Positive Mode LC-MS/MS Analysis of Chondroitin Sulfate Modified Glycopeptides Derived from Light and Heavy Chains of The Human Inter-α-Trypsin Inhibitor Complex.

The inter-α-trypsin inhibitor complex is a macromolecular arrangement of structurally related heavy chain proteins covalently cross-linked to the chondroitin sulfate (CS) chain of the proteoglycan bikunin. The inter-α-trypsin inhibitor complex is abundant in plasma and associated with inflammation, kidney diseases, cancer and diabetes. Bikunin is modified at Ser-10 by a single low-sulfated CS chain of 23-55 monosaccharides with 4-9 sulfate groups. The innermost four monosaccharides (GlcAß3Galß3Galß4Xylß-O-) compose the linkage region, believed to be uniform with a 4-O-sulfation to the outer Gal. The cross-linkage region of the bikunin CS chain is located in the nonsulfated nonreducing end, (GalNAcß4GlcAß3)(n), to which heavy chains (H1-H3) may be bound in GalNAc to Asp ester linkages. In this study we employed a glycoproteomics protocol to enrich and analyze light and heavy chain linkage and cross-linkage region CS glycopeptides derived from the IαI complex of human plasma, urine and cerebrospinal fluid samples. The samples were trypsinized, enriched by strong anion exchange chromatography, partially depolymerized with chondroitinase ABC and analyzed by LC-MS/MS using higher-energy collisional dissociation. The analyses demonstrated that the CS linkage region of bikunin is highly heterogeneous. In addition to sulfation of the Gal residue, Xyl phosphorylation was observed although exclusively in urinary samples. We also identified novel Neu5Ac and Fuc modifications of the linkage region as well as the presence of mono- and disialylated core 1 O-linked glycans on Thr-17. Heavy chains H1 and H2 were identified cross-linked to GalNAc residues one or two GlcA residues apart and H1 was found linked to either the terminal or subterminal GalNAc residues. The fragmentation behavior of CS glycopeptides under variable higher-energy collisional dissociation conditions displays an energy dependence that may be used to obtain complementary structural details. Finally, we show that the analysis of sodium adducts provides confirmatory information about the positions of glycan substituents.

Identification of chondroitin sulfate linkage region glycopeptides reveals prohormones as a novel class of proteoglycans.

Vertebrates produce various chondroitin sulfate proteoglycans (CSPGs) that are important structural components of cartilage and other connective tissues. CSPGs also contribute to the regulation of more specialized processes such as neurogenesis and angiogenesis. Although many aspects of CSPGs have been studied extensively, little is known of where the CS chains are attached on the core proteins and so far, only a limited number of CSPGs have been identified. Obtaining global information on glycan structures and attachment sites would contribute to our understanding of the complex proteoglycan structures and may also assist in assigning CSPG specific functions. In the present work, we have developed a glycoproteomics approach that characterizes CS linkage regions, attachment sites, and identities of core proteins. CSPGs were enriched from human urine and cerebrospinal fluid samples by strong-anion-exchange chromatography, digested with chondroitinase ABC, a specific CS-lyase used to reduce the CS chain lengths and subsequently analyzed by nLC-MS/MS with a novel glycopeptide search algorithm. The protocol enabled the identification of 13 novel CSPGs, in addition to 13 previously established CSPGs, demonstrating that this approach can be routinely used to characterize CSPGs in complex human samples. Surprisingly, five of the identified CSPGs are traditionally defined as prohormones (cholecystokinin, chromogranin A, neuropeptide W, secretogranin-1, and secretogranin-3), typically stored and secreted from granules of endocrine cells. We hypothesized that the CS side chain may influence the assembly and structural organization of secretory granules and applied surface plasmon resonance spectroscopy to show that CS actually promotes the assembly of chromogranin A core proteins in vitro. This activity required mild acidic pH and suggests that the CS-side chains may also influence the self-assembly of chromogranin A in vivo giving a possible explanation to previous observations that chromogranin A has an inherent property to assemble in the acidic milieu of secretory granules.

Elevated levels of protein AMBP in cerebrospinal fluid of women with preeclampsia compared to normotensive pregnant women.

PURPOSE: To investigate the cerebrospinal fluid (CSF) proteome of patients with preeclampsia (PE) and normotensive pregnant women, in order to provide a better understanding of brain involvement in PE. EXPERIMENTAL DESIGN: Ninety-eight CSF samples (43 women with PE and 55 normotensive controls) were analyzed by LC-MS/MS proteome profiling. CSF was obtained during the spinal puncture before caesarean delivery. RESULTS: Eight proteins were higher abundant and 17 proteins were lower abundant in patients with PE. The most significantly differentially abundant protein was protein AMBP (alpha-1-microglobulin/bikunin precursor). This finding was validated by performing an ELISA experiment (p = 0.002). CONCLUSIONS AND CLINICAL RELEVANCE: The current study showed a clear difference between the protein profiles of CSF from patients with PE and normotensive pregnant women. Protein AMBP is a precursor of a heme-binding protein that counteracts the damaging effects of free hemoglobin, which may be related to the presence of free hemoglobin in CSF. Protein levels showed correlations with clinical symptoms during pregnancy and postpartum. To our knowledge, this is the first LC-MS/MS proteome profiling study on a unique set of CSF samples from (severe) preeclamptic patients and normotensive pregnant women.

