Masahiko Kishikawa

Simple and defined method to detect the SOD-1 mutants from patients with familial amyotrophic lateral sclerosis by mass spectrometry

Mutations in Cu/Zn superoxide dismutase (SOD1) cause a subset of cases of familial amyotrophic lateral sclerosis (FALS). We established a simple and defined method to detect the mutant SODI in erythrocytes by electrospray ionization mass spectrometry (ESIMS) using materials precipitated with specific antiserum. Hemolysate was mixed with anti-SOD1 antiserum and the generated precipitate, which was soluble in the solvent for MS analysis, was injected on to an LC column connected to an ESI-mass spectrometer. MS spectra of the reduced SOD1 prepared from normal individuals showed ion peaks corresponding to free monomer SOD1. The spectra from FALS patients revealed doublet ion peaks corresponding to normal and mutant components. The ratios of mutant to normal SOD1 were about 1/2 in cases of (G37R) and (A4S), and about 0.15 in a case of (H46R). This method provides for the rapid diagnosis using small amount of specimens, and will contribute to elucidate the pathomechanism of FALS through the quantification of SOD1 mutants in erythrocytes and in tissues of nervous systems.

Quantification of Glycated Hemoglobin by Electrospray Ionization Mass Spectrometry

Glycated hemoglobin, considered to be the best index for the treatment of diabetes mellitus, was measured by electrospray ionization mass spectrometry (ESI/MS) according to the method proposed by Morris et al. at the 44th ASMS Conference on Mass Spectrometry and Allied Topics, 1996. They compared the values obtained by MS and affinity chromatography. Here, the values obtained by ESI/MS were compared with those obtained by high-performance liquid chromatography and by latex agglutination immunoassay. Whole blood samples were diluted 500 fold with 0.2% formic acid-50% acetonitrile solution and 5 microliters of the diluted solution was injected with the ESI/MS system (TSQ 7000) via a sample loop. The within-run and between-run relative standard deviations of the ratio of glycated and non-glycated beta-chain were less than 5%. The correlation coefficients between ESI/MS and conventional methods were higher than 0.96. However, considerable discrepancies were observed among methods. ESI/MS will allow reproducible measurements of glycated hemoglobin and will be useful in the quality control of HbA1c measurement by other principles and also in routine clinical laboratory tests.

Detection and identification of protein variants and adducts in blood and tissues: an application of soft ionization mass spectrometry to clinical diagnosis

The detection and identification of protein variants and abnormally increased modified proteins are important for clinical diagnosis. We applied soft ionization mass spectrometry (MS) to analyze proteins in blood and tissues from various patients. Over the past 8 years, we diagnosed 132 cases (55 kinds) of variant proteins including hemoglobin (Hb), transthyretin (TTR), and Cu/Zn-superoxide dismutase (SOD-1), using MS as the leading technology. Of these variants, eight were new, and nine were the first cases in Japan. Some abnormal Hb cause diseases, and most of them cause erroneous levels of glycated Hb, HbA1c, i.e., a popular index of diabetes. Most of the variant TTR causes amyloidotic polyneuropathy. Variant SOD-1 causes amyotrophic lateral sclerosis. We first showed that immunoprecipitation by a specific antiserum is a reliable and simple method to prepare protein from sera and tissues for analysis by matrix-assisted laser desorption time-of-flight MS, and liquid chromatography-electrospray ionization MS (LC-ESI-MS). The use of this technology has become widespread. Using an immunoprecipitated target protein and LC-ESI-MS, we showed that the ratios of tetra-, di- and a-sialo-transferrin from two cases of congenital glycoprotein deficient syndrome were clearly distinguishable from those of control samples. We first reported a unique modified form of TTR, that is, S-sulfonated TTR, which increased markedly and specifically in three cases with molibdenum cofactor deficiency. We proposed that S-sulfonated TTR is a useful marker for screening this disease. ESI-MS was successfully used for the accurate determination of HbA1c, and we clarified the extent of discrepancies between the HbA1c value measured by conventional methods and the accurate values for samples containing various Hb variants determined by the MS method.

