A short-term pharmacodynamic model for monitoring aggrecanase activity: injection of monosodium iodoacetate (MIA) in rats and assessment of aggrecan neoepitope release in synovial fluid using novel ELISAs.

OBJECTIVE: To develop a short-term in vivo model in rats, with an enzyme-linked immunosorbent assay (ELISA) readout for specific aggrecanase-cleaved aggrecan fragments, to facilitate testing of aggrecanase inhibitors. METHODS: Monosodium iodoacetate (MIA), a metabolic inhibitor, was injected into the right knee joint of male Lewis rats and the release of aggrecanase-cleaved fragments of aggrecan containing the NITEGE or ARGN neoepitope was measured in the synovial fluid at 7 days post MIA injection using novel ELISAs. The ELISAs utilize a commercial antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan, in combination with either an alpha-NITEGE antibody (NITEGE ELISA) or an alpha-ARGS/BC3 antibody (ARGS ELISA), to detect aggrecanase-cleavage of aggrecan within the interglobular domain (IGD). Aggrecan fragments present in in vitro digests, in cytokine-treated cartilage explant culture supernatants and in rat synovial fluid lavage samples were detected and quantified using the two ELISAs. Small molecule inhibitors of aggrecanase activity were dosed orally on days 3-7 to determine their ability to inhibit MIA-induced generation of the NITEGE and ARGN neoepitopes measured in the rat synovial fluid. RESULTS: The NITEGE assay was shown to specifically detect the N-terminal fragment of aggrecan comprising the G1 domain and the NITEGE neoepitope sequence. This assay can readily measure aggrecanase-cleaved bovine, human and rat aggrecan without the need for deglycosylation. The ARGS assay specifically detects C-terminal fragments of aggrecan comprising the ARGS/ARGN neoepitope and the G2 domain. Keratan sulfate (KS) residues of aggrecan interfere with this ELISA, and hence this assay works well with native rat articular cartilage aggrecan (that lacks KS residues) and with deglycosylated bovine and human aggrecan. Injection of MIA into the rat knee joints resulted in a time-dependent increase in the release of aggrecanase-cleaved aggrecan fragments into the synovial fluid and treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of the generation of these neoepitopes. CONCLUSIONS: We have established a short-term in vivo model in rats that involves measurement of synovial fluid biomarkers that are dependent on aggrecanase activity in the joint. The short duration of the model combined with the mechanistic biomarker readout makes it very useful for the initial in vivo screening of aggrecanase inhibitors prior to testing them in time and resource-intensive disease models of osteoarthritis (OA).

Development of a novel clinical biomarker assay to detect and quantify aggrecanase-generated aggrecan fragments in human synovial fluid, serum and urine.

OBJECTIVE: Proteolytic degradation of aggrecan in articular cartilage is a hallmark feature of osteoarthritis (OA). The present study was aimed at developing a sensitive enzyme linked immunosorbent assay (ELISA) for the detection of aggrecanase-cleaved fragments of aggrecan in human serum and urine to facilitate the clinical development of aggrecanase inhibitors for OA. METHODS: The BC3 monoclonal antibody that detects the ARGS neoepitope sequence in aggrecanase-cleaved aggrecan was engineered and optimized using complementarity determining region (CDR)-saturation mutagenesis to improve its binding affinity to the neoepitope. A sandwich ELISA (BC3-C2 ELISA) was developed using the optimized alpha-ARGS antibody (BC3-C2) as capture antibody and a commercially available antibody directed against the hyaluronic-acid binding region (HABR) of aggrecan as detection antibody. Aggrecanase-cleaved fragments of aggrecan present in in vitro digests, human cartilage explant culture supernatants and in human synovial fluid, serum and urine were detected and quantified using this ELISA. RESULTS: The optimized antibody had a 4-log improvement in affinity for the ARGS containing peptide compared to the parental BC3 antibody, while maintaining the ability to not cross-react with a spanning peptide. The BC3-C2 ELISA demonstrated the ability to detect aggrecanase-cleaved aggrecan fragments in the native state, without the need for deglycosylation. This ELISA was able to measure aggrecanase-generated ARGS containing aggrecan fragments in human articular cartilage (HAC) explant cultures in the basal state (without cytokine stimulation). Treatment with an aggrecanase inhibitor resulted in a dose-dependent inhibition of ARGS neoepitope released into the culture supernatant. The ELISA assay also enabled the detection of ARGS containing fragments in human synovial fluid, serum and urine, suggesting its potential utility as a biomarker of aggrecanase activity. CONCLUSIONS: We have developed a novel ELISA using an optimized ARGS antibody and have demonstrated for the first time, an ELISA-based measurement of aggrecan degradation products in human serum and urine. This assay has the potential to serve as a mechanistic drug activity biomarker in the clinic and is expected to significantly impact/accelerate the clinical development of aggrecanase inhibitors and other disease modifying drugs for OA.

