Thesis (Ph.D.)--Ohio University, March, 2005. / Title from PDF t.p. Includes bibliographical references (p. 163-176)

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No / The use of matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometry on intact cell microorganisms, Intact Cell MALDI (ICM), has been shown by numerous workers to yield effective species level identification. Early work highlighted the significant effect that variation in culture media, incubation conditions and length of incubation had on the spectra produced. Therefore, in order to achieve reliable and reproducible species level identification and sub-typing of microorganisms from ICM fingerprints, it has been essential to develop standardised methods. For methicillin-resistant Staphylococcus aureus (MRSA), a major nosocomial pathogen, we have developed such a standardised method. In this paper we present the experimental parameters, namely, the incubation period, the number of passages required from lyophilised or stored isolates, the method of deposition of the bacterial cells, the concentration of matrix solution, the drying time of bacterial cells prior to the addition of the matrix solution, the time between preparation of the bacterial/matrix sample and analysis and the MALDI pulsed extraction setting, which were considered during the development of defined methods.

A especiação de isolados clínicos de Aspergillus ganhou relevância nos últimos anos devido à descrição de espécies crípticas resistentes aos derivados azólicos. A identificação morfológica convencional não é capaz de discriminar as espécies de Aspergillus e o sequenciamento de DNA é uma técnica pouco adaptada a laboratórios clínicos. A espectrometria de massas por ionização/dessorção a laser auxiliada por matriz tempo-devoo (EM MALDI-TOF) é uma metodologia emergente que vem sendo explorada com intuito de fornecer identificação rápida e acurada de microrganismos, incluindo os fungos filamentosos de importância clínica. Entretanto, há poucos estudos avaliando a plataforma VITEK MS® para a identificação de espécies de Aspergillus. O presente estudo teve como objetivo fornecer dados adicionais sobre a performance dos sistemas do VITEK MS® e suas bibliotecas de espectros de referências (ER) In Vitro Diagnostics (IVD) e Research Use Only (RUO) para identificar as espécies de Aspergillus de relevância clínica. Uma biblioteca de ER in-house também foi construída e avaliada. Um total de 106 organismos foram avaliados por EM, incluindo 47 cepas provenientes das coleções de fungos do Westerdijk Fungal Biodiversity Institute (Holanda) e do LEMIUNIFESP, seis isolados ambientais (IMT-USP) e 53 isolados cínicos do HC-FMUSP. Foram utilizados dois protocolos de extração proteica, um recomendado pelo fabricante e outro empregando meio de cultura líquido para os isolados/cepas com baixa esporulação. Trinta e cinco organismos foram selecionadas para construir os ERs, e os 71 restantes foram usados para avaliação de desempenho das bibliotecas IVD, RUO e RUO+in-house. Entre os 71 organismos analisados, 91,5%, 84,5% e 100% tiveram identificação correta de gênero pelos bibliotecas IVD, RUO e RUO+in-house, respectivamente. Enquanto para identificação de espécie, as bibliotecas IVD, RUO e RUO+in-house mostraram 83,1%, 77,4% e 90,1% de identificações de espécie, respectivamente. Para as 16 espécies crípticas de Aspergillus analisadas, a identificação foi correta em 31,2%, 18,7% e 62,5%, pelos sistemas IVD, RUO e RUO+in-house, respectivamente. Entre as espécies crípticas resistentes aos derivados azólicos, o sistema IVD forneceu identificação correta para as espécies Aspergillus lentulus, Aspergillus calidoustus e Aspergillus sydowii. Entretanto, Aspergillus fumigatiaffinis e Neosartorya pseudofischeri foram erroneamente identificadas como Aspergillus fumigatus pelo sistema IVD. A biblioteca in-house demonstrou melhor performance, mas espécies filogeneticamente próximas como A. fumigatiaffinis e A. lentulus tiveram identificações cruzadas. Concluímos que o VITEK® MS demonstrou boa performance para a identificação das espécies de Aspergillus, porém para algumas espécies crípticas, há necessidade de melhoria das bibliotecas de espectro de referência comercializadas. Algumas espécies crípticas filogeneticamente relacionadas apresentaram espectros similares e são de difícil diferenciação por EM MALDI, mesmo com a construção de uma biblioteca de ER in-house com vários representantes de cada espécie / Aspergillus spp. identification has become more relevant in clinical practice since azoleresistant cryptic species have emerged in the last years. Conventional morphologic identification is not able to discriminate Aspergillus species and DNA sequencing is not feasible for clinical laboratories. Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is an emergent technology that has been explored to provide fast and accurate identification of microorganisms, including clinically relevant molds. However, only a few studies have explored the platform VITEK MS® for the identification of Aspergillus species. The present study aimed to provide additional data regarding the performance of the In Vitro Diagnostics (IVD) and Research Use Only (RUO) systems for the identification of Aspergillus species, including azoleresistant ones, and also to construct and to evaluate an in-house reference spectrum library. A total of 106 organisms were evaluated by MS, including 47 Aspergillus strains (Westerdijk Fungal Biodiversity Institute and LEMI-UNIFESP collections), six environmental (IMT-USP) and 53 clinical isolates (HC-FMUSP). Two protein extraction protocols were used, one recommended by the manufacturer and another with liquid broth for the organisms with poor sporulation. Thirty-five organisms were selected to construct the in-house reference spectrum library and the remaining 71 were used for the performance evaluation. Correct genus identification was provided in 91.5%, 84.5% and 100% by the IVD, RUO, and RUO+in-house reference spectrum libraries, respectively. Correct species identification was provided in 83.1%, 77.4% and 90.1% by the IVD, RUO, and RUO+in-house reference spectrum libraries, respectively. Among the 16 Aspergillus cryptic species, correct identification was provided in 31.5%, 18.7% and 62.5% by the IVD, RUO, and RUO+in-house reference spectrum libraries, respectively. Among the azole-resistant cryptic species, the IVD system provided correct identification for Aspergillus lentulus, Aspergillus calidoustus and Aspergillus sydowii. However, Aspergillus fumigatiaffinis and Neosartorya pseudofischeri were misidentified as Aspergillus fumigatus by the IVD system. The in-house library had better performance for the identification of Aspergillus cryptic species, but closely related taxa may be difficult to have correct differentiation by MALDI-TOF MS. In conclusion, VITEK® MS showed good performance for the identification of Aspergillus species and some azoleresistant species. However, a more robust reference spectrum library including more representatives of azole-resistant cryptic species may be necessary to achieve better identification performance of closely related taxa

O gênero Trichosporon é composto por leveduras artrosporadas do Filo Basidiomycota e é conhecido agente de infecção fúngica invasiva (IFI) em pacientes imunodeprimidos ou com outros fatores de risco. Em pacientes onco-hematológicos é a principal levedura responsável por IFI depois do gênero Candida. Entre as espécies responsáveis por infecções no homem encontram-se: T. asahii, T. inkin, T. mucoides, T. dermatis, T. jirovecii, T. ovoides, T. cutaneum, T. montevideense, T. domesticum, T. asteroides, T. coremiiforme, T. faecale, T. dohaense, T. lactis, T. japonicum. A tecnologia de identificação de fungos por espectrometria de massa (SM) MALDI-TOF ainda carece de padronização para identificação de fungos do gênero Trichosporon, mas a literatura mostra resultados encorajadores. O objetivo deste estudo é padronizar a técnica de espectrometria de massa MALDI-TOF para a identificação das espécies do gênero Trichosporon de importância médica. O estudo foi realizado em cooperação entre a Divisão de Laboratório Central do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo (DLC, HC-FMUSP), Instituto de Medicina Tropical da USP (IMT-USP), Instituto Adolfo Lutz (IAL) e Laboratoire de Parasitologie-Mycologie do Hospital Saint Antoine de Paris, vinculado ao grupo de pesquisa INSERM/UPMC UMR S945 \"Immunité et Infection\" Faculté de Medecine et Université Pierre et Marie Curie de Paris. Noventa e três cepas/isolados foram analisado(a)s, sendo dezenove cepas de referência adquiridas junto à coleção holandesa Centraalbureau Schimmelcultures (CBS), 19 isolados do HC-FMUSP e IAL, e 55 isolados de diferentes hospitais franceses. A identificação molecular foi realizada através do sequenciamento da região IGS1 do rDNA e foi considerada como método de referência. O protocolo de extração de proteínas foi estabelecido através da comparação do desempenho de três metodologias (Bruker®, Cassagne et al., Sendid et al.). Os espectros de massa foram obtidos no laboratório de bacteriologia do Hospital Saint Antoine de Paris através do aparelho Microflex LT®. A interpretação dos resultados qualitativos e quantitativos (logscore) foi realizada através do Software Biotyper 3.0®. O desempenho de identificação do banco de espectros de referência Biotyper 3.0® foi comparado a outros cinco bancos criados a partir de espectros de referência (ERs) derivados de 18 cepas de referência CBS, sete isolados clínicos e 11 ERs do banco Biotyper 3.0. O protocolo de extração de proteínas descrito por Sendid et al. foi escolhido como protocolo de referência pois os espectros produzidos tiveram logscore superiores àqueles obtidos através do método do fabricante. O banco de ERs Biotyper 3.0® apresentou 32,3% de identificações corretas das espécies, sendo que o banco de ERs in house (número 5, constituído cepas CBS e isolados clínicos) apresentou 98,5% de identificações de espécies. Espectros de referência do banco de dados Biotyper 3.0® foram submetidos à identificação com a utilização dos ERs criados a partir de cepas CBS e isolados clínicos e foram evidenciados com erros de identificação: T. mucoides (2), T. ovoides (1) e T. cutaneum (2). Após padronização do protocolo de extração e criação de banco de ERs com cepas CBS e isolados clínicos caracterizados pelo sequenciamento da região IGS, a SM por MALDI-TOF apresentou-se como potente uma ferramenta para a identificação de fungos do gênero Trichosporon. O banco de ERs Biotyper 3.0® apresentou um fraco desempenho, relacionado a ERs que foram criados a partir de cepas mal identificadas / Trichosporon spp. are arthrospored yeasts from the Filum Basidiomycota that are known to produce invasive fungal infection (IFI) in patients with immunosupression or other risk factors. After Candida, Trichosporon is the second genus of yeasts responsible for IFI in patients with onco-hematological diseases. The most important species related to human infection are: T. asahii, T. inkin, T. mucoides, T. dermatis, T. jirovecii, T. ovoides, T. cutaneum, T. montevideense, T. domesticum, T. asteroides, T. coremiiforme, T. faecale, T. dohaense, T. lactis, T. japonicum. The technology of mass spectrometry (MS) for identification of Trichosporon species has not yet been standardized. However, preliminary promising results can be found in the literature. The objective of this study is to analyse and validate MS MALDI-TOF for the identification of Trichosporon species of medical relevance. This was a multicentric study with collaboration from the Central Laboratory Section from Clinics Hospital of the Medical School from the University of São Paulo (DLC-HCFMUSP), Tropical Medicine Institute from the University of São Paulo (IMT-USP), Instituto Adolfo Lutz (IAL) and Laboratoire de Parasitologie-Mycologie from the Hospital Saint Antoine of Paris and INSERM/UPMC UMR S945 \"Immunité et Infection\", Faculté de Medecine et Université Pierre et Marie Curie of Paris. Ninety three strains/isolates belonging to sixteen Trichosporon species were analysed. Nineteen were purchased from Centraalbureau Schimmelcultures (CBS) yeast collection, 19 belonged to HC-FMUSP and IAL collections, 55 belonged to different French collections. The reference identification method was the IGS1 rDNA sequencing. A protein extraction protocol was first established after comparing