Advances in hemoglobinopathy detection and identification.

Hemoglobin disorders consist of two different groups, the structural hemoglobin variants and the thalassemias. The structural hemoglobin variants typically are based on the point mutations in the alpha- or beta-globin chain that results in a single-amino acid substitution in the corresponding globin chain, whereas thalassemias are caused by quantitative reduction in globin chain synthesis. Various techniques are applied for the laboratory investigation of these diseases, among them mass spectrometry (MS) for the detection and identification of structural hemoglobin variants and array techniques for the thalassemias. In this review, we present in the first part the most important mass spectrometric techniques applied in hemoglobin variant detection and identification and discuss several important aspects of this analysis technique in hematology. In the second part, the DNA analysis techniques used in hemoglobin analysis, such as reverse hybridization or microarray-based comparative genomic hybridization (CGH) techniques, are briefly discussed.

Deficiency in COG5 causes a moderate form of congenital disorders of glycosylation.

The conserved oligomeric Golgi (COG) complex is a tethering factor composed of eight subunits that is involved in the retrograde transport of intra-Golgi components. Deficient biosynthesis of COG subunits leads to alterations of protein trafficking along the secretory pathway and thereby to severe diseases in humans. Since the COG complex affects the localization of several Golgi glycosyltransferase enzymes, COG deficiency also leads to defective protein glycosylation, thereby explaining the classification of COG deficiencies as forms of congenital disorders of glycosylation (CDG). To date, mutations in COG1, COG4, COG7 and COG8 genes have been associated with diseases, which range from severe multi-organ disorders to moderate forms of neurological impairment. In the present study, we describe a new type of COG deficiency related to a splicing mutation in the COG5 gene. Sequence analysis in the patient identified a homozygous intronic substitution (c.1669-15T>C) leading to exon skipping and severely reduced expression of the COG5 protein. This defect was associated with a mild psychomotor retardation with delayed motor and language development. Analysis of different serum glycoproteins revealed a CDG phenotype with typical undersialylation of N- and O-glycans. Retrograde Golgi-to-endoplasmic reticulum trafficking was markedly delayed in the patient's fibroblast upon brefeldin-A treatment, which is a hallmark of COG deficiency. This trafficking delay could be restored to normal values by expressing a wild-type COG5 cDNA in the patient cells. This case demonstrates that COG deficiency and thereby CDG must be taken into consideration even in children presenting mild neurological impairments.

A new alpha-globin variant with increased oxygen affinity in a Swiss family: Hb Frauenfeld [alpha 138(H21)Ser-->Phe, TCC>TTC (alpha 2)].

A new alpha-globin mutation [alpha 138(H21)Ser-->Phe] was found in a 55-year-old male proband with an erythrocytosis known since his youth. Cation exchange high performance liquid chromatography (HPLC) revealed an additional peak eluting slightly before Hb A indicating the presence of a variant. The peak area of the variant was approximately one-third that of Hb A suggesting an alpha-globin variant. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis confirmed the mutation at the protein level. The variant is also detectable with isoelectric focusing and reversed phase HPLC. DNA analysis revealed a heterozygous sequence mutation at codon 138 of the alpha2 gene. A C>T transition at the second nucleotide of the codon indicated a Ser-->Phe exchange. The variant showed increased oxygen affinity and was named Hb Frauenfeld.

Phosphorylation regulates transcriptional activity of PAX3/FKHR and reveals novel therapeutic possibilities.

Inhibition of constitutive active signaling pathways, which are a characteristic phenomenon for many tumors, can be an effective therapeutic strategy. In contrast, oncogenic transcription factors, often activated by mutational events, are in general less amenable to small-molecule inhibition despite their obvious importance as therapeutic targets. One example of this is alveolar rhabdomyosarcoma (aRMS), in which specific translocations lead to the formation of the chimeric transcription factor PAX3/FKHR. Here, we found unexpectedly that the transcriptional activity of PAX3/FKHR can be inhibited by the kinase inhibitor PKC412. This occurs via specific phosphorylation sites in the PAX3 domain, phosphorylation of which is required for efficient DNA-binding and subsequent transcriptional activity. Consequently, we show that PKC412 exerts a potent antitumorigenic potential for aRMS treatment both in vitro and in vivo. Our study suggests that posttranscriptional modifications of oncogenic transcription factors can be explored as a promising avenue for targeted cancer therapy.

Specific citrullination causes assembly of a globular S100A3 homotetramer: a putative Ca2+ modulator matures human hair cuticle.

