Analysis of short tandem repeat polymorphisms by electrospray ion trap mass spectrometry.

The application of electrospray ionization (ESI) ion trap mass spectrometry (MS) to the analysis of short tandem repeats (STRs or microsatellites) is described. Several equine dinucleotide STR loci were chosen as a model system to evaluate ESI ion trap as a routine instrument for rapid and reliable genoytping. With the use of specific primers STR loci were amplified from different blood samples having allele sizes between 60 and 100 bp. A new purification method based on reversible binding of PCR products to magnetic particles has proven to be directly compatible with ESI ion trap MS analysis. The sense and antisense strands of the PCR products with concentrations of approximately 100 fmol/microliter were measured with a mass accuracy of 0.01%. The simplicity of the purification method and the capability for automated handling together with the precise sizing of PCR products by ESI ion trap MS facilitate the large scale analysis of polymorphic STRs. Moreover, mixtures of different allele length as obtained for heterozygous samples could accurately be assigned as well as a C-->G switch between the two strands of a PCR product.

Analysis of non-covalent protein complexes up to 290 kDa using electrospray ionization and ion trap mass spectrometry.

Non-covalently-bound subunit complexes of proteins have been measured by an ion trap mass spectrometer equipped with an orthogonal electrospray ionization source. For the analysis of the generated molecular ions with high mass/charge ratios, the mass/charge range of the ion trap was extended by increasing its radio frequency (rf) voltage to 15 kV (V(0-p)) and by resonant ion ejection. Ions of the non-covalent dimer of bovine serum albumin (BSA), as well as of subunit complexes of alcohol dehydrogenase (ADH) from bakers' yeast and from horse liver, have been detected at mass/charge values between 3000-9000 Th. The maximum observed molecular weight was that of a non-covalently-bound subunit-octamer of bakers' yeast ADH (two non-covalently-bound subunit-tetramers) at ca. 290 kDa.

In vitro immunosuppressive activity, isolation from pig liver microsomes and identification by electrospray ms-ms of a new FK-506 C19-C20 epoxide metabolite.

In order to mediate their effects, cyclosporin A and FK-506 must each bind with high affinity to a cytosolic target protein that belongs to the immunophilin group. FK-506 forms complexes with the FK-506 binding protein FKBP, mainly FKBP-12, and these complexes possess immunosuppressive activity through their ability to interact with another target, the abundant serine threonine phosphatase calcineurin. Evaluating the immunosuppressive activities of the FK-506 metabolites by comparing them with known immunosuppressive agents via mixed lymphocyte reaction is of clinical importance because some metabolites may retain the pharmacological activity of the parent drug or exhibit cytotoxic effects. FK-506 is metabolized by the cytochrome P-450-dependent mixed-function oxygenase system in different animal species, and we are reporting the isolation from pig liver microsomes, and the identification by electrospray ms-ms, of the FK-506 C19-C20 epoxide metabolite. We found that this new metabolite exhibits reduced in vitro immunosuppressive activity compared with FK-506 and has approximately the same immunosuppressive potency as other known immunosuppressive drugs, such as cyclosporin A and IMM-125, a hydroxyethyl derivative of D-serine cyclosporin A. We were able to demonstrate that after incubation of the FK-506 metabolite in human mixed lymphocyte reaction cultures for 6 days, the compound was stable under the conditions used for cell culture as evidenced by electrospray-ms data. A weak direct cytotoxic effect (< 30% cell death) was observed only at the highest concentrations (2500 and 5000 ng/ml), which shows that the mixed lymphocyte reaction inhibition cannot be due to a toxic effect.

Structural and functional characterization of a novel tumor-derived rat galectin-1 having transforming growth factor (TGF) activity: the relationship between intramolecular disulfide bridges and TGF activity.

Previously we demonstrated that overexpression of a beta-galactoside binding protein, galectin-1, caused the transformation of BALB3T3 fibroblast cells [Yamaoka, K., Ohno, S., Kawasaki, H., and Suzuki, K. (1991) Biochem. Biophys. Res. Commun. 179, 272-279]. We have now studied the structure-function relationship between the sugar-binding activity and the mitogenic activity of galectin-1 purified from an avian sarcoma virus-transformed rat NRK cell line, 77N1. The purified galectin-1 (t-galectin-1) had potent mitogenic activity in BALB3T3 cells, but no sugar-binding activity. Treatment of t-galectin-1 with 2-mercaptoethanol decreased its mitogenic activity, but resulted in the appearance of a sugar binding activity. Chemical modification of sulfhydryl groups in purified t-galectin-1 with [14C]-iodoacetamide suggested the presence of intramolecular disulfide bonds. MALDI-TOF mass spectrometric analysis of the native and reduced forms of the tryptic peptides from t-galectin-1 showed that t-galectin-1 has two intramolecular disulfide bonds (Cys2-Cys16 and Cys42-Cys60). These studies suggest that these intramolecular disulfide bonds of t-galectin-1 are essential for its mitogenic activity and that the different activities may be regulated by structural changes caused by intramolecular disulfide bond-breakage.

