Glycobiology, 2000, Vol. 10, No. 5 451-458
© 2000 Oxford University Press
Structural characterization of a xylanase from psychrophilic yeast by mass spectrometry
2International Mass Spectrometry Facilities Centre, via Pansini 5, 80131 Naples, Italy, 3CEINGE Advanced Biotechnologies scarl, via Pansini 5, 80131 Naples, Italy, 4Department of Organic and Biological Chemistry, University of Naples "Federico II," via Mezzocannone 16, 80134 Naples, Italy, and 5Laboratory of Biochemistry, University of Liege, Belgium
Received on June 18, 1999; revised on November 18, 1999; accepted on December 1, 1999.
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Abstract |
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The complete structural characterization of the xylanase, a glycoprotein constituted of 338 amino acids, from psychrophilic antarctic yeast Criptococcus albidus TAE85 was achieved both at the protein and carbohydrate level by exploiting mass spectrometric procedures. The verification of the primary structure, the definition of the S-S pattern, the assignment of glycosylation sites and the investigation of glycosylation pattern were performed. This analysis revealed the occurrence of N-glycosylation only at Asn254, modified by high-mannose structure; moreover the protein resulted to be O-glycosylated with GalGalNAc structures. The data obtained on both the N- and O-linked glycans in the cold xylanase constitute the first description of the glycosylation pattern in psychrophylic microorganisms and suggest that the glycosylation system in cold-adapted organisms might have similarities as well as differences with respect to mesophylic and thermophylic cells. The cysteine pairings were eventually identified as Cys173-Cys205 and Cys272-Cys278, with Cys89 showing a free thiol group. These data suggest that a common folding motif might occur within the entire xylanase family in which the second Cys is linked to the third one with the fourth and fifth joined together.
Key words: glycosylation/mass spectrometry/psychrophiles/xylanase/yeast
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Introduction |
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Xylan, a branched ß-D-1,4-linked xylopyranose polymer, highly substituted with acetyl, arabinosyl and uronyl groups (Biely, 1985) is a major constituent of hemicellulose, whose enzymatic hydrolysis requires the synergistic action of several activities and involves xylanases, in the first step of its bioconversion into fermentable sugars (Wong et al., 1988
) Xylan hydrolysis products are of great interest in biotechnological processes because they may be converted into liquid fuel, and organic solvents (McClear, 1986; Wong and Saddler, 1992; Paice et al., 1988). The main goal of many laboratories has been to find new cellular lines for the purification of xylanases. Thus, a number of xylanases have been purified from bacteria, yeast, or fungi. However yeast has been the favorite host organism for the production of recombinant glycoproteins. Indeed yeast, unlike bacteria, can perform certain posttranslational modifications, such as glycosylation (Runge, 1988) which particularly affects the enzymatic activity of recombinant proteins, as demonstrated for the xylanase from yeast Cryptococcus albidus (Morosoli et al., 1988).
Recently, increasing interest was addressed to the study of psychrophilic organisms which live in cold conditions, below 5°C. In order to survive and grow in cold environments, they have evolved a complex range of adaptations to all of their cellular components, including protein synthesis machinery and biodegradative enzymes. Adaptated proteins from psychrophilic organisms are currently being explored for their biotechnological potential (Russel, 1998). It is clear that the development of such systems may have a terrific impact on the biotech industry.
The production of recombinant proteins at low temperatures in psychrophilic hosts might lead to the solution of some of the major problems that affect the solubility and yield of active protein products. Furthermore, microorganisms having proteolytic enzymes active only at low temperatures might be suitable tools for the production of medically and commercially useful molecules which are very susceptible to higher temperatures in other organisms. However, this "finalized" approach to the problem of life at low temperatures suffers from dramatic lack of information about genetic and molecular biology of cold-living organisms. Therefore, the interest in the isolation and structural elucidation of enzymes from cold-adapted organisms could be useful for producing engineered proteins of biotechnological interest.
This paper reports the complete structural analysis of the xylanase, a glycoprotein secreted by the psychrophilic antarctic yeast Cryptococcus albidus TAE85. It is worth noting that this is the first structural characterization reported on a psychrophilic glycoprotein. On the basis of the cDNA sequence, psychrophilic xylanase comprises a sequence of 338 amino acids containing five cysteine residues and two putative N-glycosylation sites at Asn139 and Asn254. By using advanced mass spectrometric methodologies the verification of the primary structure, the definition of the S-S pattern, the assignment of glycosylation sites, and the investigation of glycosylation pattern were performed. These analyses revealed the occurrence of N-glycosylation only at Asn254, modified by high-mannose structure; moreover the protein resulted to be O-glycosylated with GalGalNAc structures.