S100A3 is a unique member of the Ca2+-binding S100 protein family with the highest cysteine content and affinity for Zn2+. This protein is highly expressed in the differentiating cuticular cells within the hair follicle and organized into mature hair cuticles. Previous studies suggest a close association of S100A3 with epithelial differentiation, leading to hair shaft formation, but its molecular function is still unknown. By two-dimensional PAGE-Western blot analyses using a modified citrulline antibody, we discovered that more than half of the arginine residues of native S100A3 are progressively converted to citrullines by Ca2+-dependent peptidylarginine deiminases. Confocal immunofluorescent microscopy showed that the cytoplasmic S100A3 within the cuticular layer is mostly co-localized with the type III isoform of peptidylarginine deiminase (PAD3) but not with PAD1. Recombinant PAD1 and PAD2 are capable of converting all 4 arginines in recombinant S100A3, whereas PAD3 specifically converts only Arg-51 into citrulline. Gel filtration analyses showed that either enzymatic conversion of Arg-51 in S100A3 to citrulline or its mutational substitution with alanine (R51A) promotes a homotetramer assembly. Fluorescent titration of R51A suggested that its potential Ca2+ binding property increased during tetramerization. A prototype structural model of the globular Ca2+-bound S100A3 tetramer with citrulline residues is presented. High concentrations of S100A3 homotetramer might provide the millimolar level of Ca2+ required for hair cuticular barrier formation.

Mass spectrometry: a tool for enhanced detection of hemoglobin variants.

BACKGROUND: More than 900 hemoglobin (Hb) variants are currently known. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. In our laboratory, we routinely apply chromatographic methods. To ascertain whether Hb variants are missed with our procedures, we additionally analyzed all samples with mass spectrometry (MS). METHODS: Database evaluation was performed using all entries made in the Hb variant database HbVar, and possible Hb variants were calculated based on DNA variations. During a 5-year period, we analyzed 2105 lysates with cation-exchange HPLC (PolyCAT A column) and reversed-phase HPLC and additionally with electrospray ionization or MALDI-TOF MS. Globin chains were identified by their molecular masses. RESULTS: Database evaluation revealed that 43.2% of all possible Hbalpha- and beta-chain variants were found to date (considering only single-point mutations). Currently, 68.2% of the possible charge difference variants and only 28.7% of the neutral variants are found. Among 2105 Hb samples we identified 4 samples with Hb variants that were detected only with the MS method; 2 were new Hb variants (Hb Zurich-Hottingen and Hb Zurich-Langstrasse). With cation-exchange HPLC, 1 sample was found to be a beta-thalassemia and was identified by MS to be a beta-variant (Hb Malay). More common variants, such as Hb C, Hb D, and Hb E, and thalassemias could not be detected with the MS method. CONCLUSIONS: Application of MS improves the sensitivity of Hb analysis. The combination of MS with electrophoretic and chromatographic methods is optimal for the detection of Hb variants.

Interaction of receptor-activity-modifying protein1 with tubulin.

Receptor-activity-modifying protein (RAMP) 1 is an accessory protein of the G protein-coupled calcitonin receptor-like receptor (CLR). The CLR/RAMP1 heterodimer defines a receptor for the potent vasodilatory calcitonin gene-related peptide. A wider tissue distribution of RAMP1, as compared to that of the CLR, is consistent with additional biological functions. Here, glutathione S-transferase (GST) pull-down, coimmunoprecipitation and yeast two-hybrid experiments identified beta-tubulin as a novel RAMP1-interacting protein. GST pull-down experiments indicated interactions between the N- and C-terminal domains of RAMP1 and beta-tubulin. Yeast two-hybrid experiments confirmed the interaction between the N-terminal region of RAMP1 and beta-tubulin. Interestingly, alpha-tubulin was co-extracted with beta-tubulin in pull-down experiments and immunoprecipitation of RAMP1 coprecipitated alpha- and beta-tubulin. Confocal microscopy indicated colocalization of RAMP1 and tubulin predominantly in axon-like processes of neuronal differentiated human SH-SY5Y neuroblastoma cells. In conclusion, the findings point to biological roles of RAMP1 beyond its established interaction with G protein-coupled receptors.

Effect of glycosylation on the protein pattern in 2-D-gel electrophoresis.

