DI-MS Technique for Analyzing Protein and Peptide Sequences

DI-MS Technique for Analyzing Protein and Peptide Sequences Food Industrial Application DI-MS technique has been widely applied in food industry as it is an easy and high-throughput tool for analyzing protein and peptide sequences, studying the components or structure in food, monitoring the contamination in food. FAB-MS In the study by Terzi, Boyot, Dorsselear, Luu and Trifilieff (1990), the amino acid sequence of a new 6.8 kDa proteolipid from beef heart was determined by employed FAB-MS. The study isolated and purified the 6.8 kDa proteolipid from an acidic methanol/chloroform extract of bovine cardiac muscle, and subsequently analyzed it with the application Cs atom beam of FAB-MS in 1-thioglycerol matrix. The result showed a protonated molecular ion [M+H]+ at m/z 6834.1 and indicated that it have about 60 residues. The 60 residues in the polypeptide were cleaved into three peptide fragments CN1, CN2 and CN3. To characterize these cleavage peptides from amino acid composition, the CN1 and CN2 were ionized using the FAB-MS that performed with Xe atom beam and 1-thioglycerol matrix while the CN3 which also dissolved in 1-thioglycerol was collided by Cs atom beam to produce sample ions. Table 1.0: Chemical mass of cleavage peptides Cleavage peptides Measured mass [M+H]+ [M+Na]+ CN1 1539.0 1560.9 CN2 2100.5 2121.8 CN3 3134.6 From the analysis of the cleavage peptides, the amino acid sequence and peptide fragments of the 6.8 kDa proteolipid were reported as: MLQSLIKKVWIPMKPYYTQAYQEIWVGTGLMAYIVYKIRSADKRSKALKASS 1 CN1 13 14 CN2 31 32 CN3 AAPAHGHH 60 In addition, the FAB-MS technique was applied to determine the structure of the Lycium chinense Miller fruits (Gou-Qi-Zi) in combination with NMR and IR spectroscopy (Chung, Ali, Praveen, Yu, Kim, Ahmad, 2014). L. chinense fruit is a valuable tonic or food, hence there are many researches have been reported to display the properties the L.chinense fruit. For example, the anticancer, antibacterial and antioxidant properties (Lee et al., 2005; Wang, Chang, Inbaraj. Chen, 2010; Zhang et al., 2011) and the antihepatotoxic activity and chemical constituents (Chin et al., 2003; Kim et al., 1997). In the research, the four new compounds (i, ii, iii and iv) that isolated from L. chinense fruits were characterized using the FAB-MS (JMS-700) spectrometer. The antioxidant activity of the four compounds was further studied and it demonstrated that the activity of the compounds followed the order 1423. Table 2.0: Characterization of compound (i) to (iv) Mass Species m/z Detected species Suggestion Compound (i) [M+H]+ 817 C40H65O17 -aromatic acid glycosidic ester 283 [CH3(CH2)16COO]+ -steric acid was esterified with one phenolic group 267 [CH3(CH2)16CO]+ 414 [C15H26O13]+ -three arabinose units bind to one phenolic group 265 [C10H17O8]+ 133 [C5H9O4]+ Compound (ii) [M+H]+ 1165 C50H85O30 -susquiterpene glycoside ester 265 [C10H17O8]+ -few arabinose units bind to the sesquiterpene moiety 223 [C14H27CO]+ 133 [C5H8O5]+ Compound (iii) [M]+ 1309 C58H100O32 -hexaglycoside 341 [C12H21O11]+ -six hexose sugar units bind to each other -unsaturated fatty acid located at terminal position 335 [C21H39COO]+ 325 [C12H21O10]+ 319 [C12H39CO]+ 179 [C6H11O6]+ 163 [C6H11O5]+ Compound (iv) [M+H]+ 841 C31H52O26 -five arabinose units and one glucose unit 661 [C25H41O20]+ -five pentose sugar units bind to a hexose sugar 529 [C25H41O20-C5H8O4]+ 281 [C10H17O9]+ TOF-SIMS TOF-SIMS which widely applied in surface analysis is an important technique for monitoring the contamination in food as the usage of pesticides and fungicide in the agriculture practice will alter and compromise the food quality. The study used TOF-SIMS technique to characterize and compare three different groups of Seggianese olives which