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Analysis of intact protein
Intact xylanase (50 µg) was directly analyzed by mass spectrometric methodologies in order to obtain information on the overall molecular mass and the distribution of glycoforms (Amoresano et al., 1996
). Unfortunately, attempts to measure the accurate molecular mass of the protein by electrospray mass spectrometry (ESMS) were probably unsuccessful because of the high content of glycans. The protein was then analyzed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry producing the spectrum shown in Figure 1. Two pairs of broad peaks could be identified in the spectrum, each pair corresponding to the MH+ and MH22+ ions, respectively. The broadening of the signals was very likely due both to a large heterogeneity of the glycoforms and to the presence of unresolved photochemically generated matrix adduct ions (Beavis and Chait, 1990). A molecular mass centered at about 37,453.6 Da for the unglycosylated protein could be calculated from the spectra in good agreement with the expected average mass of the protein moiety that should account for 37,426.5 Da on the basis of cDNA sequence. The major component at about 39,508.4 Da could confidently represent an average mass of the glycosylated protein due to the different contribution of the various glycoforms and could not be used to determine the real molecular mass nor to deduce the structure of each oligosaccharide chain. However, the MALDI/MS analysis confirmed that the xylanase is made up of a heterogeneous population of glycoforms which accounts for about 2000 Da.
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Structural characterization of xylanase
In the next step, an aliquot of the glycoprotein (300 µg) was submitted to an accurate structural characterization both at the protein and the carbohydrate level by exploiting different mass spectrometric methodologies. A protein sample was reduced, alkylated with iodoacetic acid and submitted to chymotrypsin digestion; the resulting peptide mixture was directly analyzed by MALDI/MS, as shown in Figure 2. Table I shows the mass signals recorded in the spectra and the corresponding peptides within the xylanase sequence identified on the basis of their mass value. The mass spectral analysis led to the verification of about 77% of the entire protein sequence and identified the occurrence of N-glycosylation at Asn 254. In the high mass region of the spectra, in fact, a series of peaks at m/z 4098.1, 4259.2, 4421.3, 4583.7, 4746.1, 4908.3, 5071.3, and 5233.9 were detected. These signals were attributed to the glycopeptides 241–265 or 241–266 modified by a high mannose glycan chains ranging from 6 to 13 or from 5 to 12 mannose residues, respectively.
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Moreover two signals at m/z 1477.9 and 2389.1 showed the expected molecular mass for the unglycosylated peptides 130–142 and 123–142, respectively, thus ruling out the occurrence of posttranslational modification at Asn139. The peptide mixture was then deglycosylated by incubation with PNGase F and re-examined by MALDI/MS. The signals at m/z 2718.4 and 2881.7 were attributed to the deglycosylated peptides 241–265 and 241–266 respectively, which exhibited a molecular mass increased by 1 Da because Asn254 was converted into an Asp residue following PNGaseF treatment. It should be underlined that the two very minor peaks at m/z 2690.5 and 2896.6 correspond to the peptide 274–282 modified by HexHexNAc2 and Hex2HexNAc2 O-linked structures, respectively.
In order to check the entire amino acid sequence of the glycoprotein, an aliquot of the reduced and alkylated xylanase was digested with endoprotease Glu-C and examined by MALDI/MS. The data combined from the two mass mapping experiments allowed us to verify about 98% of the xylanase primary structure as shown in Table II and Figure 3. Unfortunately, no evidence for the O-glycosylation was inferred by this experiment.
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Characterization of the oligosaccharide chains
The composition of monosaccharides from the glycosidic moiety of xylanase was determined by GCMS analyses. Intact protein (100 µg) was purified by a single chromatographic step and hydrolyzed under mild acid condition. The methylglycoside mixture was properly derivatized to TMS-monosaccharides and directly analyzed by GC-MS. Figure 4 shows the corresponding gas-chromatogram obtained by monitoring the total ion current as a function of time.
Apart from the relative abundance of the individual monosaccharides, this procedure provides information on the glycosidic structure linked to the protein, either complex, high mannose and/or hybrid N-linked or O-linked oligosaccharides or both. A high mannose-to-galactose ratio along with the presence of GalNAc and GlcNAc confirmed the occurrence of high-mannose type N-linked glycans and identified the presence of O-linked oligosaccharides. No sialic acid residue was detected thus suggesting that no capping occurred at the nonreducing end of N-or O-linked chains.