Single proteins, when analyzed with 2-D-PAGE, often show multiple spots due to PTMs. In gels of human body fluids, the spot patterns facilitate the assignment and identification of the proteins. We analyzed serums from patients with congenital disorders of glycosylation (CDG) in which glycoproteins are strongly impacted and exhibit highly distinguishable spot patterns compared to healthy controls. We detected a typical protein pattern for alpha1-acid glycoprotein (AGP) and transferrin (Trf) that are markers for CDG. AGP contains five glycosylation sites which results in a complex microheterogeneity of the glycoprotein. On the other hand, in Trf, a glycoprotein with only two glycosylation sites, mainly biantennary complex-type-N-linked glycans are bound. We used 2-D-PAGE, MALDI-TOF-MS, and ESI-MS for the analysis of these glycoproteins and their corresponding glycans. In AGP, the heterogenic glycosylation of the different glycosylation sites is responsible for the complex spot pattern. In contrast to AGP, the protein spots of Trf cannot be explained by glycosylation. We found strong evidence that oxidation of cysteine is responsible for the spot pattern. This study contradicts the commonly accepted assumption that the multiple protein spots of Trf observed in 2-D-PAGE are due, as in AGP, to the glycosylation of the protein.

Expression and purification of the soluble isoform of human receptor for advanced glycation end products (sRAGE) from Pichia pastoris.

RAGE is a multi-ligand receptor involved in various human diseases including diabetes, cancer or Alzheimer's disease. Engagement of RAGE by its ligands triggers activation of key cellular signalling pathways such as the MAP kinase and NF-kappaB pathways. Whereas the main isoform of RAGE is a transmembrane receptor with both extra- and intracellular domains, a secreted soluble isoform (sRAGE), corresponding to the extracellular part only, has the ability to block RAGE signalling and suppress cellular activation. Administration of sRAGE to animal models of cancer or multiple sclerosis blocked successfully tumour growth and the course of the autoimmune disease. These findings demonstrate that sRAGE may have a potential as therapeutic. We present here a fast and simple purification protocol of sRAGE from the yeast Pichia pastoris. The identity of the protein was confirmed by mass spectrometry and Western blot. The protein was N-glycosylated and 95-98% pure as judged by SDS-PAGE.

Dissimilar effect of perming and bleaching treatments on cuticles: advanced hair damage model based on elution and oxidation of S100A3 protein.

Hair treatment chemicals induce sudden and severe hair damage. In this study, we examined cuticles from untreated, permed, and bleached hair that were mechanically discriminated by shaking in water. Both perming and bleaching treatments are prone to easily delaminate cuticles. Confocal microscopy revealed that the cuticles of permed hair were delaminated with larger pieces than untreated ones. On the other hand, the cuticles of bleached hair tend to fragment into small peptides. At the minimum concentration of thioglycolate required to elute S100A3 protein from the endocuticle into the reductive permanent waving lotion, enlarged delaminated cuticle fragments were observed. Although S100A3 is retained in bleached hair, S100A3 is irreversibly oxidized upon bleaching treatment. It is likely that the oxidative cleavage of disulfide bonds between cuticle-constituting proteins, including S100A3, results in the fragile property of cuticles. Here we present a more comprehensive model of hair damage based on a diverse mechanism of cuticle delamination.

Analysis of minor hemoglobins by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

BACKGROUND: Hemoglobin (Hb) heterogeneity arises mainly from posttranslational modifications of the globin chains, and cation-exchange chromatography reveals falsely increased concentrations of some minor Hbs in the presence of abnormal Hbs. Here we describe a method for identification of the globin chains and their posttranslational modifications contained in the Hb fractions. METHODS: We used cation-exchange HPLC (PolyCAT A column) for separation of Hb fractions and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for analysis of the separated globin chains. Globin chains were identified by their molecular masses. Posttranslational modifications of globin chains were identified by digestion of the proteins with endoproteinase V8 before MALDI-TOF MS of the resulting peptides. RESULTS: Analysis of the HbA2 fractions of patients with HbS revealed 4 different globin chains. We found, in addition to the expected alpha- and delta-chains, the carbamylated alpha- and the betaS-chains. Additionally, we analyzed HbH, Hb Barts, HbA 1b, pre-HbA 1c, HbA 1c, HbF1, HbF, HbA 1d3a, HbA 1d3b, HbA2, and HbC1 fractions from control and pathologic blood samples. We identified several posttranslational modifications of the globin chains, such as pyruvatization, glycation, acetylation, carbamylation, and acetaldehyde adduct formation. CONCLUSIONS: The native and posttranslationally modified globin chains in minor and major Hbs are unambiguously identified by MALDI-TOF MS. A minor Hb containing the carbamylated alpha- and the betaS-chain elutes at the same time as normal HbA2 (alpha2delta2) and thus leads to falsely increased HbA2 values in patients with HbS when blood is analyzed with PolyCAT A chromatography.