Lipid remodeling in mouse liver and plasma resulting from delta6 fatty acid desaturase inhibition.

Electrospray/tandem mass spectrometry was used to quantify lipid remodeling in mouse liver and plasma during inhibition of polyunsaturated fatty acid synthesis by the delta6 fatty acid desaturase inhibitor, SC-26196. SC-26196 caused increases in linoleic acid and corresponding decreases in arachidonic acid and docosahexaenoic acid in select molecular species of phosphatidylcholine, phosphatidylethanolamine, and cholesterol esters but not in phosphatidylserine, phosphatidylinositol, or triglycerides. For linoleic acid-, arachidonic acid-, and docosahexaenoic acid-containing phospholipid species, this difference was, in part, determined by the fatty acid at the sn-1 position, namely, palmitic or stearic acid. An understanding of phospholipid remodeling mediated by delta6 desaturase inhibition should aid in clarifying the contribution of arachidonic acid derived via de novo synthesis or obtained directly in the diet during inflammatory responses.

Identification and characterization of falcilysin, a metallopeptidase involved in hemoglobin catabolism within the malaria parasite Plasmodium falciparum.

The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the alpha and beta chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4' positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases.

Novel, selective delta6 or delta5 fatty acid desaturase inhibitors as antiinflammatory agents in mice.

Decreased synthesis of arachidonic acid by inhibition of the Delta6 or Delta5 desaturase was evaluated as a means to mitigate inflammation. Using quantitative in vitro and in vivo radioassays, novel compounds representing five classes of Delta5 desaturase inhibitors and one class of Delta6 desaturase inhibitor were identified. The Delta6 desaturase inhibitor, SC-26196, had pharmacokinetic and pharmacodynamic profiles in mice that allowed for the evaluation of the pharmacological effects of chronic inhibition of desaturase activity. SC-26196 decreased edema to the same extent as indomethacin or essential fatty acid deficiency in the carrageenan paw edema model in the mouse. The antiinflammatory properties of SC-26196 were consistent with its mechanism of action as a Delta6 desaturase inhibitor: 1) A correlation existed between inhibition of liver Delta6 desaturase activity and decreases in edema. 2) The onset of the decrease in edema was time dependent. 3) Selective reduction of arachidonic acid occurred dose dependently in liver, plasma and peritoneal cells. 4) In the presence of SC-26196, controlled refeeding of arachidonic acid, but not oleic acid, reversed the changes resulting from desaturase inhibition. The Delta6 desaturase may be a target for development of antiinflammatory drugs whose mechanism of action is unique.

Generation of hemoglobin peptides in the acidic digestive vacuole of Plasmodium falciparum implicates peptide transport in amino acid production.

Intraerythrocytic malaria parasites avidly consume hemoglobin as a source of amino acids for incorporation into parasite proteins. An acidic organelle, the digestive vacuole, is the site of hemoglobin proteolysis. Early events in hemoglobin catabolism have been well studied. Two aspartic proteases, plasmepsins I and II, and a cysteine protease, falcipain, cleave hemoglobin into peptides. While it has been presumed that hemoglobin peptide fragments are degraded to individual amino acids by exopeptidase activity in the digestive vacuole, this hypothesis lacks experimental support. Incubation of human hemoglobin with P. falciparum digestive vacuole lysate generated a series of discrete peptide fragments with cleavage sites an average of 8.4 amino acids apart. No free amino acids could be detected and there was no evidence of peptide heterogeneity due to exopeptidase trimming. These sites correspond to points of cleavage previously established for plasmepsin I, plasmepsin II, and falcipain as well as some novel sites that suggest the existence of an additional endoproteinase. By colorimetric assay, P. falciparum has abundant aminopeptidase activity but this activity is not found in the digestive vacuoles and the parasite lacks detectable carboxypeptidase activity altogether. These data support a model for hemoglobin catabolism wherein small peptides are formed from cleavage of hemoglobin by the enzymes of the digestive vacuole and then are transported through the membrane of the digestive vacuole to the cytoplasm. There, exopeptidase activity converts the peptides to individual amino acids for parasite growth and maturation.

