Costus igneus subsp. leaves N. E. Br.
Creators: name: Hardikar, Manasi R.
name: Varma, Mokshada E.
name: Kulkarni, Anjali A.
name: Kulkarni, Prasad P.
name: Joshi, Bimba N.
Publisher: name: Zenodo
Year: 2016
Subjects: Biodiversity
Taxonomy
Plantae
Tracheophyta
Liliopsida
Zingiberales
Costaceae
Costus
Costus igneus

description: 5.7. Identification of ILP from C. igneus   
 In our attempt to identify the ILP from C. igneus MALDI TOF/TOF   
MS was performed.   5.7.1. MALDI TOF/TOF analysis of ILP   
 The ILP was resolved on 12% 1D SDS–PAGE gel and the band was excised after coomassie blue staining. The excised band was subjected to the trypsin digestion procedure as reported earlier (Shevchenko et al., 2006; Kumar et al., 2013). Briefly, the gel pieces were rinsed thrice with MQ H 2 O (Millipore) and were equilibrated with 100 mM ammonium bicarbonate (NH 4 HCO 3) for 20 min at room temperature with gentle agitation. The gel pieces were washed with 1:1 NH 4 HCO 3 and acetonitrile (CH 3 CN, ACN) and dehydrated with 100% ACN for 20 min at room temperature and vacuum dried. The protein in the gel was reduced with 10 mM dithiothreitol (DTT) in 50 mM NH 4 HCO 3 for 45 min at 56 °C . Further the protein was alkylated with 50 mM iodoacetamide (IAA) in 50 mM NH 4 HCO 3 for 30 min in dark at room temperature. Then the gel was rinsed with 1:1 NH 4 HCO 3 and ACN solution and dehydrated with 100% ACN for 20 min and were vacuum-dried . The protein was incubated with porcine trypsin (Promega) at a final concentration of 10 ng /µl in 25 mM NH 4 HCO 3, for 16 h at 37 °C.   
 The peptides were extracted with 1% trifluroacetic acid (TFA) in 60% ACN and concentrated. The pellet was dissolved in 0.1% TFA and loaded onto MALDI target plate by mixing 1 µl of each sample with the same volume of matrix solution (10 mg /ml α- cyanohydroxycinnamic acid in ACN/0.1% TFA (1:1). Samples and calibration standards with the same matrix composition were spotted adjacent to each other on the target plate for optimal calibration. Spectra were externally calibrated with the calibration standard containing the following peptide mass (M+H) + Angiotensin_II-1046.54180, Angiotensin_I-1296.684780, Substance_P-1347.735430, Bombesin-1619.822350, Renin_Substrate-1758.932610, ACTH(1-17)- 2093.086170, ACTH(18-39)-2465.198340, ACTH(1-24)- 2932.590000, Insulin b-chain-3494.650780. Measurements were performed using an Ultraflex III MALDI TOF/TOF instrument (Bruker Daltonics, Germany), in the positive ion reflector mode for acquiring the peptide mass fingerprint (PMF). Tandem mass spectrometry based fragmentation was employed to identify the protein from the observed peptide ions. Instrument parameters for PMF and MS/MS analysis were set as reported earlier (Kumar et al., 2013).   
 Fragmented peptides were analyzed using Flex analysis software version 3.0 and were sent to database search using Biotools software version 3.2. The fragment masses were searched in NCBI database with MS tolerance of up to 100 ppm and MS/MS tolerance of up to ±0.75 da. The following criteria were used for identification: (i) significance threshold was set to achieve p <0.05; (ii) the expectancy cut off was set to 0.05; and (iii) individual ion score>45 was only considered for identification (Shevchenko et al., 2006; Kumar et al., 2013). The sequence data was searched against NCBI database using the Mascot Version: 2.4.1 engine.
description: Published as part of Hardikar, Manasi R., Varma, Mokshada E., Kulkarni, Anjali A., Kulkarni, Prasad P. & Joshi, Bimba N., 2016, Elucidation of hypoglycemic action and toxicity studies of insulin-like protein from Costus igneus, pp. 99-107 in Phytochemistry 124 on page 106, DOI: 10.1016/j.phytochem.2016.02.001, http://zenodo.org/record/10485123