the performance of three different methodologies (Bruker(TM), Cassagne et al., Sendid et al.). The mass spectra were obtained through a Microflex LT(TM) mass spectrometer located at the bacteriology laboratory from Saint Antoine Hospital, Paris. Mass spectra, qualitative and quantitative results were produced through the software Biotyper 3.0(TM). The performance of the original main spectrum (MSP) library was compared to other 5 in house libraries built with the combination of MSPs derived from CBS strains (18), clinical strains (7) or (Bruker Daltonics/BD, Germany/USA) (11). The extraction protocol described by Sendid et al. showed better performance when compared to the manufacturer\'s one and was chosen for the subsequent extractions. Among the 6 different reference spectra databases tested, a specific one composed of 18 reference strains plus 7 clinical isolates (database 5) allowed the correct identification of 66 amongst 67 clinical isolates (98,5%). Biotyper 3.0 library produced only 32,3% of correct identifications. Biotyper\'s MSPs were submitted to cross-identification with MSPs derived from CBS strains and clinical isolates and misidentified original MSPs were identified: T. mucoides (2), T. ovoides (1) e T. cutaneum (2). While until now less widely applied to basidiomycetous fungi, MALDI-TOF appears to be a valuable tool for identifying clinical Trichosporon isolates at the species level. The MSP library Biotyper 3.0 showed a poorer performance which was due to misidentified strains utilized as reference for the MSPs

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Mass spectrometry is a versatile, sensitive and fast technique with which to probe biophysical properties in biological systems and one of the most important analytical tools in the multidisciplinary field of proteomics. The study of nativestate proteins and their complexes in the gas-phase is well established and direct infusion electrospray ionisation mass spectrometry (DI-ESI-MS) techniques are becoming increasingly popular as a tool for screening and determining quantitative information on protein-protein and protein-ligand interactions. However, complexes retained by ESI-MS are not always representative of those in solution and care must be taken in interpreting purely gas-phase results. This thesis details modification and advancement of solution phase techniques devised by Gross et al. utilising ESI-MS and Fitzgerald et al. applying matrix assisted laser desorption ionisation (MALDI)-MS termed PLIMSTEX (protein-ligand interactions by mass spectrometry, titration and hydrogen-deuterium-exchange)[1] and SUPREX (Stability of unpurified proteins from rates of H/D exchange)[2] to quantify these interactions with regards to high throughput analysis. The first part of this thesis describes the different developmental stages of the devised HPLC-front ends and their optimisation with myoglobin and insulin. The successfully developed HPLC-front end in conjunction with PLIMSTEX and SUPREX and ESI-MS then gets tested with self expressed and purified cyclophilin A(CypA)- cyclosporin A (CsA) system, followed by a test screen with potential CypA binding ligands. Dissociation constants (Kd’s) within one order of magnitude to reported values are determined. In the third part of this thesis the application of the devised ESI-SUPREX methodology has been applied to anterior gradient 2 (AGr2) and the factor H complement control proteins module 19-20 (fH19-20) exhibiting binding potential to a taggedhexapeptide and a synthetic pentasaccharide, respectively, resulting in thermodynamical data for these protein-ligand interactions. For the AGr2 system another dimension of investigation has been added by temperature controlling the devised ESI-SUPREX approach, revealing a phase transition in the protein at higher temperatures. The final part of this thesis describes the application of the ESI-SUPREX methodology to probe folding properties of CypA in the presence of the self expressed and purified E. coli chaperonin groEL. Thereby the denaturing properties of groEL have been emphasised along with the stabilisation of a denatured CypA species.

