A Simple Protein Extraction Method for Proteomic Analysis of Diverse Biological Specimens

Reddy, Panga Jaipal; Malhotra, Deepti; Sharma, Samridhi; Kumar, Romesh; Jain, Rekha; Gollapalli, Kishore; Pendharkar, Namita; Rapole, Srikanth; Srivastava, Sanjeeva

doi:10.2174/15701646113106660004

Cited by 31 publications

(29 citation statements)

References 53 publications

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“…Total protein was extracted from FC2 cells grown for 40 h, 88 h and 120 h following N-starvation and 0 h (control) using TRIzol reagent following standard protocol with slight modification95. Briefly, the algal pellet was incubated in PBS (pH 7.4) containing lysozyme (1 mg mL −1 ) and protease inhibitor cocktail at ambient temperature for 3 h, followed by sonication for 10 min (30 s ON and 30 s OFF cycle) and centrifuged at 12,000× g for 15 min.…”

Section: Methodsmentioning

confidence: 99%

Real-time iTRAQ-based proteome profiling revealed the central metabolism involved in nitrogen starvation induced lipid accumulation in microalgae

Rai

Muthuraj

Gandhi

et al. 2017

Sci Rep

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To understand the post-transcriptional molecular mechanisms attributing to oleaginousness in microalgae challenged with nitrogen starvation (N-starvation), the longitudinal proteome dynamics of Chlorella sp. FC2 IITG was investigated using multipronged quantitative proteomics and multiple reaction monitoring assays. Physiological data suggested a remarkably enhanced lipid accumulation with concomitant reduction in carbon flux towards carbohydrate, protein and chlorophyll biosynthesis. The proteomics-based investigations identified the down-regulation of enzymes involved in chlorophyll biosynthesis (porphobilinogen deaminase) and photosynthetic carbon fixation (sedoheptulose-1,7 bisphosphate and phosphoribulokinase). Profound up-regulation of hydroxyacyl-ACP dehydrogenase and enoyl-ACP reductase ascertained lipid accumulation. The carbon skeletons to be integrated into lipid precursors were regenerated by glycolysis, β-oxidation and TCA cycle. The enhanced expression of glycolysis and pentose phosphate pathway enzymes indicates heightened energy needs of FC2 cells for the sustenance of N-starvation. FC2 cells strategically reserved nitrogen by incorporating it into the TCA-cycle intermediates to form amino acids; particularly the enzymes involved in the biosynthesis of glutamate, aspartate and arginine were up-regulated. Regulation of arginine, superoxide dismutase, thioredoxin-peroxiredoxin, lipocalin, serine-hydroxymethyltransferase, cysteine synthase, and octanoyltransferase play a critical role in maintaining cellular homeostasis during N-starvation. These findings may provide a rationale for genetic engineering of microalgae, which may enable synchronized biomass and lipid synthesis.

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Section: Methodsmentioning

confidence: 99%

Real-time iTRAQ-based proteome profiling revealed the central metabolism involved in nitrogen starvation induced lipid accumulation in microalgae

Rai

Muthuraj

Gandhi

et al. 2017

Sci Rep

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“…The in-house standardized protein extraction protocol was followed for this study (Reddy et al, 2013). In brief, plumbagin was added to the freshly growing B. subtilis cultures at OD 0.2 and grown further for 2 h to reach midexponential phase, whereas DMSO was added to the control.…”

Section: Whole Cell Protein Extractionmentioning

confidence: 99%

“…IEF was carried out with the same parameters as reported previously (Reddy et al, 2013). Focused IPG strips were equilibrated with DTT and IAA for 15 min each.…”

Section: Whole Cell Protein Extractionmentioning

confidence: 99%

“…Differentially expressed (plumbagin treated vs. control) statistically significant ( p £ 0.05) protein spots were excised from the 2-DE gels and in-gel digestion was performed following the same protocol reported previously (Shevchenko et al, 2006;Reddy et al, 2013;Rao et al, 2014). In brief, the gel pieces were washed with 25 mm ABC and Sol-A (1:2 of 25 mM ABC and acetonitrile), followed by reduction (10 mM DTT in 100 mM ABC) for 1 h at 65°C and alkylation (50 mM IAA in 100 mM ABC) for 30 min in dark.…”