classified as untreated (UT), treated with insecticide (dimethoate) and fungicide (copper oxychloride) without washing (TU) and treated with insecticide and fungicide with washing with cold water (TW) (Focardi, et al., 2006). Before measuring the molecular masses of three different groups of olives, the mass calibration was done by using CH3+, C2H3+ and C3H5+ peaks as calibration compounds for positive ions C, CH and C2H peaks as calibration compounds for negative ions. The SIMS data proved the chemical treatments modify the surface composition of olive, resulting in higher intensity signals in TU compared with TW or UT olive spectra. Besides, the intensity of UT and TW olives showed a small variance between each other, demonstrating the washing process was no effective in removing of insecticides and fungicides that will stimulate the composition alterations of olives. Table 3.0: Intensity of few relevant peaks from three different olive samples Peaks Mass peak m/z Attribution Intensity TU olive TW olive UT olive Positive 31.018 CH3O+ 4.23 10-4 6.93 10-5 1.12 10-4 57.074 C4H9+ 1.18 10-2 5.92 10-3 5.08 10-3 73.051 C4H9O+ 7.83 10-4 3.34 10-4 3.08 10-4 147.082 C6H14NOP+ / C6H13NOS+ 2.42 10-4 7.37 10-5 2.93 10-5 Negative 15.994 O 1.13 10-4 2.30 10-3 7.20 10-3 17.002 OH 1.19 10-4 4.42 10-3 5.44 10-3 31.972 S 5.24 10-4 1.99 10-5 1.47 10-5 MALDI-TOF/TOF MS MALDI-MS is a widespread analytical tool for proteins, peptides and oligonucleotides as it offers high ion yields of the intact analyte samples, high sensitivity and accuracy (Lewis, Wei, Siuzdak, 2000). In year 2012, a research about the peptide of peanut hydrolysate has the properties of umami taste had reported by Su, Cui, Zheng, Yang, Ren Zhao. Umami taste is known as the fifth basic taste and it usually described as savory or MSG-like taste. In the study, two novel umami and umami-enhancing peptides were isolated from peanut hydrolysate, purified using chromatography and identified by MALDI-TOF/TOF MS. The analysis of peptides was performed by MALDI-MS equipped with 337nm of UV nitrogen laser and matrix solution as sinapic acid saturated in 0.1% trifluoroacetic acid and acetonitrile. The MALDI-TOF/TOF MS was used to sequence the two novel peptides that termed as P3 and P4 as they produce umami taste. It found that the molecular mass of P3 and P4 was 1091.419 Da and 965.595 Da respectively and the amino sequence of P3 is EGSEAPDGSSR while P4 is SSRDEQSR. Bibliography Chung, I. M., Ali, M., Praveen, N., Yu, B. R., Kim, S. H., Ahmad, A. (2014). New polyglucopyranosyl and polyarabinopyranosyl of fatty acid derivatives from the fruits of Lycium chinense and its antioxidant activity. Food Chemistry, 435-443. Focardi, S., Ristori, S., Mazzuoli, S., Tognazzi, A., Leach-Scampavia, D., Castner, D. G., et al. (2006). ToF-SIMS and PCA studies of Seggianese olives and olive oil. Colloids and Surfaces, 225-232. Lewis, J. K., Wei, J., Siuzdak, G. (2000). Matrix-assited laser desorption/iomization mass spectrometry in peptide and protein analysis. Chichester: John Wiley Sons Ltd. Su, G., Cui, C., Zheng, L., Yang, B., Ren, J., Zhao, M. (2012). Isolation and identification of two novel umami and umami-enhancing peptides from peanut hydrolysate by consecutive chromatography and MALDI-TOF/TOF MS. Food Chemistry, 479-485. Terzi, E., Boyot, P., Dorsselear, A. V., Luu, B., Trifilieff, E. (1990). Isolation and amino acid sequence of a novel 6.8-kDa mitochondrial proteolipid from beef heart. FABS Letters, 122-126. Conclusion In food industry, the analysis of protein and peptide can be performed well by using MALDI-MS and FAB-MS while the application of SIMS is mostly used for detecting the surface of contaminated food. Besides, the structure of the components in foods can also be identified by using the FAB-MS combined with NMR and IR spectroscopies.