Altered arachidonic acid metabolism in urate crystal induced inflammation.

Gout is an acute rheumatic disorder that occurs in connection with the deposition of monosodium urate (MSU) crystals in the joints. This disease is characterized by intermittent episodes of severe pain and inflammatory joint swelling which are seemingly driven by prostaglandins. In this study we investigated the effect of MSU crystals on arachidonic acid (AA) metabolism in the mouse. We have demonstrated that prostaglandins and other AA metabolites were transiently formed after MSU crystal injection with peak levels occurring after 10 min. In contrast, free AA levels remained high for 2-4 hours after MSU crystal injection. By contrast, when exogenous AA was administered instead of MSU crystals, both the eicosanoids and AA diminished at the same high rates. The metabolism of exogenously administered AA to eicosanoids was inhibited by pretreatment with MSU crystals. No inhibition of AA metabolism was observed when mice were pretreated with AA itself, Ca2+ ionophore (A23187), or zymosan. We conclude that the MSU crystal treatment of mice results in a transient eicosanoid production which is followed by attenuated AA metabolism. It could be that MSU crystals similarly inhibit AA metabolism in gout and thereby limit the duration of gout attacks.

Rapid quantitation of a large scope of eicosanoids in two models of inflammation: development of an electrospray and tandem mass spectrometry method and application to biological studies.

Assessment of eicosanoid levels in biological systems is important for understanding their role in cell function and pathophysiological events. Current methods of eicosanoid quantitation are limited by sensitivity, scope, or throughput. The development of a new method for eicosanoid assessment in biological samples by electrospray and tandem mass spectrometry (MS/MS) in the multiple reaction monitoring mode is described here. In this study, 14 biologically significant eicosanoids were quantitated in a single sample. Complete sample analysis required two repeated injections of 5 microliters with an analysis time of 1.5 min/injection. Limits of detection ranged from 0.5 pg for thromboxane B2 (TxB2) to 10 pg for 6-keto prostaglandin F1 alpha (6-keto PGF1 alpha). The reliability, reproducibility, sensitivity, and cross-detection of the method is also described. The MS/MS method was used to explore eicosanoid production in two inflammation models: lipopolysaccharide (LPS)-stimulated human whole blood and carrageenan-challenged rat air pouch. The most abundant metabolites in LPS-stimulated whole blood were prostaglandin E2 (PGE2), TxB2, and 6-keto PGF1 alpha; prostaglandins E1, D2, and F2 alpha and leukotrienes B4 and C4 were detected in lower amounts. Eicosanoid levels determined by MS/MS were similar to those obtained by immunoassay and GC-MS. The most abundant metabolites detected in carrageenan-challenged rat air pouch were PGE2, 6-keto PGF1 alpha, and TxB2. The method described in this work is accurate and rapid and should greatly aid in evaluating the role of multiple eicosanoids in future biological studies.

Naturally processed peptides from rheumatoid arthritis associated and non-associated HLA-DR alleles.

Naturally processed peptides from immunoaffinity-purified HLA-DRB1*0401, -DRB1*0404 (rheumatoid arthritis (RA)-associated), and -DRB1*0402 (non-RA-associated) molecules were analyzed by capillary liquid chromatography and mass spectrometry. The molecular weights observed for more than 60 eluted peptides from each HLA-DR protein ranged from 788 to 3535 atomic mass units, corresponding to peptides 7 to 32 amino acids in length. Sequencing of more than 60 of the abundant peptides revealed nested sets of peptides that were derived from only 12 different proteins. The majority of these proteins were membrane-associated (HLA class I, class II, and Ig molecules). Synthetic peptides, corresponding to endogenous peptide sequences, bound with high affinity (5 to 80 nM) to the HLA-DR molecules from which they were eluted. In addition, most were promiscuous binding peptides in that they also bound to other HLA-DR molecules. Truncations of eluted peptide sequences and alanine scanning mutational analysis of a Mycobacterium leprae peptide were used to identify the peptide residues involved in binding to DRB1*0404 and DRB1*0402 molecules. Furthermore, an invariant chain peptide was eluted from the DRB1*0402 molecules but not from the RA-associated molecules. The lack of invariant chain peptides from DRB1*0401 and DRB1*0404 molecules may contribute to the loading of autoantigen peptides into these molecules and to their association with disease.