The incidence of breast cancer became the most common female cancer, and the fourth cause of female cancer death. In this study, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/MS) have been combined with multivariate statistics to investigate breast cancer tissues and cell lines. Core needle biopsy and fine needle aspiration (FNA) are techniques largely applied in the diagnosis of breast cancer. In this study, we have established an efficient protocol for detecting breast tissue and FNA samples with MALDI-TOF/MS. With the help of statistical analysis software, we can find the lipid-derived ion signals which can be use to distinguish breast cancer tumor tissues from non-tumor parts. This strategy can differentiate normal and tumor tissue, which is potential to apply in clinical diagnoses. The analysis of breast cancer tissue is challenging as the complexity of the tissue sample. Direct tissue analyses by matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) allows us to investigate the molecular structure and their distribution while maintaining the integrity of the tissue and avoiding the loss of signals from extraction steps. Combined MALDI-IMS with statistic software, tissues can be analyzed and classified based on their molecular content which is helpful to distinguish tumor regions from non-tumor regions of breast cancer tissue. Our result shows the differences in the distribution and content of lipids between tumor and non-tumor tissue which can be supplements of current pathological analysis in tumor margins. In this study, MALDI-TOF/MS combined with multivariate statistics were used to rapidly differentiate breast cancer cell lines with different estrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2) status. The protocol for efficiently detecting peptides and proteins in breast cancer cells with MALDI-TOF/MS was established, two multivariate statistics including principle component analysis (PCA) and hierarchical clustering analysis were used to process the obtaining MALDI mass spectra of six different breast cancer cell lines and one normal breast cell lines. Based on the difference of the peptide and protein profiles, breast cancer cell lines with same ER and HER-2 status were grouped in nearby region on the PCA score plot. The results of hierarchical cluster analysis also revealed high conformity between breast cancer cell protein profiles and respective hormone receptor types.

Elucidation of the primary structure of peptides and proteins de novo by mass spectrometry (MS) has become possible with the advent of tandem MS methods. The most widely used chemical method due to Edman (Edman & Begg, 1967) has shortcomings with regard to N- terminal blocked peptides, cyclic peptides and posttranslational modifications, for example phosphorylation (Metzger, 1994). However, mass spectrometric sequencing methods are increasingly becoming applicable for a variety of peptides and proteins, including N- and C- termini modified peptides and cyclic peptides (Jegorov et al., 2003; Sabareesh & Balaram, 2006; Sabareesh et al., 2007). Further, conventional and tandem mass spectrometry have proven useful in the detection of post-translational modifications (Hansson et al., 2004; Nair et al., 2006; Mandal et al., 2007). This thesis details mass spectrometric sequencing of acyclic and cyclic peptides, involving tandem MS methods carried out using both electrospray ionization (ESI) ion trap (Esquire 3000 plus, Bruker Daltonics) and matrix assisted laser desorption and ionization time-of-flight/time-of-flight (MALDI TOF/TOF) (Ultraflex TOF/TOF, Bruker Daltonics) instruments. The peptides are either chemically synthesized or isolated from diverse natural sources. Synthetically designed peptides possessing modified N- and C- termini and peptaibols from the soil fungus Trichoderma constitute the acyclic peptides. The cyclic peptides include backbone cyclized depsipeptides from the fungus Isaria and disulfide bonded peptides from the venom of marine cone snails. Chapter 1 gives an account of various concepts of mass spectrometry, tandem mass spectrometry and peptide fragmentation chemistry, providing necessary background information for the following chapters. Chapter 2 describes the fragmentation studies of [M + H]+ and [M + Na]+ adducts of six neutral peptides with blocked N- and C- termini investigated using an electrospray ion trap mass spectrometer. The N- terminus of these synthetically designed peptides is blocked with a tertiarybutyloxycarbonyl (Boc) group and