Mass spectrometric analysis of human transferrin in different body fluids.

In this study, we present a versatile new procedure for the analysis of transferrin and its isoforms isolated from human body fluids such as serum, plasma, and cerebrospinal fluid. This method is based on a three-step procedure: (i) isolation of transferrins using anion-exchange chromatography with UV detection; (ii) concentration of the transferrin fraction; (iii) detection of the transferrins with liquid chromatography-electrospray mass spectrometry. Pre-analytical sample procedures can be omitted and no immunoaffinity columns or transferrin-specific immunoassays were used. Anticoagulants such as heparin, EDTA, citrate, and oxalate do not interfere with our analysis. According to their respective molecular masses, up to ten different isoforms of transferrin could be identified in a serum sample from a patient with a congenital disorder of glycosylation type Ia (CDG-Ia). The method was successfully applied to different pathological samples from patients with CDG-Ia, CDG-Ib, CDG-Ic, CDG-Ie, CDG-If, and CDG-IIa. Additionally, samples from alcohol consumers that were found with turbidimetric immunoassay to contain increased levels of carbohydrate-deficient transferrin were analyzed.

Characterization of the cysteine-rich calcium-binding S100A3 protein from human hair cuticles.

S100A3, a unique protein among all members of the calcium-binding S100 family, is specifically expressed at the inner endocuticle of human hair fibers. Upon hair damage, S100A3 is released from hair fibers and possibly destabilizes the hair tissue architecture. This study describes the purification and characterization of native S100A3 isolated from human hair fibers. We extracted native S100A3 from cuticles and purified the protein by anion-exchange chromatography. The results of 2D gel electrophoresis showed that cuticle S100A3 has a slightly lower isoelectric point compared to the recombinant protein. Tandem mass spectrometry of the peptides resulting from endoproteinase digest of cuticle S100A3 revealed that the N-terminal methionine is replaced with an acetyl group. This is the first report on biochemical characteristics of S100A3 in hair cuticle.

Polar fuel constituents: compound identification and equilibrium partitioning between nonaqueous phase liquids and water.

Groundwater contamination by fuel constituents from nonaqueous phase liquids (NAPLs) on top of the groundwater table is a widespread problem. While leaching of classical fuel constituents such as benzene, toluene, ethylbenzene, and xylenes (BTEX) from NAPLs into groundwater has been studied extensively, little is known about the identity and partitioning of polar fuel components. Our work shows that gasoline commonly contains appreciable amounts of aniline, phenol, and their alkyl-substituted homologues as well as a suite of other polar compounds. To assess the potential for leaching of such compounds from NAPLs into groundwater we measured the equilibrium fuel/water partitioning coefficients, Kfw, of some representative constituents in batch systems. Kfw values for the investigated phenols, anilines, benzotriazoles, and S-heterocycles ranged from 0.2 to 1700. These values are up to 3 orders of magnitude lower than the Kfw of benzene. The NAPL--water partitioning of anilines and phenols strongly depends on the compounds' structure as well as on pH and the gasoline composition (e.g., MTBE content). Linear free energy relationships (LFERs) using Kow or Cwsat failed to predict measured Kfw values of polar solutes. In contrast, a polyparameter approach taking into account molecular interactions (van der Waals forces, hydrogen bonds) between solutes and major gasoline components allows precise a-priori predictions of Kfw values of both polar and BTEX fuel constituents without any fit parameters. Since most of the polar fuel constituents studied here are extracted from NAPLs by groundwater much more efficiently than BTEX, such compounds could form contaminant plumes threatening receiving wells before detectable concentrations of BTEX are present.

Detection of a novel variant human hemoglobin by electrospray ionization mass spectrometry.

A novel hemoglobin variant was detected by electrospray ionization mass spectrometry. Hb Zurich-Hottingen is characterized by an Asn --> Ser replacement in the alpha-chain at position 9 as confirmed by DNA analysis. This hemoglobin variant is silent in isoelectric focusing, reversed-phase chromatography, and cation-exchange chromatography. The mutant alpha-chain was detectable only with electrospray mass spectrometry by its mass shift of -27 Da. The carrier was found to be heterozygous for the new hemoglobin variant. These results illustrate the power of ESI mass spectrometry for hemoglobin analysis.