Separation and characterisation of bovine histone H1 subtypes by combined ion-exchange and reversed-phase chromatography and mass spectrometry.

In order to separate and identify histone H1 subtypes from calf thymus we used both electrospray mass spectrometry (ES-MS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) after a three-step chromatographic procedure consisting of reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC) and ion-exchange chromatography (IEC). Under the RP-HPLC conditions described, we obtained two baseline-separated H1-fractions which were characterised by MALDI-TOF-MS. The determined masses ranged from 22,850 to 22,590 for the first fraction and from 22,070 to 21,250 for the second fraction. Further, it was shown that the first fraction contained at least four and the second one at least five subtypes of the histone class H1. Four homogeneous pure H1 subtypes were obtained by a combination of IEC followed by SEC and RP-HPLC. The molecular masses of these four subtypes determined by ES-MS were 22,606, 22,761, 21,347 and 21,263. We obtained six additional molecular masses of histone H1 subtypes from three heterogeneous fractions, namely 22,066, 21,802, 20,586 and 19,817 by ES-MS and 22,800 and 22,675 by MALDI-TOF-MS. The retention times of these fractions and the molecular masses were in agreement with the data obtained from RP-HPLC fractions by MALDI-TOF-MS.

A novel amino acid modification in sulfatases that is defective in multiple sulfatase deficiency.

Multiple sulfatase deficiency (MSD) is a lysosomal storage disorder characterized by a decreased activity of all known sulfatases. The deficiency of sulfatases was proposed to result from the lack of a co- or posttranslational modification that is common to all sulfatases and required for their catalytic activity. Structural analysis of two catalytically active sulfatases revealed that a cysteine residue that is predicted from the cDNA sequence and conserved among all known sulfatases is replaced by a 2-amino-3-oxopropionic acid residue, while in sulfatases derived from MSD cells, this cysteine residue is retained. It is proposed that the co- or posttranslational conversion of a cysteine to 2-amino-3-oxopropionic acid is required for generating catalytically active sulfatases and that deficiency of this protein modification is the cause of MSD.

Analysis of serum protein precipitated with antiserum by matrix-assisted laser desorption ionization/time-of-flight and electrospray ionization mass spectrometry as a clinical laboratory test.

Serum transferrin precipitated with specific antisera was analyzed by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI/TOF-MS) and electrospray ionization-mass spectrometry (ESI-MS). When analyzed by MALDI, transferrin showed signal peaks that clearly could be separated from ions of IgG present in the immunoprecipitate. By ESI-MS, when the immunoprecipitates were loaded through a microcapillary polymeric reversed-phase column connected to the electrospray ionization probe, the mass spectra of transferrin were observed with a high signal-to-noise ratio and good resolution. By MALDI/TOF-MS, the observed molecular weight of normal transferrin was ∼ 1.2 ku smaller when analyzed in the reflectron mode than in the linear mode. The observed molecular weight of transferrin treated with sialidase was approximately the same in both modes. A comparison between the results obtained in both modes may help to estimate the number of sialic acids on the protein molecule. A transferrin isoform with a molecular weight of ∼2.2 ku less than the normal species was identified in the serum of patients with a carbohydrate-deficient glycoprotein syndrome as well as in heavy alcohol consumers.

Glycosylation and phosphorylation of arylsulfatase A.

The glycosylation and phosphorylation of the lysosomal enzyme arylsulfatase A was analyzed by a combination of metabolic labeling, tryptic fragmentation, mass spectrometry, and radiosequencing. The results demonstrate that all three potential N-glycosylation sites at Asn residues 158, 184, and 350 are utilized in arylsufatase A and carry high mannose or hybride type oligosaccharides. Phosphorylation of mannose residues is restricted to oligosaccharides at the first and third N-glycosylation site (Asn-158 and Asn-350). Both are phosphorylated with comparable efficiency. An earlier study had shown that a mutant arylsulfatase A containing only the second N-glycosylation site at Asn-184 folds correctly and is phosphorylated (Gieselmann, V., Schmidt, B., and von Figura, K. (1992) J. Biol. Chem. 267, 13262-13266). The lack of phosphorylation at Asn-184 in wild type arylsulfatase A therefore indicates that in vivo the presence of oligosaccharides can interfere with phosphorylation of other sites or that phosphorylation occurs in an ordered manner whereby phosphorylation of one site can affect the phosphorylation of another site.