The structure of the glycosidic moieties of xylanase was further investigated by direct mass analysis of the permethylated oligosaccharide mixtures. The O-linked glycans were released from the peptide moiety by reductive elimination in basic conditions (Carlson, 1968). The oligosaccharide mixture was then peracetylated and directly analyzed by MALDI/MS showing the occurrence of mass signals at m/z 748.7, 1036.2, and 1324.6. These mass values were associated to HexNAcHex, HexNAc2Hex, and HexNAc2Hex2 structures as indicated in Figure 5A.
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The intact N-linked oligosaccharides were released from the peptide backbone by PNGaseF treatment of the xylanase peptide digests. The glycan mixture was separated from the peptides by a Sep-pak reverse phase chromatographic step, permethylated and analyzed by MALDI mass spectrometry. The corresponding MALDI spectrum is reported in Figure 5B; the N-linked glycosidic moiety of xylanase was found to be a heterogeneous mixture of high mannose glycans, in fact Hex6–12HeNAc2 structures were detected. As in MALDI/MS peak intensity could be related to the relative abundance of the corresponding structures, Hex9HeNAc2 (MNa+ 2396.83) and Hex10HeNAc2 (MNa+ 2601.47) structures seem to represent the most abundant glycoforms (see Figure 5B). These data were confirmed by a sugar analysis carried out on the N-linked glycan moiety showing the occurrence of Man and GlcNAc residues.
In order to define the glycan structure in the mixture, the permethylated derivatives were submitted to linkage analysis. The mixture of oligosaccharides was hydrolyzed to the individual monosaccharides, which were then reduced and acetylated prior to analysis by GC-MS. Terminal (t-Man, r. t. 0.681) and monosubstituted monosaccharides (2-Man, r. t. 0.774; 6-Man, r. t. 0.805) were easily separated from disubstituted monosaccharides (2,6-Man, r. t. 0.899; 3,6-Man, r. t. 0.912) and from hexosamine (4-GlcNAc, r. t. 0.927). The myo-inositol was added as internal standard (r. t. 1.000). The unambiguous identification of the individual components was reached through their characteristic EI spectra. The linkage analysis experiments confirmed the occurrence of the branching pattern peculiar of high-mannose glycans.
Assignment of disulfide pattern
The assignment of S-S bridges in the xylanase was accomplished by combining chemical and enzymatic hydrolyses of the native protein with different mass mapping procedures. Putative free cysteine residues were previously alkylated with iodoacetamide under native conditions. The glycoprotein (200 µg) was then digested with CNBr and deglycosylated by PNGaseF treatment. The peptide mixture was then fractionated by HPLC (Figure 6) and the individual fractions were collected and analyzed by ESMS; the results are summarized in Table III.
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Fraction 3 showed the presence of a single component whose mass value was measured as 5865.3 ± 0.3 Da and assigned to the peptide pairs (171 – 189) + (190 – 225) revealing the occurrence of an S-S bridge joining Cys173 and Cys205. Two components were detected in the ESMS spectrum of fraction 6. The major species displayed a molecular mass of 10232.9 ± 0.9 Da, 2 Da lower than that expected for the peptide 238–332, indicating the occurrence of an intramolecular S-S bridge involving Cys272 and Cys278. The minor component with a mass value of 10962.9 ± 0.4 Da corresponded to a O-glycosylated form of the same peptide bearing Hex2HexNAc2 structures, which represented the major O-linked glycan structure thus confirming the previous results. The single component observed in fraction 5 exhibited a molecular mass of 13,549.1 ± 1.3 Da and was identified as the peptide 53–170 in which Cys89 was alkylated by iodoacetamide. In order to obtain a more rigorous assignment of the S-S bridge involving Cys 272 and Cys278, fraction 6 was digested with endoproteinase AspN and the mixture was directly analyzed by MALDIMS. The signal at m/z 2763.1 attributed to the peptide 263–287 confirmed the occurrence of an intramolecular S-S bridge between Cys272 and Cys278. Therefore, the S-S bridges pattern in the xylanase resulted to be Cys173-Cys205, Cys272-Cys278, with Cys89 having a free thiol group. Finally, the ES spectrum of fraction 1 showed the presence of two components, showing a molecular mass of 699.6 ± 0.1 and 601.1 ± 0.1 Da, that were attributed to the C-terminal fragments 333–338 and 333–337, respectively, thus indicating the occurrence of a partial proteolytic processing at the C-terminus.