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified from Escherichia coli.

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified from Escherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified from Escherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.

Proteolytic cleavage of insulin-like growth factor binding protein 4 (IGFBP-4). Localization of cleavage site to non-homologous region of native IGFBP-4.

Insulin-like growth factor binding protein 4 (IGFBP-4) is a 24-kDa protein that binds insulin-like growth factor 1 (IGF-1) and IGF-2 with high affinity and inhibits IGF action in vitro. We recently described a protease produced by the B104 neuronal cell line that cleaves IGFBP-4, yielding an approximate 16-kDa immunoreactive protein that binds IGFs with reduced affinity. We analyzed fragments produced by exposing pure IGFBP-4 to the protease to determine potential cleavage sites. Electrospray mass spectrometry and amino acid sequencing indicated the 16-kDa fragment spanned the NH2 terminus of native IGFBP-4 through Lys-120. There was evidence for an additional proteolytic fragment beginning at amino acid 132 and continuing to the COOH terminus. Proteolysis could be blocked by a synthetic peptide that spanned amino acids 117-126 but not by peptides that contained flanking sequences 111-120 or 125-135. Mutagenesis was used to alter the basic residue at position 120. The expressed mutant IGFBP-4 (K120A) was relatively resistant to cleavage, strongly suggesting that residues 120-121 represent the cleavage site. This region of IGFBP-4 is not homologous with other IGFBPs, explaining the apparent specificity of the protease for IGFBP-4. The 16-kDa IGFBP-4 fragment no longer inhibited IGF-1-stimulated thymidine uptake in vitro, suggesting that proteolytic processing of IGFBP-4 may have important functional consequences in vivo.

Enzymatic synthesis of a 6'-sulphated sialyl-Lewisx which is an inhibitor of L-selectin binding to peripheral addressin.

A sulphated form of sialyl-Lewisx, NeuAc alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNAc6OSO3 beta 1-3Gal, was synthesized enzymatically from a precursor disaccharide, GlcNAc6OSO3 beta 1-3Gal, using sequential steps involving beta 1,4-galactosyltransferase, alpha 2,3-trans-sialidase and recombinant alpha 1,3-fucosyltransferase, respectively. Successful enzymatic fucosylation at the 3 position of the GlcNAc6OSO3 residue demonstrated that fucosyltransferase are capable of generating, in situ, sulphated sialyl Lewisx structures containing sulphate at the 6 position of GlcNAc. The sulphated sialyl-Lewisx pentasaccharide produced by this procedure inhibited binding of a soluble form of L-selectin to 35SO4-labelled peripheral addressin with an IC50 of 0.8 mM, whereas sialyl-Lewisx tetrasaccharide was a weaker inhibitor, displaying an IC50 of 3.2 mM. Hemmerich and Rosen (Biochemistry, 33, 4820-4829, 1994) recently reported the presence of Gal beta 1-4GlcNAcO6SO3 structures on murine peripheral addressin Sgp50, in addition to sialyl Lewisx structures sulphated at the 6-O-galactose position. Based on our data, we suggest that sialyl Lewisx sulphated at the 6-O-GlcNAc position may also exist on receptors and function as a ligand for L-selectin.

Activity of two-chain recombinant human cytomegalovirus protease.

The human cytomegalovirus UL80 protease was expressed in Escherichia coli and purified by metal-chelate chromatography using a histidine tag engineered at the amino terminus. Cleavage of the 30-kDa protease at an internal site, VEA/A144, resulted in the recovery of 16- plus 14-kDa two-chain protease. The amino-terminal 16-kDa chain and the carboxyl-terminal 14-kDa chain remained associated as an active enzyme that was modified specifically at Ser132 on the 16-kDa chain by [3H]diisopropyl fluorophosphate. Disruption of the cleavage site by mutation from VEA/A to AEA/A facilitated the recovery of active 30-kDa one-chain enzyme that could be similarly modified at Ser132 by [3H]diisopropyl fluorophosphate. Both one- and two-chain enzymes cleaved recombinant assembly protein at the maturation site, VNA/S, and a peptide, GVVNASARL, mimicking this site. Internal processing does not inactivate the protease but forms a two-chain enzyme that retains activity.

Heterogeneity of plasma tissue factor pathway inhibitor.