the C- terminus is esterified. These peptides do not possess sidechains that are capable of complexation and hence the backbone amide units are the sole sites of protonation and metallation. The cleavage pattern of protonated adducts is strikingly different from that of sodium adducts. While the loss of the N- terminal blocking group happens quite readily in the case of MS/MS of [M + Na]+, the cleavage of C- terminal methoxy group seems to be a facile process in the case of MS/MS of [M + H]+. Fragmentation of the protonated adducts yields only bn ions, while yn and an type ions are predominantly formed from the fragmentation of sodium adducts. The an ions arising from the fragmentation of [M + Na]+ lack the N-terminal Boc group (termed as an*). MS/MS of [M + Na]+ species also yields bn ions of substantial lower intensities, that lack the N- terminal Boc group (bn*). Comparison of the fragmentation of [M + H]+ with [M + Na]+ of the peptides chosen in this study reveal that the combined use of both protonated and sodium adducts should prove useful in de novo sequencing of peptides that possess modified N- and C- termini, particularly naturally occurring neutral peptides, for example, peptaibols. Chapter 3 describes about the ESI-MS/MS investigation of an HPLC fraction from the soil fungus Trichoderma, which aided in identification of microheterogeneous trichotoxin peptaibols in that fraction. Dramatic differences were noted between the fragmentation spectra of [M + H]+ and [M + Na]+ species. While b-type ions were noted from the former, the latter yielded a-, b-and y- type ions (the same feature was noted in the cases presented in the previous chapter). Inspection of the isotope pattern of b-ions yielded from the dissociation of H+ species, clearly revealed the presence of three microheterogeneous trichotoxin sequences; two isobars (1718 Da), each possessing one Glu residue and another completely neutral peptide (1717 Da). The microheterogeneity is due to Gly ↔ Ala, Iva ↔ Aib and Gln ↔ Glu replacements and exchanges (Iva: DIva: R-Isovaline; Aib: α-aminoisobutyric acid). The MS/MS of [M + Na]+ adduct predominantly yielded product ions from the neutral peptaibol. Further, the fragmentation patterns of H+ and Na+ adducts of two N-acetyl peptide esters were found to be very similar to that of the neutral peptaibol component. The results presented in this chapter establish that under the electrospray ion trap conditions, the fragmentation patterns of the H+ and Na+ adducts of model peptides that possess modified N- (Boc and acetyl) and C- termini are indeed very similar to that of the neutral trichotoxin. Chapter 4 delineates the applicability of liquid chromatography coupled to conventional and tandem electrospray ionization mass spectrometry (LC-ESI-MS, LC-ESI-MS/MS, LC-ESI-MS3) for the screening of novel cyclic hexadepsipeptide metabolites directly from the crude hyphal extract of the fungus Isaria. The fungal strain was grown on a solid medium (potato carrot agar), which yields aerial hyphae growing erect from the basal mycelial colony (Ravindra et al., 2004). A total of ten microheterogeneous components were identified to belong to the isariin class of cyclodepsipeptides from the LC-ESI-MS and LC-ESI-MS/MS analysis of the crude hyphal extract. Out of ten, six are determined to be new and the remaining four are previously reported isariins A-D. The primary structures of isariins A-D were from the fungi Isaria cretacea and Isaria felina (Vining & Taber 1962; Deffieux et al., 1981) and the fungal strain used in this study resembles Isaria felina (Sabareesh et al., 2007). Isariins are backbone cyclized hexadepsipeptides composed of a D-β-hydroxy acid possessing a hydrocarbon sidechain and five α-amino acids; one of the α-amino acids is a D-amino acid (Vining & Taber 1962; Deffieux et al., 1981). The detection of fragment ions due to loss of CO concomitant with the loss of H2O from the protonated precursor ion ([M + H]+) ascertained the cyclic depsipeptide nature of both the known and the new components. The fragmentation behavior of the [M + H]+ of known isariins facilitated sequence determination of the new components. Therefore, the configuration of the amino acids and the β-hydroxy acid of the new components is assumed to be same as that of the reported peptides. The microheterogeneity of the ten sequences is due to changes in the D-β-hydroxy acid (residue 1) and the adjoining α-amino acid (residue 6), whose carbonyl is linked to the hydroxyl function by an ester