Quantitative analysis of UV-A shock and short term stress using iTRAQ, pseudo selective reaction monitoring (pSRM) and GC-MS based metabolite analysis of the cyanobacterium Nostoc punctiforme ATCC 29133 Nishikant Wase 1 , Trong Khoa Pham, Saw Yen Ow, Phillip C. Wright ⁎ Biological and Environmental Systems Group, ChELSI Institute, Department of Chemical and Biological Engineering, The University of Sheffield, Mappin St., Sheffield, S1 3JD, United Kingdom ARTICLE INFO ABSTRACT Article history: Received 10 January 2014 Accepted 22 June 2014 A quantitative proteomics and metabolomics analysis was performed using iTRAQ, HPLC and GC–MS in the filamentous cyanobacterium Nostoc punctiforme ATCC 29133 to understand the effect of short and long term UV-A exposure. Changes in the proteome were measured for short-term stress (4–24 h) using iTRAQ. Changes in the photosynthetic pigments and intracellular metabolites were observed at exposures of up to 7 days (pigments) and up to 11 days (intracellular metabolites). To assess iTRAQ measurement quality, pseudo selected reaction monitoring (pSRM) was used, with this confirming underestimation of protein abundance levels by iTRAQ. Our results suggest that short term UV-A radiation lowers the abundance of PS-I and PS-II proteins. We also observed an increase in abundance of intracellular redox homeostasis proteins and plastocyanin. Additionally, we observed statistically significant changes in scytonemin, Chlorophyll A, astaxanthin, zeaxanthin, and β-carotene. Assessment of intracellular metabolites showed significant changes in several, suggesting their potential role in the Nostoc's stress mitigation strategy. Cyanobacteria under UV-A radiation have reduced growth due to intensive damage to essential functions, but the organism shows a defense response by remodeling bioenergetics pathway, induction of the UV protection compound scytonemin and increased levels of proline and tyrosine as a mitigation response. Biological significance The effect of UV-A radiation on the proteome and intracellular metabolites of N. punctiforme ATCC 29133 including photosynthetic pigments has been described. We also verify the expression of 13 iTRAQ quantified protein using LC-pSRM. Overall we observed that UV-A radiation has a drastic effect on the photosynthetic machinery, photosynthetic pigments and intracellular amino acids. As a mitigation strategy against UV-A radiation, proline, glycine, and tyrosine were accumulated. © 2014 Published by Elsevier B.V. Keywords: Nostoc punctiforme iTRAQ Selected reaction monitoring Metabolomics UV induction UV-A stress JOURNAL OF PROTEOMICS 109 (2014) 332 – 355 ⁎ Corresponding author. E-mail address: p.c.wright@shef.ac.uk (P.C. Wright). 1 Present Address: Department of Biochemistry, Beadle Center, 1901 Vine St., University of Nebraska, Lincoln, NE 68588-0664, United States. http://dx.doi.org/10.1016/j.jprot.2014.06.024 1874-3919 © 2014 Published by Elsevier B.V. Available online at www.sciencedirect.com ScienceDirect www.elsevier.com/locate/jprot