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Discussion |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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The complete structural characterization of the xylanase from psychrophilic antarctic yeast Criptococcus albidus TAE85 was achieved both at the protein and carbohydrate level by exploiting mass spectrometric procedures. On the basis of the cDNA sequence (personal communication) the cold-adapted xylanase consisted of 338 amino acid residues containing five cysteine residues and two putative consensus sequence for the N-glycosylation, namely Asn139 and Asn254. The mass spectral analysis led to the verification of the entire primary structure of the enzyme, the assignment of the S-S pattern, and the determination of the N-linked glycan structures. Moreover, a single O-glycosylation site was identified and the structure of the O-linked glycans was defined.
The MALDI mass spectra of the intact xylanase showed the occurrence of a heterogeneous mixture of molecular species in which the major components corresponded to glycosylated forms of the enzyme, while nonglycosylated xylanase constituted a minor species. Although the MALDI resolution did not allow the separation of individual components, the mass values extrapolated from the spectra suggested the occurrence of a single N-glycosylation site. Peptide mass mapping of the reduced and carboxymethylated protein demonstrated that the amino acid sequence is essentially identical to that predicted by the cDNA sequence. However, a partial processing was detected at the C-terminus where the last Val residue was proteolytically processed. The presence of this one-residue shorter xylanase component could not be detected in the intact enzyme due to both the resolution of the MALDI spectra and the large heterogeneity of the glycosidic moiety.
The mass mapping analysis revealed the occurrence of a N-glycosylation site at Asn254 bearing high-mannose type glycans ranging from 6 to 12 residues, while Asn139 was found to be unmodified. However, higher mannose structures might also be present in the xylanase extract as reported for other yeast glycoproteins (Zeng and Biemann, 1999). These results were confirmed and complemented by the MALDI analysis of the permethylated intact glycans released from the glycoprotein by PNGase F treatment. The classical pattern of signals corresponding to high-mannose oligosaccharides with the antennae ranging from 6 to 12 residues in length were observed. As judging from signal intensities, the major components showed 9 or 10 mannose residues whereas the minor component had only three hexoses linked to the pentasaccharide core.
These data are in good agreement with previous data on glycoproteins from other yeasts (Herscovics and Orlean, 1993), confirming that the glycosylation pattern is mainly dictated by the enzymatic machinery of each species (Kukuruzinska et al., 1987). Yeast glycoproteins, in fact, constitute a unique class of glycoconjugate molecules whose N-linked glycan structures differ significantly from those found in other eukaryotic glycoproteins. These glycans exclusively belong to the high-mannose type with a large variety in the number of mannose residues that can be as high as 150 or more (Kuo and Lampen, 1974).
The GCMS analysis of the Me3Si-monosaccharides revealed the presence of GalNAc and galactose and the absence of sialic acid residues, thus suggesting the occurrence of peculiar O-glycans in cold yeast Xylanase. These data were confirmed by the MALDI analysis of peracetylated intact O-linked oligosaccharides after reductive elimination in basic condition. O-linked HexHexNAc structures as well as dimeric (HexHexNAc)2 glycans were detected in the spectra indicating the presence of non-capped O-linked glycans. These results are rather unexpected since HexHexNAc structures are peculiar of higher eukaryotic glycoproteins whereas the O-linked glycans found in mesophylic yeasts only consist of mannose residues directly attached to the polypeptide chain (Kuo and Lampen, 1974). A more complex enzymatic machinery should then exist in psychrophilic yeast for the synthesis of O-linked oligosaccharides as compared to the more extensively studied mesophilic microorganisms.
Altogether, the data obtained on both the N- and O-linked glycans in the cold xylanase constitute the first description of the glycosylation pattern in psychrophilic microorganisms and suggest that the glycosylation system in cold-adapted organisms might have similarities as well as differences with respect to mesophilic and thermophilic cells. Moreover, although xylanases from mesophilic yeasts are known to be glycosylated, no data on the structure of the glycosidic moiety have been reported so far (Morosoli et al., 1988). The role of glycan in xylanases is still not clearly understood. However the deglycosylation or the underglycosylation can results in the inhibition of the enzyme synthesis and activation of the protein secretion (Hickman et al., 1977; Morosoli, 1985). It has been shown that glycosylation of this enzyme might contribute to the stability of protein conformation thus increasing enzymatic activity (Morosoli et al., 1986). When xylanase was depleted in carbohydrate content in fact, the enzymatic activity was largely reduced. Moreover, in the last step of the secretion process, xylanase is retained by the cell wall by carbohydrate interactions, thus keeping the active protein in close proximity to the xylan degradation product which in turn supports cell growth (Morosoli et al., 1988).