Section: In-gel Digestion and Protein Identificationmentioning

confidence: 99%

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Proteomics Analyses ofBacillus subtilisafter Treatment with Plumbagin, a Plant-Derived Naphthoquinone

Reddy

Sathe

et al. 2015

OMICS: A Journal of Integrative Biology

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Infectious diseases and increasing antibiotic resistance among diverse classes of microbes are global health concerns and a prime focus of omics systems science applications in novel drug discovery. Plumbagin is a plant-derived naphthoquinone, a natural product that exhibits antibacterial activity against gram-positive bacteria. In the present study, we investigated the antimicrobial effects of plumbagin against Bacillus subtilis using two complementary proteomics techniques: two-dimensional electrophoresis (2-DE) and isobaric tag for relative and absolute quantification (iTRAQ). Comparative quantitative proteomics analysis of plumbagin treated and untreated control samples identified differential expression of 230 proteins (1% FDR, 1.5 fold-change and ‡ 2 peptides) in B. subtilis after plumbagin treatment. Pathway analysis involving the differentially expressed proteins suggested that plumbagin effectively increases heme and protein biosynthesis, whereas fatty acid synthesis was significantly reduced. Gene expression and metabolic activity assays further corroborated the proteomics findings. We anticipate that plumbagin blocks the cell division by altering the membrane permeability required for energy generation. This is the first report, to the best of our knowledge, offering new insights, at proteome level, for the putative mode(s) of action of plumbagin and attendant cellular targets in B. subtilis. The findings also suggest new ways forward for the modern omics-guided drug target discovery, building on traditional plant medicine.

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“…The TRIzol protocol was designed using protocol with minor modifications [32]. In brief, to the cell lysate 1 mL of TRIzol reagent and 200 lL of chloroform were added, mixed vigorously and centrifugation was carried out at 14,0009g for 15 min at 4°C.…”

Section: Protein Extractionmentioning

confidence: 99%

Differential proteomic analysis of respiratory samples from patients suffering from influenza

et al. 2016

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The exact molecular pathways involved in the pathogenesis of influenza are yet unclear. In the present study we investigated the upper respiratory proteome in influenza patients. 200 nasal and throat swab samples were collected from patients suffering from acute respiratory illness. These samples were confirmed for influenza pandemic A/H1N1/2009 and influenza type B using qRT-PCR. 10 similar swabs were collected from healthy individuals and were used as controls. Proteins were extracted from the cell pellets and were subjected to 2-D gel electrophoresis. The differentially expressed proteins were identified using MALDI-TOF. Identified proteins were classified into different functional groups based on functions reported in the databases. 25 % of these proteins were involved in cytoskeletal formation, whereas 14 % were involved in signal transduction. Proteins involved in anti-viral responses, Ca-signaling, transport, and tumor suppression constituted 10 % each, where as 5 % of proteins each belong to Nicotinic acetylcholine receptor, Protein Synthesis and anti-bacterial proteins. 10 % of the proteins have not been described previously. This is the first report on respiratory proteome profile in Influenza patients. The study emphasizes the role of such profiling studies using multiple platforms for bio-marker discoveries, especially non-invasive diagnostic marker in Influenza and other infectious diseases.

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A Simple Protein Extraction Method for Proteomic Analysis of Diverse Biological Specimens

Cited by 31 publications

References 53 publications

Real-time iTRAQ-based proteome profiling revealed the central metabolism involved in nitrogen starvation induced lipid accumulation in microalgae

Real-time iTRAQ-based proteome profiling revealed the central metabolism involved in nitrogen starvation induced lipid accumulation in microalgae

Proteomics Analyses ofBacillus subtilisafter Treatment with Plumbagin, a Plant-Derived Naphthoquinone

Differential proteomic analysis of respiratory samples from patients suffering from influenza

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