D. Promé

Differentiation of O-Acetyl and O-Carbamoyl esters of N-Acetyl-Glucosamine by decomposition of their oxonium ions. application to the structure of the nonreducing terminal residue of Nod factors

Nod factors are substituted N-acyl chito-oligomers secreted by plant symbiotic bacteria of the Rhizobium family. Substitutions on the oligosaccharide core specify their recognition by host plants. A method using tandem mass spectrometry is proposed to locate the O-acetyl and O-carbamoyl substituents on the nonreducing terminal residue of the chito-oligomers. As model compounds, all the positional isomers of monoacetyl and monocarbamoyl esters of 1-O-methyl-N-acetyl-α-D-glucosamine were synthesized. Oxonium ions (MH − CH3OH)+ were generated by liquid secondary ion mass spectrometry (LSIMS) and their decomposition was recorded on a tandem magnetic instrument. Large differences were observed in the relative abundances of ions resulting from elimination of water and of the O-ester substituent from metastable oxonium ions. Deuterium exchange reactions indicated parallel elimination pathways involving either exchangeable or carbon-linked hydrogens. The intensity ratios of some of the ions generated by collisions with helium atoms allowed the isomers to be distinguished. The main dissociation routes were identified. Metastable and collision-induced decomposition of the B1 ions from Nod factors of Sinorhizobium meliloti and Azorhizobium caulinodans resembled that of the 6-O-substituted N-acetylglucosamine models. Decomposition of the B1 ion from Mesorhizobium loti and Rhizobium etli Nod factors, was similar to that of 3-O-carbamoyl N-acetyl-glucosamine and different to that of the 4-O isomer. 6-O- and 3-O-carbamoylation specified by the nodU and nolO genes, respectively, of Rhizobium. sp. NGR234 were confirmed.

Use of tandem mass spectrometry (MS-MS) for aldosterone assay at the nanogram level in complex biological mixtures

Aldosterone in biological samples was measured comparatively by two methods: radioimmunoassay (RIA) and tandem mass spectrometry. The last method is described in this paper. Aldosterone was derivatized as butane boronate cyclic ester. This derivative gives an intense molecular ion under 70-eV electron impact conditions. This ion exhibits an abundant metastable decomposition in the second field free region by loss of formic acid, which can be used for the assay. A linear relationship was observed between the ionic current and the amount of aldosterone from 10 to 200 ng. A good correlation between this new method and the radioimmunological assay is observed. Aldosterone can be readily detected at the nanogram level.

Association of unstable hemoglobin variants and heterozygous β-thalassemia: Example of a new variant Hb acharnes or [β53(D4) Ala Thr]

We report here the functional and structural characterization of Hb Acharnes [β53(D4) Ala → Thr], an unstable and electrophoretically silent variant, that was found associated in trans with a β0‐thalassemic mutation (IVSI‐1 G → A), in a patient with thalassemia intermedia syndrome. This case is discussed in comparison with other sporadic cases that we have previously investigated, resulting from the co‐inheritance of a β0‐thalassemic mutation (CD39 C → T) with two other types of unstable hemoglobins, Hb Köln [β98(FG5) Val → Met], and Hb Arta [β45(CD4) Phe → Cys]. It may be concluded that, in these associated forms, both the degree of instability of the variant and the altered oxygen binding properties (affecting the degree of tissue hypoxia) are major determinants of their clinical expression. Am. J. Hematol. 62:186–192, 1999.

The pivotal role of tandem mass spectrometry in structural determinations of Nod factors produced by Rhizobia: Nod factors produced by wild-type strains of Mesorhizobium huakii and Rhizobium sp. mus10

Abstract Nod factors are signalling molecules secreted by bacteria of the Rhizobium family that plays an important role in initiating the early events of the nitrogen-fixing symbiosis between Rhizobia and legumes. They possess a lipochitooligomeric structure decorated by several functional groups. The structural details are essential for their specific recognition by plants and for switching on a genetic mechanism leading to the formation of nodules. Mass spectrometry is now able to fully characterise these molecules, even within mixtures and on minute amounts. It opens the possibility to study Nod factors from wild-type low-producing strains. It was demonstrated that Nod factors from the wild-type strain of Mesorhizobium huakii are almost the same as those produced by a genetically engineered over-producing strain. Rhizobium sp. mus10 is an Indian strain that nodulates the plant Sesbania. It was found that several Nod factors produced by this strain are identical to those produced by African strains that nodulate Sesbania species. However, two other Nod factors possessed new structural features. This synthetic possibility was probably acquired by the Rhizobium sp. mus10 strain by evolution in a different environment.

Two New Gγ Chain Variants: Hb F-Clamart [γ17(A14)Lys→Asn] and Hb F-Ouled Rabah [γ19(B1)Asn→Lys]

Two new fetal hemoglobin variants affecting the Gγ chain are reported. Hb F-Clamart was found during investigation of a French newborn who presented with a mild microcytemia. the second variant was found during neonatal screening for hemoglobinopathies of 30,000 babies from a population-at-risk living in the Paris region. It was named Hb F-Ouled Rabah because its structural modification and ethnic distribution is similar to that of Hb D-Ouled Rabah [Gγ19(B1)AsnLys]. Hb F-Ouled Rabah is clinically silent and occurs at a frequency of ca. 0.1% in newborns originating from Maghreb. Structural characterization of both variants was done by protein chemistry methods, including amino acids analysis and mass spectrometry.