linkage. The number of methylene units ((-CH2)n) in the hydrocarbon sidechain of the residue 1 differs between 2 and 8 and the variability of the residue 6 is limited to Ala/Val. The ester oxygen atom was chosen as the preferable site of protonation causing ring-opening, based on the observed distribution of the fragment ions. Chapter 5 demonstrates the utility of the LC-ESI-MS and LC-ESI-MS/MS methods in the identification and characterization of six microheterogeneous backbone cyclized hexadepsipeptides, isaridins, directly from the crude hyphal extract of the fungus Isaria. Among the six components, four were found to be novel. The other two peptides, isaridins A and B were identified earlier from this laboratory (Ravindra et al., 2004). The isaridins are characterized by the presence of unusual amino acids such as N-methylated residues, β-methylproline (β-MePro) and hydroxyleucine (HyLeu) (Ravindra et al., 2004). The cyclic nature of both the known and the new peptides were confirmed from the observation of peaks due to loss of CO and H2O from the protonated precursor ion ([M + H]+). However, unlike isariins (Chapter 4), the intensity of the peak corresponding to [M + H - H2O]+ was noted to be of very low intensity, in the case of isaridins. Detection of product ion peak due to [M + H - CO2]+ suggests an additional dissociation pathway involving cleavage at the depsipeptide linkage and is supportive of the cyclic depsipeptide nature (Eckart, 1994). The sequencing of the newly detected components was enabled by understanding the fragmentation mechanism of the known isaridins. The tertiary amide nitrogens of the N-methylated residues were regarded as the preferable sites of protonation leading to ring-opening, as noted from the fragmentation spectra. The microheterogeneity in the sequences was identified using the diagnostic product ions obtained from the protonated precursor of the known isaridins. The microheterogeneity can be attributed to the variations of two residues; Pro ↔ β-MePro and N-MePhe ↔ N-MeLxx (Lxx: Leu, Ile, alloIle). The recently reported ‘isarfelins’ from the fungus Isaria felina (Guo et al., 2005) were reassigned as ‘isaridins’. The reassignment was based on very similar fragmentation profiles observed for the [M + Na]+ adduct of isaridins and isarfelins; further, the fungal strain used in this study resembles Isaria felina (Sabareesh et al., 2007). Chapter 6 presents mass spectrometric sequencing of disulfide bonded peptides from marine cone snails (conopeptides), using the MALDI LIFT MS/MS method. Lo959, a single disulfide bonded octapeptide isolated from Conus loroisii, was identified to belong to the class of contryphans (Sabareesh et al., 2006). Contryphans are small single disulfide bonded conopeptides, whose length is in the range of 7-11 residues and are rich in tryptophan. A significant feature of the contryphans is the presence of conserved DTrp (DW) at the 3rd residue within the disulfide loop (Sabareesh et al., 2006). Lo959 displays an unusual behavior under reverse phase chromatographic conditions, typical of the DW containing contryphans (Jacobsen et al., 1998). It undergoes slow conformational interconversion on the chromatographic time scale exhibiting two distinct peaks. The presence of DW at the 4th position in Lo959 was established by comparing the chromatographic profiles of natural peptide with that of two chemically synthesized peptides, one containing LW (4) and another possessing DW (4). De novo sequencing of the two peptides Ar1446 and Ar1430 from Conus araneosus established that they belonged to M-superfamily of conotoxins, in particular m-2 branch. M-superfamily conotoxins are three-disulfide bonded peptides characterized by the consensus cysteine framework, CC…C…C…CC (Corpuz et al., 2005). Ar1446 and Ar1430 are fourteen residue long peptides, each possessing three disulfide bonds. The peptides have the cysteine scaffold typical of the M-superfamily, as shown above. Specifically, the peptides belong to m-2 branch of M-superfamily, where the fourth and fifth cysteines are separated by two residues (Corpuz et al., 2005). The sequences of the peptides were derived following chemical and enzymatic modifications. The carboxamidomethylation reaction established the presence of three disulfide bonds. Indeed, the sequences were deduced from the MALDI LIFT MS/MS of [M + H]+ of the tryptic peptides. The sequences of the two peptides are almost identical and they differ only at residue 12; hydroxyproline in Ar1446, proline in Ar1430.