Analysis of phospho- and glycopolypeptides with infrared matrix-assisted laser desorption and ionization.

The analytical characteristics of infrared (IR) matrix-assisted laser desorption and ionization (MALDI) were investigated for the analysis of phosphopeptides, a phosphopolypeptide, and glycopeptides. Two commercially available instruments, a high-resolution delayed extraction (DE) reflectron time-of-flight (RETOF) mass spectrometer and a high-power pulsed Er:YAG laser, were interfaced to produce a high-resolution MALDI-DE-RETOF instrument that is easy to use and can be switched between UV- and IR-MALDI mode within seconds. In the interface design, particular attention was paid to maintaining the same professional operating environment for the new IR-MALDI mode as exists for the commercial UV-MALDI mode. This instrument configuration facilitates comparative observation and investigation of the relative analytical merits of IR- and UV-MALDI. The results of studies of the tryptic alpha-casein phosphopeptides, RP1 (a Thr45-monophosphorylated congener of the recombinant protein hirudin variant 1), and fetuin Asn81 tryptic glycopeptides are presented. The elimination of labile substituents such as phosphoric acid and sialic acid is suppressed in IR-MALDI-RETOF mass spectrometry, with concomitant higher analyte ion yields. These results reflect the advantages that accrue from deposition of significantly less internal energy in the case of IR-MALDI.

Characterization of a beta-Asp33 isoform of recombinant hirudin sequence variant 1 by low-energy collision-induced dissociation.

A new low-concentration congener (Ib) of recombinant hirudin sequence variation 1 was structurally characterized as a beta-Asp33 isoform of the parent protein (Ia). alpha-beta Isomerization at the 33-position was expected in view of the previous isolation of a potential precursor (Asp33-Gly34-anhydro-hirudin (Ic)), i.e., a succinimide-type dehydration product liable to undergo facile hydrolysis with ring opening, yielding beta- (along with alpha-) aspartates. In order to identify and locate the modified site in Ib, a sufficiently small peptide ([28-35]-octapeptide IIIb) was prepared by disulfide bond reduction, S-alkylation (pyridylethylation) and twofold enzymatic degradation (Glu-C protease followed by trypsin). When [M + H] + ions of IIIb were analyzed by electrospray ionization tandem mass spectrometry (ESIMS/MS) and low-energy collision-induced dissociation (CID), a singular [bn + H2O]+ ion indicative of beta-Asp in the neighboring 'n + 1' position was observed for n = 5. This located the beta-Asp residue unambiguously in the 6-position of IIIb and thus, as expected, in the 33-position of Ib. The formation of this highly diagnostic [bn + H2O]+ ion, for which precedents had only been reported for CID under high-energy conditions, requires net OH migration from one to another amino acid position. Confirmatory results from 18O-labeling of the suspected migratory oxygen atom (beta-Asp33-CO18OH) together with the low-energy genesis suggest a specific charge-triggered rather than charge-remote mechanism for the formation of the ion. The analogy of this process to the ejection of the C-terminal amino acid similarly involving net OH rearrangement is discussed.

Spectroscopic and protein chemical analyses demonstrate the presence of C-mannosylated tryptophan in intact human RNase 2 and its isoforms.

Recently, the C-mannosylation of a specific tryptophan residue in RNase 2 from human urine has been reported [Hofsteenge, J., et al. (1994) Biochemistry 33, 13524-13530; de Beer, T., et al. (1995) Biochemistry 34, 11785-11789]. In those studies, identification of this unusual modification was accomplished by mass spectrometric and NMR spectroscopic analysis of peptide fragments. The evidence for the occurrence of C2-alpha-mannosyltryptophan [(C2-Man-)Trp] in the intact protein relied exclusively on the detection of the same phenylthiohydantoin derivatives during Edman degradation. In this paper, we have (1) excluded the possibility that (C2-Man-)Trp arose artificially under the acidic conditions previously employed for protein and peptide isolation and analysis, by maintaining the pH > 5 throughout these procedures, (2) demonstrated the occurrence of (C2-Man-)Trp in the intact protein, by NMR spectroscopy, (3) showed that (C2-Man-)Trp is not unique for RNase 2 from urine but that it is also present in the enzyme isolated from erythrocytes, and (4) found also that high-molecular mass isoforms of urinary RNase 2 are C-mannosylated. These observations firmly establish C-mannosylation as a novel way of post-translationally attaching carbohydrate to protein, in addition to the well-known N- and O-glycosylations. Furthermore, the NMR data, in combination with molecular dynamics calculations, indicate that in the native protein the mannopyranosyl residue is in a different conformation than in the glycopeptide or denatured protein, due to protein-carbohydrate interactions.