A simple and reliable method of detecting variant transthyretins by multidimensional liquid chromatography coupled to electrospray ionization mass spectrometry.

Transthyretin (TTR) variants cause amyloidosis. A method, originally reported by us, of detecting the variants by high performance liquid chromatography/electrospray ionization mass spectrometry (ESIMS) using materials precipitated with anti-TTR antiserum, has been successfully applied by several institutions. The method is simple and reliable, but some variants may not precipitate with the antiserum or may precipitate in different yields compared to normal TTR. Moreover, unidentified minor peaks were observed, which may have been derived from cross reactive materials. We have now devised a new procedure to overcome these problems. An anion exchange and reversed phase liquid chromatography system and ESI mass spectrometer were connected in a tandem fashion using a 6 port valve and a protein trap cartridge. The profile of ion peaks by the method was the same as that by MS with immunoprecipitates. The minor peaks were proved not to be derived from cross reactive materials, and the molecular species of these peaks were characterized. This method is faster than immunoprecipitation method and using no antibody is a great benefit. The method can be applied widely to the study various proteins, when antibodies are not available.

The peak height ratio of S-sulfonated transthyretin and other oxidized isoforms as a marker for molybdenum cofactor deficiency, measured by electrospray ionization mass spectrometry

Molybdenum cofactor deficiency is a fatal neurological disorder, which follows an autosomal-recessive trait and is characterized by combined deficiency of the enzyme, sulfite oxidase, xanthine dehydrogenase and aldehyde oxidase. Early detection of molybdenum cofactor-deficient patients is essential for their proper care and genetic counseling of families at risk. We demonstrate the use of S-sulfonated transthyretin (TTR) as a marker for molybdenum cofactor deficiency. Plasma or sera obtained from 4 patients with molybdenum cofactor deficiency and 57 controls were studied by electrospray ionization mass spectrometry (ESIMS) following selective enrichment of TTR by immunoprecipitation using protein G/A agarose. The data obtained from molybdenum cofactor deficiency samples indicated a strong increase in the peak height of S-sulfonated TTR. A more significant difference was revealed if the peak height ratio of S-sulfonated TTR and the sum of the other oxidized TTR were determined. By accurate determination of the ratio, the samples of molybdenum cofactor deficiency patients could clearly be distinguished from controls without molybdenum cofactor deficiency.

Enhanced amyloidogenicity of sulfonated transthyretin in vitro, a hypothetical etiology of senile amyloidosis

Genetic variants of transthyretin (TTR) cause systemic amyloidosis and wild-type TTR may also in some situations produce amyloid fibrils. We have analyzed wild-type and variant TTRs by mass spectrometry and found that TTR preparations from all individuals demonstrated free TTR, TTR conjugated with thiol compounds and several minor components. We previously described a component which had a molecular mass 80 Da larger than free TTR and was proved to be TTR conjugated with sulfite. Here, the amyloid fibril formation of the TTR isoforms was monitored by the turbidity at 330 nm, and by a Congo red-binding assay as a function of pH, according to the method of Lai et al. The S-sulfonated TTR showed the highest level of amyloid fibril formation. In contrast, TTR reduced by dithiothreitol, which was free of the S-sulfonated component, showed neither elevation of the turbidity nor the Congo red binding. Commercially purchased TTR without further treatment containing free, S-sulfonated and other species of TTR molecules showed an intermediate elevation. These results suggested that the S-sulfonated wild-type TTR is highly amyloidogenic. Although further experiments are needed to apply the observation to in vivo phenomenon, exogenous sulfite may be a cause of senile systemic amyloidosis.

Detection by mass spectrometry of highly increased amount of S-sulfonated transthyretin in serum from a patient with molybdenum cofactor deficiency.