Much of tissue factor pathway inhibitor (TFPI) in plasma is bound to lipoproteins. The major form of TFPI associated with low density lipoproteins (LDL) is 34 kDa, whereas that associated with high density lipoproteins (HDL) is 41 kDa and appears in part to represent a mixed disulphide complex between TFPI and apolipoprotein AII. The native and recombinant TFPI produced by mammalian cells in tissue culture and the TFPI released by heparin in vivo, however, are 34 kDa. Western blotting with antibodies raised against specific TFPI peptides and cation exchange chromatography under denaturing conditions of partially purified plasma TFPI suggest that only a fraction of TFPI circulating in plasma is in the form of the full length molecule, the remainder consisting of variably carboxyl-terminal truncated forms. Electrospray mass spectrometry of the isolated 34 kDa form of plasma TFPI, which predominantly circulates bound to LDL, confirms that it lacks a substantial portion of the carboxyl-terminus including at least a portion of the third Kunitz-type domain.

Isolation of Escherichia coli synthesized recombinant eukaryotic proteins that contain epsilon-N-acetyllysine.

Recombinant porcine (rpST) and bovine somatotropins (rbST) synthesized in Escherichia coli contain the amino acid, epsilon-N-acetyllysine. This amino acid was initially discovered in place of the normal lysine144 in a modified reversed-phase HPLC (RP-HPLC) species of rpST. Mass spectrometry and amino acid sequencing of a tryptic peptide isolated from this RP-HPLC purified protein were used to identify this altered residue as epsilon-N-acetyllysine. Ion-exchange chromatography was utilized to prepare low isoelectric point (pI) forms of rpST and rbST, which are enriched in epsilon-N-acetyllysine. Electrospray mass spectrometry demonstrated that the majority of the protein in these low pI fractions contained species 42 Da larger than normal. Immobilized pH gradient electrophoresis (IPG) of the ion-exchange purified low pI proteins was used to isolate several monoacetylated species of rpST and rbST. The location of the acetylated lysine in each IPG-purified protein was determined by tryptic peptide mapping and amino acid sequencing of the altered tryptic peptides. Amino acid analyses of enzymatic digests of rpST and rbST were also used to confirm the presence of epsilon-N-acetyllysine in these recombinant proteins. These data demonstrate that a significant portion of rpST and rbST produced in E. coli contain this unusual amino acid.

Order and specificity of the Plasmodium falciparum hemoglobin degradation pathway.

The human malaria parasite, Plasmodium falciparum, degrades nearly all its host cell hemoglobin during a short segment of its intraerythrocytic development. This massive catabolic process occurs in an acidic organelle, the digestive vacuole. Aspartic and cysteine proteases have been implicated in this pathway. We have isolated three vacuolar proteases that account for most of the globin-degrading activity of the digestive vacuole. One is the previously described aspartic hemoglobinase that initiates hemoglobin degradation. A second aspartic protease is capable of cleaving hemoglobin with an overlapping specificity, but seems to prefer acid-denatured globin. The third is a cysteine protease that does not recognize native hemoglobin but readily cleaves denatured globin. It is synergistic with the aspartic hemoglobinase, both by in vitro assay of hemoglobin degradation, and by isobologram analysis of protease inhibitor-treated parasites in culture. The cysteine protease is highly sensitive to chloroquine-heme complex, suggesting a possible mechanism of 4-aminoquinoline antimalarial action. The data suggest an ordered pathway of hemoglobin catabolism that presents an excellent target for chemotherapy.

Characterization of human uroguanylin: a member of the guanylin peptide family.

Guanylin, a peptide homologue of the bacterial heat-stable enterotoxins (ST), is an endogenous activator of guanylate cyclase C (GC-C). We have initiated a search for other members of the guanylin peptide family and in the current study describe a "guanylin-like peptide" from human urine. Bioactivity was monitored by determining the effect of urine extracts on T84 cell guanosine 3',5'-cyclic monophosphate (cGMP) levels. Purification yielded two bioactive peaks of peptides that, when sequenced by NH2-terminal analysis, possessed 15 and 16 amino acids. The sequence of the smaller peptide represented an NH2-terminal truncation of the larger peptide. We have termed the larger peptide human uroguanylin; it has the following amino acid sequence: NDDCELCVNVACTGCL. Human uroguanylin shares amino acid sequence homology with guanylin and ST. Synthetic uroguanylin increased cGMP levels in T84 cells, competed with 125I-labeled ST for receptors, and stimulated Cl- secretion as reflected by an increased short-circuit current. Thus we report the isolation from human urine of a unique peptide, uroguanylin, that behaves in a manner similar to guanylin and appears to be a new member of this peptide family.

