Comprehensive Overview of Bottom-up Proteomics using Mass Spectrometry

Proteomics is the large scale study of protein structure and function from biological systems through protein identification and quantification. "Shotgun proteomics" or "bottom-up proteomics" is the prevailing strategy, in which proteins are hydrolyzed into peptides that are analyzed by mass spectrometry. Proteomics studies can be applied to diverse studies ranging from simple protein identification to studies of proteoforms, protein-protein interactions, protein structural alterations, absolute and relative protein quantification, post-translational modifications, and protein stability. To enable this range of different experiments, there are diverse strategies for proteome analysis. The nuances of how proteomic workflows differ may be challenging to understand for new practitioners. Here, we provide a comprehensive overview of different proteomics methods to aid the novice and experienced researcher. We cover from biochemistry basics and protein extraction to biological interpretation and orthogonal validation. We expect this work to serve as a basic resource for new practitioners in the field of shotgun or bottom-up proteomics

The Need for Next-Generation Antivenom for Snakebite Envenomation in India

The limitations posed by currently available antivenoms have emphasized the need for alternative treatments to counteract snakebite envenomation. Even though exact epidemiological data are lacking, reports have indicated that most global snakebite deaths are reported in India. Among the many problems associated with snakebite envenomation, issues related to the availability of safer and more efficient antivenoms are of primary concern. Since India has the highest number of global snakebite deaths, efforts should be made to reduce the burden associated with snakebite envenoming. Alternative methods, including aptamers, camel antivenoms, phage display techniques for generating high-affinity antibodies and antibody fragments, small-molecule inhibitors, and natural products, are currently being investigated for their effectiveness. These alternative methods have shown promise in vitro, but their in vivo effectiveness should also be evaluated. In this review, the issues associated with Indian polyvalent antivenoms in neutralizing venom components from geographically distant species are discussed in detail. In a nutshell, this review gives an overview of the current drawbacks of using animal-derived antivenoms and several alternative strategies that are currently being widely explored

Additional file 5: Figure S3. of Weight control interventions improve therapeutic efficacy of dacarbazine in melanoma by reversing obesity-induced drug resistance

Effect of inhibition of FASN, Cav-1, and P-gp on response of B16F1 cells to DTIC. B16F1 cells were chronically grown in medium containing 5% serum collected from experimental ND or HFD C57BL/6J mice for 15 days. Thereafter, these cells were subjected to long-term survival assay. First, cells were treated with respective inhibitors followed by treatment of DTIC for 48 h. Then, the medium was changed and fresh medium was added. The medium was changed every 2–3 days. After 10 days, the cells were stained with 0.05% crystal violet and images were taken using Olympus digital camera. Data were quantitated using ImageJ software. The data are representative of experiments performed three times; Ceru or C = cerulenin; MCD or M = methyl β-cyclodextrin; Vera or V = verapamil. The results are given as means ± standard deviation; *, p < 0.05. (PDF 666 kb

Additional file 8: Figure S6. of Weight control interventions improve therapeutic efficacy of dacarbazine in melanoma by reversing obesity-induced drug resistance

Effect of inhibiting FASN, Cav-1, and P-gp on response of B16F1 cells to DTIC upon culture in CM collected from 3T3-L1 cells. 3T3-L1 cells were induced to differentiate with 500 μM 3-isobutyl-1-methylxanthine (IBMX) and 250 μM dexamethasone (DEX). The medium was changed every alternate day. After 10 days, cells were washed twice with DMEM and fresh DMEM without serum was added to the cells. After 18 h, conditioned medium (CM) was collected from undifferentiated or differentiated 3T3-L1 cells. Thereafter, B16F10 or B16F1 cells were cultured in these CM for 48 h. First, cells were treated with respective inhibitors followed by treatment of DTIC for 48 h. Then, the medium was changed and fresh medium was added. The medium was changed every 2–3 days. After 10 days, the cells were stained with 0.05% crystal violet and images were taken using Olympus digital camera. Data were quantitated using ImageJ software. The data are representative of experiments performed three times; PA = preadipocytes; ID = differentiated 3T3-L1 cells induced by IBMX and DEX; Ceru or C = cerulenin; MCD or M = methyl β-cyclodextrin; Vera or V = verapamil. The results are given as means ± standard deviation; *, p < 0.05. (PDF 538 kb

Additional file 6: Figure S4. of Weight control interventions improve therapeutic efficacy of dacarbazine in melanoma by reversing obesity-induced drug resistance

Effect of adipocyte-secreted factors on the protein level of P-gp, Cav-1, and FASN in B16F1 cells. 3T3-L1 cells were induced to differentiate with 500 μM 3-isobutyl-1-methylxanthine (IBMX) and 250 μM dexamethasone (DEX). The medium was changed every alternate day. After 10 days, cells were washed twice with DMEM and fresh DMEM without serum was added to the cells. After 18 h, conditioned medium (CM) was collected from undifferentiated or differentiated 3T3-L1 cells. Thereafter, B16F1 cells were cultured in these CM for 48 h, and these cells were subjected to immunofluorescence confocal staining for the indicated molecules. The data were recorded using Zeiss LSM510 META Confocal Microscope (Scale bar = 20 μm); PA = preadipocytes; ID = differentiated 3T3-L1 cells induced to differentiate by IBMX and DEX. (PDF 215 kb

Additional file 9: Figure S7. of Weight control interventions improve therapeutic efficacy of dacarbazine in melanoma by reversing obesity-induced drug resistance

MS analysis on the distribution of DTIC in tumors and organ samples collected from experimental ND or HFD mice. Lysates of tumors and organ samples from the experimental ND or HFD mice were prepared and subjected to liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI MS/MS). The spectrum shows protonated mass of DTIC at 183.0984, m/z and the product ions at m/z, 166.0733, 138.0396, and 123.0427 (similar fragmentation patterns were observed in tumors and tissue samples of mice treated with DTIC). The inset contains chemical structure of DTIC with the fragmentation patterns marked. All the MS data were collected in the presence of internal standards, where mass tolerance of DTIC was maintained well within 3 ppm. (PDF 122 kb

Additional file 1: Table S1. of Weight control interventions improve therapeutic efficacy of dacarbazine in melanoma by reversing obesity-induced drug resistance

Composition of diets used in the study. Normal diet (ND) was procured from Amrut Laboratory Animal Feed, Pune, India, and high fat diet (HFD) was purchased from Provimi Animal Nutrition Pvt. Ltd., Bangalore, India. *, HFD was also supplemented with 400 g groundnut and 200 g dried coconut per kg body weight of mice. (PDF 169 kb

Additional file 7: Figure S5. of Weight control interventions improve therapeutic efficacy of dacarbazine in melanoma by reversing obesity-induced drug resistance

Effect of adipocyte-secreted factors on Rh-123 efflux in B16F1 cells. 3T3-L1 cells were induced to differentiate with 500 μM 3-isobutyl-1-methylxanthine (IBMX) and 250 μM dexamethasone (DEX). The medium was changed every alternate day. After 10 days, cells were washed twice with DMEM and fresh DMEM without serum was added to the cells. After 18 h, conditioned medium (CM) was collected from undifferentiated or differentiated 3T3-L1 cells. Thereafter, B16F10 or B16F1 cells were cultured in these CM for 48 h. Further, these cells were subjected to Rh-123 efflux assay. Data were acquired on FACS Calibur and analyzed using BD CellQuest Pro software. The data are representative of experiments performed three times; PA = preadipocytes; ID = differentiated 3T3-L1 cells induced by IBMX and DEX; Vera = verapamil. (PDF 150 kb