A slightly different mass spectrometric procedure was employed to assign the disulfide bridge pattern of xylanase in that the peptide mixture from proteolytic digestion of the native protein was first fractionated by HPLC and the individual fractions analyzed by ESMS. The cysteine pairings were eventually identified as Cys173–Cys205 and Cys272–Cys278, with Cys89 showing a free thiol group.
Cold-adapted xylanase shows about 60% homology with the mesophilic enzyme from Cryptococcus albidus (Schmidt et al., 1998), whereas the sequence homology with respect to other xylanases from fungi is only 35% (Maccabe et al., 1996). Figure 7 shows the alignment of the amino acid sequence of the two yeast enzymes. A higher number of cysteine residues occur in the mesophilic xylanase as compared to the cold enzyme; however, the positions of the first five cysteine residues appear to be strictly conserved in the two proteins. Within this subset of Cys residues, an identical S-S bridge pattern exists in which the second Cys is linked to the third one with the fourth and fifth joined together. Moreover, this last disulfide bridge is conserved among various xylanases isolated from fungi (Maccabe et al., 1996). These data suggest that a common folding motif might occur within the entire xylanase family.
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Materials and methods |
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Materials
Psychrophilic yeast xylanase was produced and purified as already reported (personal communication). Chymotrypsin, dithiothreitol, iodoacetic acid were purchased from Sigma. Iodoacetamide was from Fluka. Peptide N-glycosidase F (PNGase F) and endoprotease GluC were obtained from Boehringer. Cyanogen bromide was purchased from Pierce. Pre-packed PD-10 gel filtration cartridges were from Pharmacia; prepacked Sep Pak C18 cartridges were purchased from Waters. All other reagents and solvents were of the highest purity available from Carlo Erba.
Chemical and enzymatic procedures
The glycoprotein was further purified by RP-HPLC on a Vydac C4 column (25 x 0.46 cm, 5 µm) using 0.1% trifluoroacetic acid (solvent A) and 0.07% trifluoroacetic acid in 95% acetonitrile (solvent B). The glycoprotein was eluted by means of a linear gradient from 15% to 95% solvent B over 10 min. Fraction containing the protein was manually collected, dried in a Speed-Vac centrifuge (Savant), lyophilized twice, and stored at –20°C.
Purified protein samples were reduced and alkylated with iodoacetic acid according to the procedure previously described (Nitti et al., 1995). Chymotryptic and GluC digestions were carried out in 0.4% ammonium bicarbonate, pH 8.5 at 37°C overnight using an enzyme to substrate ratio of 1:50 (w/w). Release of the N-linked glycans from the peptide mixtures by PNGase F treatment and purification of the oligosaccharide chains were performed as already described (Amoresano et al., 1996). Briefly, N-linked oligosaccharide chains released by PNGase F were separated from peptides and O-glycopeptides by reverse phase chromatography on prepacked Sep-pak C18 cartridges by using 5% AcOH and 40% PrOH solution.
Xylanase was incubated with a 15-fold excess CNBr (w/w) in 70% trifluoroacetic acid, overnight, at room temperature in the dark. The protein sample was then diluted 10-fold with water, evaporated in a Speed-Vac centrifuge (Savant), and lyophilized.
CNBr peptide mixture was fractionated by RP-HPLC on a Vydac C18 column (25 x 0.46 cm, 5 µm) using 0.1% trifluoroacetic acid (solvent A) and 0.07% trifluoroacetic acid in 95% acetonitrile (solvent B) by means of a linear gradient from 5% to 70% solvent B over 65 min.
O-Linked oligosaccharides were released from the glycopeptides by treating the 40% propanol fraction with 400 µl of 1 M sodium borohydride (NaBH4)in 0.05 M NaOH at 45°C overnight. The sample was then neutralized with glacial acetic acid and desalted on a Dowex 50W-X8(H+) column; the borates were removed by co-evaporation under nitrogen with 10% v/v acetic acid in methanol (Carlson, 1968).
