Identification and molecular characterization of a novel protein Saglin as a target of monoclonal antibodies affecting salivary gland infectivity of Plasmodium sporozoites.

Molecular mechanisms underlying the interaction between malarial sporozoites and putative receptor(s) on the salivary glands of Anopheles gambiae remain largely unknown. In previous studies, a salivary gland protein of ~100 kDa was identified as a putative target based on recognition of the protein by a monoclonal antibody (mAb) 2A3 that caused a >/= 70% reduction in the average number of sporozoites per infected salivary gland when fed to mosquitoes. Using affinity purification we purified the target of this mAb from extracts of female A. gambiae salivary glands and it was found to be a novel protein by tandem mass spectrometric analysis. Biochemical and molecular characterization of the 100 kDa protein showed that this molecule, designated Saglin, exists as a disulphide-bonded homodimer of 50 kDa subunits. The ability to form homodimers was retained even in the recombinant Saglin expressed in mammalian cells (HEK293). The amino acid sequence of Saglin contains a signal peptide suggesting that Saglin is a secreted protein. If Saglin is indeed involved in the process of invasion of A. gambiae salivary glands by sporozoites of Plasmodium, it could provide a novel target for future investigations aimed at interruption of malaria transmission.

Investigation of tyrosine nitration in proteins by mass spectrometry.

In vivo nitration of tyrosine residues is a post-translational modification mediated by peroxynitrite that may be involved in a number of diseases. The aim of this study was to evaluate possibilities for site-specific detection of tyrosine nitration by mass spectrometry. Angiotensin II and bovine serum albumin (BSA) nitrated with tetranitromethane (TNM) were used as model compounds. Three strategies were investigated: (i) analysis of single peptides and protein digests by matrix-assisted laser desorption/ionization (MALDI) peptide mass mapping, (ii) peptide mass mapping by electrospray ionization (ESI) mass spectrometry and (iii) screening for nitration by selective detection of the immonium ion of nitrotyrosine by precursor ion scanning with subsequent sequencing of the modified peptides. The MALDI time-of-flight mass spectrum of nitrated angiotensin II showed an unexpected prompt fragmentation involving the nitro group, in contrast to ESI-MS, where no fragmentation of nitrated angiotensin II was observed. The ESI mass spectra showed that mono- and dinitrated angiotensin II were obtained after treatment with TNM. ESI-MS/MS revealed that the mononitrated angiotensin II was nitrated on the side-chain of tyrosine. The dinitrated angiotensin II contained two nitro groups on the tyrosine residue. Nitration of BSA was confirmed by Western blotting with an antibody against nitrotyrosine and the sites for nitration were investigated by peptide mass mapping after in-gel digestion. Direct mass mapping by ESI revealed that two peptides were nitrated. Precursor ion scanning for the immonium ion for nitrotyrosine revealed two additional partially nitrated peptides. Based on the studies with the two model compounds, we suggest that the investigation of in vivo nitration of tyrosine and identification of nitrated peptides might be performed by precursor ion scanning for the specific immonium ion at m/z 181.06 combined with ESI-MS/MS for identification of the specific nitration sites.

Characterization of a recombinant monoclonal antibody by mass spectrometry combined with liquid chromatography.

In this report, we present the characterization of a humanized monoclonal antibody specific for the human epidermal growth factor receptor (hEGFR). Direct analysis by matrix assisted laser desorption ionization mass spectrometry (MALDI-MS) of peptide mixtures and chromatographically isolated fractions allowed identification of 94.0% and 85.4% of the amino acid sequence of light and heavy chains, respectively. Microheterogeneity sources were identified in light and heavy chains and a previously unreported posttranslational modification for immunoglobulins was found. One N-glycosylation site was identified in the heavy chain with non-sialylated bianntenary fucosylated structures. This study is one of the first to assess the potential of MALDI-MS in combination with more conventional protein chemistry techniques for the characterization of monoclonal antibodies.

Proteomics of the chloroplast: systematic identification and targeting analysis of lumenal and peripheral thylakoid proteins.

The soluble and peripheral proteins in the thylakoids of pea were systematically analyzed by using two-dimensional electrophoresis, mass spectrometry, and N-terminal Edman sequencing, followed by database searching. After correcting to eliminate possible isoforms and post-translational modifications, we estimated that there are at least 200 to 230 different lumenal and peripheral proteins. Sixty-one proteins were identified; for 33 of these proteins, a clear function or functional domain could be identified, whereas for 10 proteins, no function could be assigned. For 18 proteins, no expressed sequence tag or full-length gene could be identified in the databases, despite experimental determination of a significant amount of amino acid sequence. Nine previously unidentified proteins with lumenal transit peptides are presented along with their full-length genes; seven of these proteins possess the twin arginine motif that is characteristic for substrates of the TAT pathway. Logoplots were used to provide a detailed analysis of the lumenal targeting signals, and all nuclear-encoded proteins identified on the two-dimensional gels were used to test predictions for chloroplast localization and transit peptides made by the software programs ChloroP, PSORT, and SignalP. A combination of these three programs was found to provide a useful tool for evaluating chloroplast localization and transit peptides and also could reveal possible alternative processing sites and dual targeting. The potential of proteomics for plant biology and homology-based searching with mass spectrometry data is discussed.

Structure determination of two conotoxins from Conus textile by a combination of matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry and biochemical methods.

Two highly modified conotoxins from the mollusc Conus textile, epsilon-TxIX and Gla(1)-TxVI, were characterized by matrix-assisted laser desorption/ionization and electrospray mass spectrometry and also by electrospray ionization tandem and triple mass spectrometry in combination with enzymatic cleavage and chemical modification reactions. The mass spectrometric studies allowed the confirmation of the sequence determined by Edman degradation and assignment of unidentified amino acid residues, among which bromotryptophan residues and an O-glycosylated threonine residue were observed. Methyl esterification was found necessary for the site-specific assignment of the Gla residues in the peptides.

A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx.

Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a gamma-carboxyglutamic acid-containing peptide, conotoxin epsilon-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic Ca2+ channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of Ca2+ in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional 1H NMR spectroscopy, identifies an electronegative patch created by the side chains of two gamma-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region.

Purification, characterization, sequence determination, and mass spectrometric analysis of a trypsin inhibitor from seeds of the Brazilian tree Dipteryx alata (Leguminosae).

Dipteryx alata trypsin inhibitor (DATI) has been purified and completely sequenced. It showed homology to members of the Bowman-Birk family of inhibitors. The last step of DATI purification by RP-HPLC (narrow-borc C18 column) suggested the existence of some isoforms of the inhibitor due to the presence of a cluster of very close peaks in the chromatogram. By using electrospray ionization mass spectrometry (ESIMS) and laser desorption mass spectrometry (LDIMS), the identification of DATI isoforms was made possible. From the ESIMS data, the following molecular masses were found: 6803.22 +/- 0.92 for isoform a; 6890.94 +/- 0.73 for b; 6977.58 +/- 0.39 for c; 7065.07 +/- 0.67 for d; 7151.42 +/- 0.86 for e; and 7291.70 +/- 0.43 for f. Similar masses were found when using LDIMS. Isoform b was the most abundant and its molecular mass matched the molecular mass of 6893 calculated from the sequence of DATI. The mass differences between a and b, b and c, c and d, and d and e were equal to 87, which corresponds to Ser. Isoform a might not have the N-terminal Ser present in isoform b, while the other additional Ser residues might comprise a row localized in the C- or N-terminal. The appearance of all these isoforms could result from posttranslational N- and C-terminal processing.