New type of linkage between a carbohydrate and a protein: C-glycosylation of a specific tryptophan residue in human RNase Us.

We report a new type of linkage between a carbohydrate and a protein, involving the rarely modified side chain of a tryptophan residue. An aldohexopyranosyl residue was found to be linked via a C-C bond to the indole ring of the tryptophan residue at position 7 of human RNase Us. Mass spectrometric analysis of peptides containing this residue showed a molecular mass 162 Da higher than that expected for tryptophan. The fragmentation pattern of the modified amino acid side chain was reminiscent of that of aromatic C-glycosides, suggesting a direct attachment of a hexose residue to a C-position of the tryptophan indole moiety. 1H and 13C NMR spectroscopic data confirmed this inference and unequivocally demonstrated the substituent to be an aldohexopyranosyl residue, C-glycosidically linked to the C2 atom of the indole. This mode of attachment differs from the ones known so far, in which carbohydrates are linked to an amino acid side chain by N- or O-glycosidic bonds.

Characterization and direct glycoform profiling of a hybrid plasminogen activator by matrix-assisted laser desorption and electrospray mass spectrometry: correlation with high-performance liquid chromatographic and nuclear magnetic resonance analyses of the released glycans.

Mass spectrometric analysis of the recombinant hybrid plasminogen activator K2tu-PA at the native glycoprotein and corresponding protein level (high mass) is described. For gross structural characterization of the major glycotypes present, a combination of enzymatic degradation and matrix-assisted laser desorption mass spectrometric analysis of the products proved convenient. In this way, mono- and di-N-glycosylated one- and two-chain molecules, unresolved in the spectra of native material, were identified, with the one-chain type monoglycosylated at Asn247 representing the major component. Actual detection of resolved original glycoforms, or unresolved groups thereof when composed of isobaric species, and their assignments regarding antennicity and degree of sialylation were possible by electrospray mass spectrometry for the major monoglycosylated one-chain species. Desialation also allowed detection of the diglycosylated one-chain species as a minor constituent. The electrospray mass spectrometric results were correlated with structural and quantitative data available from a parallel high-performance liquid chromatographic and 1H nuclear magnetic resonance study performed at the glycan (low mass) level on liberated individual carbohydrate components. The results of the two studies showed full consistency in all respects amenable to evaluation, i.e. excellent agreement for the assignments of antennicity and degree of sialylation of the major glycan components, and good agreement for the determination of proportions in which these were present in the major monoglycosylated glycoprotein. This provided a sound basis for direct glycoform profiling by electrospray mass spectrometry. Typical applications to batch quality control and to structural characterization of non-standard material are shown.

Characterization of succinimide-type dehydration products of recombinant hirudin variant 1 by electrospray tandem mass spectrometry.

Two congeners Q4 and Q5 inferred in earlier analyses to be cyclic succinimide-type dehydration products of recombinant hirudin (variant 1) were structurally fully characterized. After isotopic labeling by ring-opening with H2(18)O, the suspected anhydro-positions Asp53 and Asp33 were confirmed by tandem mass spectrometry (MS/MS) sequencing of relevant smaller peptides directly in the enzymatic hydrolysates (V8 protease) using electrospray MS/MS. The chosen strategy proved highly efficient and sensitive.

Cysteine phosphorylation of the glucose transporter of Escherichia coli.