N-Linked oligosaccharides were permethylated using the sodium hydroxide mixtures procedure (Dell, 1990) and directly analyzed by mass spectrometric techniques. O-linked oligosaccharides were acetylated by adding 50 µl of pyridine and 50 µl of acetic anhydride and incubating at room temperature for 18 h.
The glycoprotein was further purified by a chromathographic step using the same eluents previously described. The xylanase was eluted by means of a rapid linear gradient from 15% to 95% solvent B over 5 min but the isocratic step was extended for 15 min. Fraction containing the protein was manually collected, dried in a Speed-Vac centrifuge (Savant) and lyophilized twice An aliquot of the further purified xylanase was then dissolved in 500 µl 1M methanolic-HCl at 80°C for 16 h. After neutralization by adding Ag2CO3, the re-N-acetylation was achieved with 50 µl acetic anhydride and incubating at room temperature overnight. The trimethylsilylation was carried out in 500 µl SIGMA-SIL-A at 80°C for 20 min. The sample was dried down under nitrogen, dissolved in 50 µl hexane, and centrifuged to remove the excess of solid reagents. The hexane supernatant (1/50) was used for the GC-MS analysis.
The methylated glycans were purified by CHCl3 extraction and acid hydrolysate by H2SO4/AcOH at 100°C for 9 h. After neutralization the crude reaction was reduced with NaBD4, acetylated, and GLC-MS analyzed. Inositol was added as internal standard.
Mass spectrometry
MALDI mass spectra were recorded using a Voyager DE MALDI-TOF mass spectrometer (PerSeptive Biosystem): a mixture of analyte solution, sinapinic acid, 
-cyano-4-hydroxycinnamic acid or 2,5-dihidroxybenzoic acid (Sigma) and bovine insulin was applied to the sample plate and air dried. Mass calibration was performed using the molecular ions from the bovine serum albumin at 66,437 Da, bovine insulin at 5734.5 Da, and the matrix at 379.1 or a peptide at 1209.3 Da as internal standards. Raw data were analyzed by using computer software provided by the manufacturer and are reported as average masses.
Electrospray mass spectra were performed on a Bio-Q triple quadrupole instrument (Micromass). Peptide samples (100 pmol) were dissolved in 1% acetic acid in 50% acetronitrile and injected into the ion source at a flow rate of 10 µl/min. Spectra were acquired and elaborated using the MASS LYNX software (Micromass). Calibration of the mass spectrometer was achieved by means of a separate injection of horse heart myoglobin (MW 16,951.5 Da). All masses reported are average masses.
The GC-MS analysis was performed on a Trio 2000 quadrupole mass spectrometer equipped with a Fisons Instruments 8060 gas chromatograph by using a DB5 fused silica capillary column (30 m, 0.5 mm ID, 0.25 µm ft) from Supelco. The injection temperature was 250°C; the oven temperature was increased to 90°C over 1 min and held for 1 min before increasing to 140°C at 25°C/min and then to 200°C at 5°C/min and finally to 300°C at 10°C/min. Electron Ionization (EI) mass spectra were recorded by continuous quadrupole scanning at 70eV ionization energy.
Partially methylated alditol acetates were analyzed by GLC-MS on a Hewlett-Packard 5890 instrument, in the following conditions: ZB-5 capillary column (Phenomenex, 30 m x 0.25 mm i.d., flow rate 0.8 ml/min, He as carrier gas), with the temperature program: 100° for 2 min, 100°
240° at 2°/min.
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This work was supported by European Community contracts ERBBIO4CT950017 "EUROCOLD" Concerted Action, ERBBIO4CT960051 "COLDZYME," ERBFMRXCT970131 "COLDNET," and CNR 97.01138.PF49 "BIOTECNOLOGIE," MURST PRIN 1999.
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Abbreviations |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsAcknowledgmentsAbbreviationsReferences |
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DHB, 2,5 dihydroxybenzoic acid; a-cyano,
-cyano-4-hydroxycinnamic acid, r. t., retention time; EI, electron ionization; GC, gas chromatography; MS, mass spectrometry; MALDI, matrix-assisted laser desorption ionization; Hex, hexose; HexNAc, N-acetylhexosamine; peptide N-glycosidase F, PNGase F; HSer, homoserine; CAM; carboxyamidomethyl; AcOH, acetic acid; PrOH; isopropilic alcol.
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Footnotes |
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1 To whom correspondence should be addressed at: International Mass Spectrometry Facilities Centre, via Pansini 5, 80131 Napoli, Italy
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References |
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