Serum transthyretin has several isoforms, most of which are caused by disulfide linkage with cysteine residue at position 10. We found an ion peak 80 D larger than unmodified transthyretin by electrospray ionization mass spectrometry and assigned it to S- sulfonated transthyretin. The peak height was <2% of total transthyretin in control sera from more than 200 individuals including infants. Transthyretin from a patient with molybdenum cofactor deficiency was analyzed, and the peak was prominent, higher than 85% of total transthyretin. In patients with this disease, the presence of elevated levels of sulfite leads to the formation of S-sulfonated cysteine. The peak can be used as a diagnostic marker for molybdenum cofactor deficiency, although more sera from patients with this disease should be tested.

A new amyloidogenic transthyretin variant, [D38A], detected by electrospray ionization/mass spectrometry.

A new variant of transthyretin (TTR) was detected by mass spectrometry (MS) in a 63-year-old Japanese female patient suffering from amyloidosis. TTR was analyzed by 2-dimensional liquid chromatography coupled with electrospray ionization MS. Variant TTR showed extra peaks in addition to normal TTR peaks. The extra peaks were about 44 Da smaller than normal TTR peaks, and the abundance of variant peaks showed about 80% of the corresponding normal free and adduct peaks. Direct genomic DNA sequencing of TTR exon 2 showed both adenine and cytosine in the position corresponding to the second base of codon 38. This codes for a variant alanine (GCT) as well as the normal aspartic acid (GAT), indicating that the case is heterozygous for the substitution, [D38A].

HB Peterborough [β111(G13)VALPHE] in Japan; Rapid Identification by ESI/MS Using Proteolytic Digests of Oxidized Globin

A variant hemoglobin was found in a Japanese female whose hemoglobin was studied to clarify the cause of a low Hb A1c value, found during a routine medical examination. The detection and identification of the variant was performed by electrospray ionization mass spectrometry. Its structure was revealed to be the same as Hb Peterborough [beta 111(G13)Val-->Phe]. For sequence determination, oxidized globin as well as non-derivatized globin were cleaved by trypsin and lysyl endopeptidase. An abnormal peptide was found in digests of oxidized globin, as shown by electrospray ionization mass spectrometry. Cysteic acid in oxidized peptides enhanced the abundance of fragment ions in tandem mass spectrometry, which helped to quickly and accurately determine the substitution in beta T-12, a peptide in the core region. Electrospray ionization mass spectrometry analysis of the hemolysate also showed a low level of glycated hemoglobin. The patients hemolysate showed decreased stability in the isopropanol test. An abnormal band was detected on isoelectrofocusing on the cathodic side of normal Hb A. This is the second report of Hb Peterborough and the first of its occurrence in Japan.

Electrospray ionization-tandem mass spectrometry analysis of peptides derived by enzymatic digestion of oxidized globin subunits: An improved method to determine amino acid substitution in the hemoglobin “core”

The determination of an amino acid substitution in the “core” region of abnormal hemoglobin is technically difficult and is time- and labor-intensive both by conventional and by mass spectrometry techniques. Underivatized subunits cleaved with trypsin and lysyl endopeptidase were analyzed by high-performance liquid chromatography-electrospray ionizationmass spectrometry. The core peptides showed a series of multiply charged ions of homo- and heterodimers. Abnormal peptides in the core region could be detected in dimeric form. The sequence of core peptides was determined by product ion spectra of the peptides from oxidized globin digested with trypsin and lysyl endopeptidase. Oxidation of cysteine residues to cysteic acids in the core region resulted in the strong promotion of y-series ions by product ion analysis with a tryptic peptide from apoprotein B-100 as previously reported by Burlet, Yang, and Gaskell (J. Am. Soc. Mass Spectrom.1992, 3, 337–344). These techniques were used to prove that substitution of an unstable hemoglobin, known as Hb Santa Ana (β88 leucine → proline), occurred in a patient with congenital hemolytic anemia. The tandem mass spectrometry analysis with oxidized globin digested with trypsin and lysyl endopeptidase offers a novel method to detect substitutions in the core region of hemoglobin.