Although within the last few years good number of S-nitrosylated proteins are identified but information on endogenous targets is still limiting. as target repeatedly due to its abundance. It also competes out low abundant proteins which are important NO signaling components. Therefore, RuBisCO was removed (over 80%) using immunoaffinity purification. Purified S-nitrosylated RuBisCO depleted proteins were resolved on 2-D gel as 110 spots, including 13 new, which were absent in the crude S-nitrosoproteome. These were identified by nLC-MS/MS as thioredoxin, fructose biphosphate aldolase class I, myrosinase, buy MK-2461 salt responsive proteins, peptidyl-prolyl cis-trans isomerase and malate dehydrogenase. Cold showed differential S-nitrosylation of 15 spots, enhanced superoxide dismutase activity (via S-nitrosylation) and promoted the detoxification of superoxide radicals. Increased S-nitrosylation of glyceraldehyde-3-phosphate dehydrogenase sedoheptulose-biphosphatase, and fructose biphosphate aldolase, indicated regulation of Calvin cycle by S-nitrosylation. The results showed that RuBisCO depletion improved proteome coverage and provided clues for NO signaling in cold. (Lindermayr et al., 2005), (Abat et al., 2008), (Abat and Deswal, 2009), (Kato et al., 2012), (Lin et al., 2012) and (Camejo buy MK-2461 et al., 2013). S-nitrosoproteome analysis is mostly done using NO donor because of the low concentration of endogenous S-nitrosothiols (SNOs). It is mandatory to identify and validate the endogenously S-nitrosylated proteins not only to confirm the targets identified using donors but also to understand their physiological relevance. For the identification of S-nitrosylated proteins, biotin switch technique (BST, Jaffrey and Snyder, 2001) is used. It involves the selective reduction of the SNOs by ascorbate, their substitution with biotin and their purification by avidin-affinity chromatography. A major drawback of this procedure is the masking of the low abundant S-nitrosylated proteins by the abundant ones like Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO, Abat and Deswal, 2009), RuBisCO activase (Tanou et al., 2012), glyceraldehydes-3-phosphate dehydrogenase (GAPDH, Maldonado-Alconada et al., 2010) and heat shock proteins (Maldonado-Alconada et al., 2010). These protein saturate the avidin column and contend out the reduced abundant S-nitrosylated protein. Besides hindering the recognition of the reduced abundant goals, these waste valuable commitment during MS identification also. This prompted us to eliminate RuBisCO to boost the probability of obtaining the regulatory S-nitrosylated goals. Lately, a NO-cold crosstalk was suggested at genes, protein and lipid level, however the regulatory systems involved remain elusive (Sehrawat et al., 2013). As a result, to obtain a better knowledge of these signaling pathways, id from the regulatory goals is vital. Previously, cool mediated inhibition of RuBisCO by S-nitrosylation was proven (Abat and Deswal, 2009), in the similar lines other signaling buy MK-2461 goals have to be validated functionaly. Therefore, the purpose of this research was to show if the repertoire of cool responsive S-nitrosoproteome could possibly be enriched by detatching RuBisCO. Furthermore, the result of S-nitrosylation in the superoxide dismtase (SOD) activity, a cool responsive S-nitrosylated focus on (determined in this research), was validated to comprehend its legislation by NO. Furthermore, to determine the NO signaling in Layn cold, NO production and modulation of the thiol pool by NO was measured. Materials and methods Herb material and growth conditions var. pusa jaikisan seeds were obtained from The Indian Agricultural Research Institute, New Delhi, India. Seeds were surface sterilized with 70% ethanol for 10 min and soaked overnight in double distilled water. Seeds buy MK-2461 were placed in the wet germination paper rolls and kept overnight in dark. These were transfered to a growth chamber at 25C under white fluorescent light (270 mol/m2/s, 16 h light/8 buy MK-2461 h dark) for 7 days. Cold stress, SNP (sodium nitroprusside) and cPTIO (2-pHENYL-4,4,5,5-TETREMETHYL-IMIDAZOLINE-1-OXYL-3-OXIDE) treatment For cold stress, 7 days old seedlings were kept in a cold chamber at 4C for 2C96 h under the same conditions as mentioned in the above section. Control seedlings were kept at 25C. Seedlings were treated with SNP (a NO donor, 50, 100, 250 M) or cPTIO (a NO scavenger, 100 M). Following the treatment, the seedlings were rinsed with the double distilled water and blotted onto a filter paper and were.