High-level expression and purification of human leukotriene A4 hydrolase from insect cells infected with a baculovirus vector.

Leukotrienes constitute a group of bioactive compounds derived from arachidonic acid which play important roles in immediate hypersensitivity and inflammation. Leukotriene A4 hydrolase (LTA4H) is an epoxide hydrolase, catalyzing the hydration of LTA4 to LTB4, and also acts an aminopeptidase, with the ability to cleave amides of p-nitroaniline. The cDNA for LTA4H was cloned using oligonucleotide-directed amplification of the cDNA sequence by polymerase chain reaction and by oligonucleotide-based screening of a bacteriophage lambda gt11 cDNA library derived from human placental tissue. High levels of biologically active LTA4H were expressed in cultured Spodoptera frugiperda insect cells infected with a baculovirus expression vector containing the LTA4H cDNA. Expression levels were approximately 100 mg per liter of cell-free culture media. LTA4H was recovered from the medium and purified to > 95% purity by ion-exchange and gel-filtration chromatography, with an overall yield of 76%. LTA4H produced by insect cells exhibits both hydrolase and aminopeptidase activities and has kinetic properties similar to those reported for enzyme isolated from human lung. Two major isoforms, with pI's of 5.3 and 5.1, were isolated by preparative chromatofocusing chromatography. NH2-terminal sequence analysis revealed that the two different by an NH2-terminal blocking group. Electrospray ionization mass spectrometry indicates that the two isoforms differ by a molecular mass of 42, indicating that the blocking group is an acetyl group.

Identification and oligosaccharide structure analysis of rhodopsin glycoforms containing galactose and sialic acid.

The N-linked oligosaccharides of frog (Rana pipiens) rhodopsin were analysed by sequential exoglycosidase digestion and gel filtration chromatography, following reductive tritiation. In addition, selected tryptic glycopeptides obtained from frog retinal rod outer segment membranes were examined by electrospray mass spectrometry (ES-MS), fast atom bombardment mass spectrometry (FAB-MS), amino acid sequence and composition analysis, and carbohydrate composition analysis. The amino acid sequence data demonstrated that the glycopeptides were derived from rhodopsin and confirmed the presence of two N-glycosylation sites, at residues Asn2 and Asn15. The predominant glycan (approximately 60% of total) had the structure GlcNAc beta 1-2Man alpha 1-3(Man alpha 1-6) Man beta 1-4GlcNAc beta 1-4GlcNAc-(Asn), with the remaining structures containing 1-3 additional hexose residues, as reported previously for bovine rhodopsin. Unlike bovine rhodopsin, however, a sizable fraction of the total glycans of frog rhodopsin also contained sialic acid (NeuAc), with the sialylated oligosaccharides being present exclusively at the Asn2 site. FAB-MS analysis of oligosaccharides released from the Asn2 site gave, among other signals, an abundant quasimolecular ion corresponding to a glycan of composition NeuAc1Hex6HexNAc3 (where Hex is hexose and HexNAc is N-acetylhexosamine), consistent with a hybrid structure. The potential biological implications of these results are discussed in the context of rod outer segment membrane renewal.

Identification of components in waste streams by electrospray and tandem mass spectrometry.

Highly polar, non-gas-chromatographable compounds have few unambiguous analysis protocols for environmental applications. A recent environmental investigation, concerning the identification of a non-gas-chromatographable yellow component in chemical waste water and in effluents from a biological wastewater treatment plant required the use of a number of analytical approaches. Electrospray mass spectrometry, tandem mass spectrometry, high-performance liquid chromatography, nuclear magnetic resonance, and molecular spectroscopy of commercial and synthesized chlorodinitrophenol isomers were required in order to identify the specific isomer causing the color. The present report summarizes the electrospray ionization and tandem mass spectrometric studies that were used. The mass spectrometric study shows that two different isomers of chlorodinitrophenol exhibit very different collision-induced dissociation (CID) spectra. Differences in the tandem mass spectra can be attributed to the different structures of the anions formed from these two different isomers. Instrumentation that uses electrospray ionization and produces CID mass spectra and optical absorption spectra in a single analysis may be required in order to produce highly specific information on non-gas-chromatographable compounds found in the environment.