The glucose transporter (IIBCGlc/IIAGlc complex) of the bacterial phosphotransferase system couples vectorial translocation to phosphorylation of the transported sugar. The IIAGlc subunit transfers the phosphoryl group from the phosphoryl carrier protein P-HPr to the IIBCGlc subunit. IIBCGlc translocates and phosphorylates glucose. The site of IIBCGlc phosphorylation is cysteine 421 as shown by mass spectrometric and biochemical analyses of phosphorylated peptides. Site-directed mutagenesis of Cys421 (C421S) afforded a stable but completely inactive protein (Nuoffer, C., Zanolari, B., and Erni, B. (1988) J. Biol. Chem. 263, 6647-6655). Cys421 is located in the C-terminal cytoplasmic domain of the IIBCGlc subunit in a sequence context (LDACITRL) which is well conserved in other transporters of the bacterial phosphotransferase system. Phosphocysteine has been shown previously to be the catalytic intermediate of the mannitol transporter (Pas, H. H., Meyer, G. H., Kruizinga, W. H., Tamminga, K. S., van Weeghel, R. P., and Robillard, G. T.

1H-NMR and mass spectrometric studies of tetrahydropterins. Evidence for the structure of 6-pyruvoyl tetrahydropterin, an intermediate in the biosynthesis of tetrahydrobiopterin.

The conversion of dihydroneopterin triphosphate in the presence of 6-pyruvoyl tetrahydropterin synthase was followed by 1H-NMR spectroscopy. The interpretation of the spectra of the product is unequivocal: they show formation of a tetrahydropterin system carrying a stereospecifically oriented substituent at the asymmetric C(6) atom. The spectra are compatible with formation of a (3')-CH3 function, and with complete removal of the 1' and 2' hydrogens of dihydroneopterin triphosphate. The fast-atom-bombardment/mass spectrometry study of the same product yields a [M + H]+ ion at m/z 238 compatible with the structure of 6-pyruvoyl tetrahydropterin. The data support the proposed structure of 6-pyruvoyl tetrahydropterin as a key intermediate in the biosynthesis of tetrahydrobiopterin.

Location of disulphide bonds in human insulin-like growth factors (IGFs) synthesized by recombinant DNA technology.

Natural human insulin-like growth factor (IGF) I is a relatively large single chain peptide (mol. wt 7649) with a known sequence of 70 amino acids. C6----C48, C47----C52 and C18----C61 assignments have been previously proposed for the three disulphide bonds linking six cysteine residues (C6, C18, C47, C48, C52 and C61), on the basis of analogy (and homology) with proinsulin. In this work, IGF I synthesized by recombinant DNA technology (r-IGF I, with identical biological activity and chromatographic behaviour) was subjected to a three-step mass spectrometric analysis in combination with degradation methods for structural verification. Firstly, the correct molecular weight of the intact peptide was determined by high-mass fast atom bombardment (FAB) analysis. Secondly, twofold enzymatic degradation (chymotrypsin followed by V8 protease, 'FAB mapping' of the cleavage products) was employed in order that fragments with 'isolated' S-S bonds would be produced which allow an unambiguous assignment. This immediately established the C18----C61 linkage as it was contained in a singly bridged two-chain peptide. The two other S-S bonds, which cross-link C6 and the 'tight' C47 to C52 segment, remained 'unresolved' within a more complex, doubly bridged triple-chain peptide. Thirdly, further degradation of this structural block, in which cleavage of the C47-C48 bond was required to discern these bonds, was carried out by using FAB tandem mass spectrometry and (for additional corroboration) manual Edman degradation. Both procedures confirmed the original C6----C48/C47----C52 prediction.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct stereochemical assignment of sugar subunits in naturally occurring glycosides by low energy collision induced dissociation. Application to papulacandin antibiotics.

A tandem mass spectrometric method is described which allows the assignment of stereochemistry to fragment ions comprising intact sugar subunits of larger glycosides without chemical degradation and product isolation by chromatography. The approach relies on the mass selection of the 'sugar ion' of interest followed by analysis of stereoselective fragmentation induced by low-energy collisional activation. The daughter ion spectra provide configurational fingerprints of the selected sugar ions which can be matched for identity with reference spectra obtained from suitable precursors of known stereochemistry. Glucose, mannose and galactose furnished the required set of the most important reference ions. By using peracetyl (and perdeuterioacetyl) derivatives, galactose was readily identified as the glycosidic sugar constituent of the (known) antibiotic papulacandin B and a further (unknown) congener.

