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Proceedings of 12th ISMAS Symposium cum Workshop on Mass Spectrometry

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Tandem Mass Spectrometric Studies of Natural Peptides

V. Sabareesh and P. Balaram Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012 Email: [email protected] 1. Introduction Over the last few years, there has been a rapid progress in the mass spectrometric determination of the primary structures of peptides and proteins. In particular, tandem mass spectrometric procedures have facilitated de novo sequencing. In this presentation, studies on protonated ([M+H]+) and sodiated ([M+Na]+) adducts of natural peptides are described. Linear and cyclic peptides from the fungi, Trichoderma and Isaria, respectively, have been investigated. Fragmentation in an ion trap under electrospray ionization (ESI) conditions enables identification of new microheterogeneous components. 2. Peptaibols from the soil fungus Trichoderma Peptaibols are linear peptides that are rich in -aminoisobutyric acid (Aib), possessing C-terminal alcohol and acylated N-terminus [1, 2]. The sequences reported thus far are of length 5-20 amino acid residues with majority are of length 17-20 residues. An interesting feature of peptaibols is the observation of microheterogeneity in their sequences, leading to mass differences of 1, 14 and 15 Da, due to amino acid exchanges and replacements; Gly Ala (G A), Ala Aib (A U), Aib DIva (U J) (J: DIva: Risovaline), Gln Glu (Q E). Further, there are also several microheterogeneous sequences of identical molecular masses (isobars). Comparison of the fragmentation pattern of [M+H]+ and [M+Na]+ species aided in establishing the microheterogeneity of an apparently homogeneous HPLC fraction [3]. Particularly, examination of isotope pattern of b type product ions from the protonated adduct facilitated identification of three microheterogeneous components, one peptide of mass 1717 Da and two isobaric peptides of mass 1718 Da (Figure 1). In the case of fragmentation of [M+Na]+ adduct product ions were observed only from the neutral peptaibol component that lacked any glutamic acid residues.

Figure 1. Microheterogeneous peptaibol sequences, along with the calculated m/z values of product ions, b6 - b12 and y6.

12th ISMAS-WS-2007, March 25-30, 2007, Cidade de Goa, Dona Paula, Goa

Proceedings of 12th ISMAS Symposium cum Workshop on Mass Spectrometry

IT-15

3. Cyclohexadepsipeptides from the fungus Isaria Two distinct classes of cyclic hexadepsipeptides are known from the fungus Isaria, namely isariins and isaridins [4-6]. Isariins are characterized by a -hydroxyacid residue with a hydrocarbon sidechain and are devoid of aromatic residues [4, 5], while isaridins possess an -hydroxyacid, an -amino acid and N-methylated residues [6]. The two classes of peptides are conveniently distinguished by their drastically different fragmentation patterns. In the case of cyclic peptides, the initial event in fragmentation is the ring opening step upon charging. The availability of multiple sites of charging suggests alternative ring opening pathways that result in the formation of several kinds of linear peptide ions of the same mass, but with cyclically permuted sequences. Subsequently, the fragment ions can arise from any linear ion, depending on the ring opening pathway. The favoured protonation site and a plausible ring opening mechanism can thus be determined based on the observed product ion spectrum. The MS2 and MS3 experiments aid in the identification of two isobaric isariins that are found to coelute during HPLC separation. Among the two coeluting isobars, the primary structure of one peptide is known [5], while another is a newly identified component (Figure 2). The microheterogeneity of isariin sequences is due to the variation of the residue that is bound to the hydroxyl oxygen of the -hydroxyacid (Ala/Val) and the concomitant change in the number of methylene units (-(CH2)n-) of the -hydroxyacid residue's sidechain (Figure 2).

Figure 2. General primary structure of isariins, indicating the variable regions of the sequence responsible for microheterogeneity. The sequences of coeluting, isobaric isariins: C and C2 are also shown. References 1. T. Degenkolb, A. Berg, W. Gams, B. Schlegel, U. Gräfe, J. Pept. Sci. 9 (2003) 666. 2. http://www.cryst.bbk.ac.uk/peptaibol/home.shtml 3. V. Sabareesh, P. Balaram, Rapid Commun. Mass Spectrom. 20 (2006) 618. 4. L.C. Vining, W.A. Taber, Can. J. Chem. 40 (1962) 1579. 5. G. Deffieux, D. Merlet, R. Baute, G. Bourgeois, M.-A. Baute, A. Neveu, J. Antibiot. 34 (1981), 1266. 6. G. Ravindra, R.S. Ranganayaki, S. Raghothama, M.C. Srinivasan, R.D. Gilardi, I.L. Karle, P. Balaram, Chem. Biodivers. 1 (2004), 489.

12th ISMAS-WS-2007, March 25-30, 2007, Cidade de Goa, Dona Paula, Goa

Proceedings of 12th ISMAS Symposium cum Workshop on Mass Spectrometry

IT-15

1. Name: Varatharajan Sabareesh 2. Date of birth: August 6, 1979 3. Address for correspondence: Molecular Biophysics Unit Indian Institute of Science Bangalore 560 012, India

Email:[email protected]

4. Academic profile: a. M.Sc Chemistry (5 year Integrated course) : Pondicherry University (1996-2001) b. Summer Research Fellow : Chemistry and Physics of Materials Unit, Jawaharlal Nehru Centre for Advanced Scientific Research (JNCASR), Bangalore, India (2000) c. Summer Research Fellow (of JNCASR) : Regional Sophisticated Instrumentation Centre, Indian Institute of Technology, Chennai, India (2001) d. Ph.D : Molecular Biophysics Unit (from August 2001) e. DST award for participation in the Meeting of Nobel Laureates and Students in Lindau (Germany) : 53rd meeting in the field of Physiology and Medicine (2003) (DST: Department of Science and Technology, Government of India) 5. List of publications 1. Gowd KH, Sabareesh V, Sudarslal S, Iengar P, Franklin B, Fernando A, Dewan K, Ramaswami M, Sarma SP, Sikdar SK, Balaram P, Krishnan KS. Novel peptides of therapeutic promise from Indian Conidae. Ann. N. Y. Acad. Sci. (2005) 1056: 462473. 2. Sabareesh V, Balaram P. Tandem electrospray mass spectrometric studies of proton and sodium ion adducts of neutral peptides with modified N- and C- termini: synthetic model peptides and microheterogeneous peptaibol antibiotics. Rapid Commun. Mass Spectrom. (2006) 20: 618-628. 3. Sabareesh V, Gowd KH, Ramasamy P, Sudarslal S, Krishnan KS, Sikdar SK, Balaram P. Characterization of contryphans from Conus loroisii and Conus amadis that target calcium channels. Peptides (2006) 27: 2647-2654.

12th ISMAS-WS-2007, March 25-30, 2007, Cidade de Goa, Dona Paula, Goa

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Tandem mass spectrometric studies of natural peptides

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