Hb Harrow [β118(GH1)Phe→Cys]: A New Neutral Hemoglobin Variant

We report here on a new hemoglobin (Hb) variant found in a newborn baby boy of Indian Gujerati origin, living in the Harrow area of London, England. This variant was observed during a systematic program of neonatal screening for the main hemoglobinopathies performed at the Central Middlesex Hospital in the North Thames (West) Region of London. In this program, the samples are collected from a heelprick onto filter paper when the child is 7-10 days old. A dried blood spot of 0.3 cm diameter is obtained from which Hb is eluted by a 5 mM KCN solution to be analyzed by isoelectrofocusing (IEF).

Hb SITIA [β128(H6)Ala→Val]: AN UNSTABLE VARIANT WITH A SUBSTITUTION IN THE α1β1 INTERFACE

Hb Sitia [β128(H6)Ala → Val] was found in a Greek female with slightly reduced red blood cell indices. The abnormal hemoglobin was indistinguishable from Hb A by electrophoresis but eluted after Hb A on cation exchange high performance liquid chromatography. DNA sequence analysis revealed a GCT → GTT mutation at codon 128, which is predicted to encode an Ala → Val substitution. This was confirmed by mass spectrometry analyses of the β-globin chain. Since alanine at β128(H6) interacts with several amino acids of the α1β1 contact, its replacement by a larger residue results in a mild instability of the molecule and slight modifications of the oxygen binding properties.

Hb Diamant [α119(H2)Pro→Leu]: A New Variant with a Modification at the α1β1 Interface

The hemoglobin (Hb) tetramer results from the association of the two functional dimers; a l p 1 and a2p2, which are formed by subunits tightly bound through an interface almost unchanged when oxygen binds to the Hb molecule (1). Here we describe a new variant in which the proline residue at position a 1 19(H2), located within this contact, is replaced by leucine. This variant, named Hb Diamant for the place of origin of the proband, was found in a 30-year-old man from Antilles during a Hb study for genetic counseling purposes. His RBC parameters were normal (Hb 15.0 g/dL, RBC 5.2 x 10l2/L, MCV 86.3 fL) and no abnormal clinical feature was observed. The variant was observed by isoelectrofocusing

Hb Neuilly-Sur-Marne, a New Human Hemoglobin Variant with Ser-Asp-Leu Inserted between α86(F7) Leu and α87(F8) His: Characterization by High-Energy Collision-Induced Dissociation Liquid Secondary Ion Mass Spectrometry and Low Energy Collision-Induced Dissociation Tandem Mass Spectrometry in An Ion Trap Fitted with a Nanospray Ionization Source:

Hemoglobin (Hb) Neuilly-sur-Marne is a new α-chain variant found during a systematic screening. Electrospray mass measurements showed the presence of an abnormal α-chain displaying a shift of +315 u relative to the normal value. Tryptic cleavage of this chain and molecular weight determination of the peptides indicated that the 315 u shift was located into the αT-9 peptide, the molecular weight of which is higher than 3000 Da. High-energy collision spectra of MH+ ions generated by liquid secondary ion mass spectrometry from the normal and abnormal αT-9 afforded mainly amino-terminal containing ions. They indicated that these two peptides have an identical amino acid sequence from their 1st to 25th residues, the mass increase being thus located beyond this point. Too few ions were formed to establish reliably the sequence forward. It was hypothesized that this mass shift could result from a repeated sequence since the sum of the mass of the three residues—leucine, serine and aspartic acid—preceding position 25 is exactly 315 u. To get sequence information above position 25, decomposition of multicharged species was attempted. An ion trap fitted with a nanospray ionization source was used. It produced mainly triply- and quadruply-charged ions. Decomposition of the triply-charged ion afforded a series of singly-charged Y-ions in the expected region, giving a readily interpretable sequence. It confirmed the insertion of a Ser-Asp-Leu sequence above position 25. Surprisingly, decomposition of the quadruply-charged molecular ion gave too few ions to provide sequence information in the expected region. Spectra were dominated by some multicharged Y ions arising from cleavages close to the amino end. Tandem mass spectrometry experiments were performed on the abundant Y303+ ion and produced again a singly-charged Y ion series in the suitable domain which confirmed the above result. In Hb Neuilly-sur Marne this insertion of the Ser-Asp-Leu residues. between positions α-86 and α-87 is very likely due to a slipped strand mispairing mechanism.

Hb Aubenas [β26(B8)GLU→GLY]: A new Variant Normally Synthesized, Affecting the Same Codon as in Hb E

Hb Aubenas [β26(B8)Glu → Gly] is a mildly unstable variant that was found in a French family without hematological or clinical features. The structural abnormality was determined by protein chemistry methods, including tandem mass spectrometry, and was confirmed by Apa I digestion of a polymerase chain reaction-amplified DNA fragment. Although the substitution involves the same residue as in Hb E, the new nucleotide sequence does not create an additional out-of-frame splice site. The mutated chain is therefore normally synthesized.