N-terminal variants of fatty acid-binding protein from bovine heart overexpressed in Escherichia coli.

An expression vector for bovine heart fatty acid-binding protein (H-FABP) was constructed by introducing the coding part of the cDNA into the pET-3d vector. Transformed Escherichia coli strain BL21 (DE3)pLysS produced functional recombinant H-FABP up to 40% of the soluble proteins. The expression of fatty acid-binding protein was under the control of the T7-phi 10 promoter and the corresponding T7-RNA-polymerase in turn was induced by isopropyl beta-D-thiogalactopyranoside. By combination of cation exchange chromatography and gel filtration pure recombinant protein was obtained exhibiting isoelectric heterogeneity. Recombinant H-FABP was resolved into at least six variants with isoelectric points between 5.1 and 5.6. After separation by preparative isoelectric focusing the four major variants were digested with trypsin and the resulting peptides were characterized by high performance liquid chromatography (HPLC), matrix assisted laser desorption/ionization (MALDI) mass spectrometry, amino acid sequencing and chemical modification. The structural differences were traced back to the N-termini beginning with either methionine, as expected from the cDNA, or methionine sulfoxide, valine and N-formyl methionine. The latter three arise from oxidation, cleavage of N-terminal methionine and incomplete deformylation, respectively.

Matrix-assisted laser desorption/ionization mass spectrometry of nucleic acids with wavelengths in the ultraviolet and infrared.

A number of different matrices have been tested and compared for ultraviolet and infrared (UV and IR) matrix-assisted laser desorption/ionization (MALDI) of oligodeoxyribonucleotides and mixtures thereof, as well as ribonucleic acids (tRNA from yeast and rRNA from E. coli). A new technique for removing alkali cations from nucleic acid samples during sample preparation on the sample support is demonstrated. The amount of oligonucleotide sample consumed during a typical measurement in IR-MALDI-MS was determined.

Application of cross-flow filtration to the purification of biologically active peptides in human plasma after incubation with a protease-rich extract.

The aim of this study was to find an experimental procedure to purify biologically active peptides from a complex biological matrix (plasma), which was incubated with a protease-rich extract (submandibular gland extract). Special interest was focused on the practicability of cross-flow filtration for this purpose. Therefore, peptides in the incubation mixture were purified with a combination of high-performance liquid chromatographic steps. Purification of biologically active peptides was monitored by a sensitive bioassay and by laser desorption/ionization mass spectrometry. This permitted not only purity control at each purification step but also identification of one of the peptides with vasoconstrictor properties as angiotensin II. This result demonstrates the practicability of cross-flow filtration for extracting enzymatic reaction products from complex substrate-enzyme mixtures during the incubation.

Influence of muzolimine and other diuretics on human red cell Na+, K+-cotransport.

It is known that the diuretic activity of so-called loop diuretics is characterized by the ability of such compounds to inhibit the Na+, K+-cotransport system of the cell membrane. A good correlation has been demonstrated of the inhibitory activity of several furosemide- and ethacrynic acid-related compounds with their natriuretic activity in man. It was the aim of the study to examine, wether muzolimine has Na+, K+-cotransport inhibitory activity, and if so, if this occurs in a dose range which might be relevant for its natriuretic activity. For reasons of comparison, several other diuretics with high ceiling activity were studied. Cotransport inhibitory activities were compared with natriuretic activities in rats. Na+, K+-cotransport was measured by determination of furosemide-sensitive, ouabain-insensitive Na+-extrusion from Na+-loaded human red blood cells. Whereas the natriuretic activity of muzolimine is equal to that of furosemide in rats and superior in man, its cotransport inhibitory activity is merely 1/13 of that of furosemide. Additionally, only 50% inhibition is reached. The inhibitory activity of muzolimine, in contrast to ethacrynic acid, is not enhanced in the presence of equimolar cysteine. Muzolimine therefore appears to act via other mechanisms than furosemide-like diuretics. However, it cannot be excluded that muzolimine might act by an active metabolite.