3-Hydroxyrifamycin S and further novel ansamycins from a recombinant strain R-21 of Nocardia mediterranei.

The structures of 3-hydroxyrifamycin S and six further novel ansamycins isolated from the recombinant strain R-21 of Nocardia mediterranei were identified by spectroscopic methods. Three types of structure were distinguished: Type 1: Ansamycins of the rifamycin S type Type 2: Ansamycins of the rifamycin G type Type 3: Ansamycins of the rifamycin W type.

A genetic approach to the biosynthesis of the rifamycin-chromophore in Nocardia mediterranei. III. Isolation and identification of an early aromatic ansamycin-precursor containing the seven-carbon amino starter-unit and three initial acetate/propionate-units of the ansa chain.

A number of rifamycin non-producing UV-mutants derived from Nocardia mediterranei strains N813 (rifamycin B producer) and A10 (aro--mutant excreting shikimate derived from strain N813) were found to accumulate an identical complex of aromatic components instead of rifamycin B. The main component of this aromatic complex, product P8/1-OG, was isolated from six of these P--mutant strains and identified spectroscopically as a very early precursor in the biosynthesis of rifamycins.

Papulacandins, a new family of antibiotics with antifungal activity. Structures of papulacandins A, B, C and D.

The structures of the papulacandins A, B, C and D, new antibiotics of Papularia sphaerosperma have been established by means of spectral analysis and degradation reactions. Base catalysed hydrolysis of the main product papulacandin B (1) gave two new hydroxylated long-chain unsaturated fatty acids 5 and 6 along with a hitherto unknown spirocyclic diglycoside 7. The structure of 7 was determined by further degradation reactions. The positions of attachment of the two fatty acids to the spirocyclic diglycoside 7 through ester-bonds were established by selective base catalysed hydrolysis of 1 and spectral analysis of 1 and some derivatives and degradation products thereof. The structures of papulacandin A (2), papulacandin C (3) and papulacandin D (4) were determined in an analogous way.

Early intermediates in the biosynthesis of ansamycins. III. Isolation and identification of further 8-deoxyansamycins of the rifamycin-type.

A number of minor compounds were isolated from fermentations of the protorifamycin I producing strain Nocardia mediterranei F 1/24(1,2)) and identified by means of chemical and spectroscopic methods. Two types of structures were identified: Type 1: modified protorifamycins (derived from protorifamycin I) Type 2: defective rifamycins (8-deoxyrifamycins).

Early intermediates in the biosynthesis of ansamycins. II. Isolation and identification of proansamycin B-M1 and protorifamycin i-M1.

Proansamycin B-M1 and protorifamycin I-M1 were isolated as minor compounds from fermentations of the protorifamycin I producing strain Nocardia mediterranei F 1/24, identified by means of chemical and spectroscopic methods and shown to be degradation products of the hypothetical proansamycin B postulated in part I of this series of papers and of protorifamycin I, respectively.

Biotransformation of diclofenac sodium (Voltaren) in animals and in man. I. Isolation and identification of principal metabolites.

1. The anti-inflammatory agent diclofenac sodium (o-[(2,6-dichlorophenyl)amino]phenylacetic acid sodium salt) is extensively metabolized by rat, dog, baboon and man. The main metabolites were isolated from the urine of all species and from the bile of rat and dog and identified by spectroscopy. 2. Metabolism involves direct conjugation of the unchanged drug, or oxidation of the aromatic rings usually followed by conjugation. Sites of oxidation are either position 3' or 4' of the dichlorophenyl ring or, alternatively, position 5 of the phenyl ring attached to the acetic acid moiety. 3. In the urine of rat, baboon and man conjugates of the hydroxylated metabolites predominate, but the major metabolite in dog urine is the taurine conjugate of unchanged diclofenac. 4. In the bile of rat and dog, the main metabolite is the ester glucuroniade of unchanged diclofenac.

Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I.

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.