIN, Vitamin D binding protein etc which were differentially expressed and actively involved in pregnancy associated events such as embryo implantation, establishment and maintenance of pregnancy. Thus, we have identified a set of potential protein biomarkers for early detection of pregnancy.
Read more
...Read less
|
Proteomics and Structural Biology Lab, Animal biotechnology Center, National Dairy Research Institute, Karnal, Haryana, India |
Shotgun proteomics, Gel-based experiment |
2025-08-15 |
27429603
|
| 126 |
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.
Read more
...Read less
|
Proteomics and Structural Biology Lab, Animal biotechnology Center, National Dairy Research Institute, Karnal, Haryana, India |
Shotgun proteomics |
2025-08-15 |
32179766
|
| 127 |
IPD2682 |
Profiling of cow urinary proteins using various extraction methods reveals more than 1550 proteins |
Dr. Ashok Kumar Mohanty |
Urine can help in diagnosis of different diseases including cancer and other patho-physiological conditions. Urine proteome studies have been mainly human- centric. No information is available on urinary proteome from bovine till date. In the present study, we have used 3 protein extraction methods such as ammonium sulphate precipitation, ProteoSpin...
Urine can help in diagnosis of different diseases including cancer and other patho-physiological conditions. Urine proteome studies have been mainly human- centric. No information is available on urinary proteome from bovine till date. In the present study, we have used 3 protein extraction methods such as ammonium sulphate precipitation, ProteoSpin column and diafiltration method from bovine urine for identification of urinary proteome. The tryptic peptides generated after In-gel and In-solution method were identified using LC/MS/MS (ESI-qTOF) which resulted in identification of 1582 proteins. In-gel trypsin digestion method revealed more protein (1191) in comparison to in-solution digestion method (541). Maximum proteins were identified in ammonium sulphate precipitation method (938) followed by ProteoSpin (606) and diafiltration (444) methods respectively. The profile of the identified proteins were compared with human urinary proteome of which 311 bovine urinary proteins matched with human. An exclusive list of 38 bovine urinary proteins with high protein scores were listed which are absent in human urine. All identified proteins were analyzed according to Gene Ontology which were classified according to cellular component, biological processes and molecular functions. This study reports for the first time an exclusive evidence of more than 1550 proteins in urine of healthy cow donors.
Read more
...Read less
|
Proteomics and Structural Biology Lab, Animal biotechnology Center, National Dairy Research Institute, Karnal, Haryana, India |
Shotgun proteomics, Gel-based experiment |
2025-08-15 |
26021477
|
| 128 |
IPD7996 |
Vibrio cholerae (p)ppGpp and DksA mutants SWATH |
Prof.Bhabatosh Das |
This study elucidates the whole proteome changes in the wild type and gnetically modified strains of Vibrio cholerae. Label-free SWATH-MS is used for protein quantitation following genetic modifications in alarmone (p)ppGpp metabolizing genes and transcription factor DksA.
|
DBT-Translational Health Science and Technology Institute, Faridabad, Haryana, India |
SWATH MS |
2025-11-27 |
39992161
|
| 129 |
IPD8604 |
MDR K. penumoniae, A. baumannii, E. coli, and P. aeruginiosa SWATH |
Prof.Bhabatosh Das |
This study elucidates the whole proteome changes in the MDR isolates of K. penumoniae, A. baumannii, E. coli, and P. aeruginiosa in the presence of Ampicillin, Kanamycin, and Nalidixic acid. Label-free SWATH-MS is used for protein quantitation of antibiotics treated and untreated samples.
|
DBT-Translational Health Science and Technology Institute, Faridabad, Haryana, India |
SWATH MS |
2025-11-27 |
|
| 130 |
IPD6285 |
Temporal proteome profiling of Botrytis cinerea reveals proteins involved in plant invasion and survival |
Dr. Nidhi Adlakha |
Botrytis cinerea is a necrotrophic fungal pathogen that poses a significant threat to many
crops. Understanding the proteome dynamics of phytopathogens during infection can help
combat plant diseases. However, most proteomics studies in phytopathogens face
interference from abundant host proteins. Here, we optimized a solid media that better
mimics in-planta conditions and used it...
Botrytis cinerea is a necrotrophic fungal pathogen that poses a significant threat to many
crops. Understanding the proteome dynamics of phytopathogens during infection can help
combat plant diseases. However, most proteomics studies in phytopathogens face
interference from abundant host proteins. Here, we optimized a solid media that better
mimics in-planta conditions and used it to perform the temporal protein dynamics in Botrytis
cinerea. An agar media with 20% tomato fruit extract and 2% deproteinised leaf extract was
utilized for label-free quantitative proteomics at 12, 36, 72 and 120 hpi. Out of 3244
quantified proteins, 2045 showed differential regulation. Glycosyl hydrolases, pectin
esterases, stress protein DDR48, RhoGEF and essential transcription factors were found to be
upregulated during the early phase, highlighting their role in fungal virulence. Meanwhile,
pathways such as macromolecule synthesis, purine, and carbohydrate metabolism were
upregulated in the late-growth phase. Overall, the study provides a comprehensive
understanding of proteome dynamics during Botrytis infection.
Read more
...Read less
|
Department of Biotechnology, Central University Of Haryana |
Bottom-up |
2025-11-28 |
|