Identification of bikunin as an endogenous inhibitor of dynorphin convertase in human cerebrospinal fluid.

Dynorphin-converting enzymes constitute a group of peptidases capable of converting dynorphins to enkephalins. Through the action of these enzymes, the dynorphin-related peptides bind to delta-opioid instead of kappa-opioid receptors, leading to a change in the biological function of the neuropeptides. In this article, we describe the identification of the protein bikunin as an endogenous, competitive inhibitor of a dynorphin-converting enzyme in human cerebrospinal fluid. This protein is present together with its target enzyme in the same body fluids. The K(M) value of the convertase was found to be 9 microm, and the K(i) value of the inhibitor was 1.7 nm. The finding indicates that bikunin may play a significant role as a regulatory mechanism of neuropeptides, where one bioactive peptide is converted to a shorter sequence, which in turn, can affect the action of its longer form.

Cerebrospinal fluid proteins in multiple sclerosis.

Various CSF proteins were studied in 255 definite multiple sclerosis patients at various disease stages and compared with corresponding values obtained from 174 controls. The CSF changes in acute multiple sclerosis patients included a significant increase of total proteins and of gamma globulin, IgG, IgA, IgM, alpha-2 ceruloplasmin, 7S-gamma-1, and cytotoxic index for nerve cells in tissue culture, and significant decreases of pre-albumin, alpha-1, and alpha-2 and of the beta/gamma globulin ratio. The CSF levels of IgG, IgA, and IgM remained significantly higher in steroid-treated multiple sclerosis patients than in controls, but the levels often were significantly reduced while patients were on treatment or in remission. During remission or treatment with ACTH and/or steroids, the alpha-2 ceruloplasmin, 7S-gamma-1, and cytotoxic index were significantly reduced and the pre-albumin, alpha-1, and alpha-2 globulin classes and the beta/gamma ratio showed a tendency to return to normal.

Adsorption of alpha-1-microglobulin from biological fluids onto polymer surfaces.

A recent study in our laboratory has identified the potential role of urine-derived alpha-1-microglobulin (alpha-1-m) in mediating Pseudomonas aeruginosa adhesion to polystyrene, while other workers have suggested a possible role of the protein in the immunological response. Due to the ubiquitous presence of alpha-1-m in body fluids, the adsorption of the protein from serum, cerebrospinal fluid, urine and used continuous ambulatory peritoneal dialysis fluid onto polystyrene was investigated. The treated surfaces were sequentially immersed in water and increasingly concentrated isopropanol-water solutions in order to selectively desorb bound proteins on the basis of their binding strength. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the wash supernatants showed different protein desorption profiles for each biological fluid, despite the qualitative similarity between the protein composition of the fluids, and highlighted the uptake of alpha-1-m from each fluid to the surface. In the case of urine, the analysis was extended to commercial polyurethane and silicone stents. The ease of desorption of urine-derived alpha-1-m could be correlated with surface hydrophobicity of the stent biomaterial.

Isoelectric focusing and electrophoresis of cerebrospinal fluid proteins in muscular dystrophies and spinal muscular atrophies.

In the very few previous investigations of the CSF-proteins in muscular dystrophies the results have generally been reported as normal. In spinal muscular atrophies a barrier-damage pattern of CSF-proteins has been found in amyotrophic lateral sclerosis (ALS). In the present investigation the CSF-proteins were examined by isoelectric focusing and quantitative paper electrophoresis in 13 patients with muscular dystrophies and in 11 patients with spinal muscular atrophies. On isoelectric focusing, CSF-protein abnormalities were found in 85% of the cases with muscular dystrophies and in all patients with spinal muscular atrophies. Differences in the CSF-protein patterns were observed within the group of muscular dystrophies and between these and the cases of spinal muscular atrophies. In ALS and in myotonic dystrophy, abnormal CSF-protein fractions occurred mainly in the alkaline pH-range, while in limb-girdle dystrophy and the patient with facioscapulohumeral dystrophy, aberrant fractions appeared mainly in the acidic region. CSF-protein abnormalities were found in both the alkaline fractions (HAFs) with pI 9.2-9.6 and a fraction with PI 7.1 were found in half of the patients with myotonic dystrophy. The CSF electrophoresis in myotonic dystrophy showed increased levels of beta1-globulin in all cases examined. Signs of barrier-damage were commonly encountered in ALS in contrast to the muscular dystrophies, except for myotonic dystrophy. The results are discussed in terms of possible diagnostic value and with regard to pathogenetic significance, particularly in relation to the current hypothesis of a neural involvement in muscular disorders.

Analysis of potential diagnostic biomarkers in cerebrospinal fluid of idiopathic normal pressure hydrocephalus by proteomics.

BACKGROUND: The pathogenesis of idiopathic normal pressure hydrocephalus (INPH) is unknown, and the syndrome of INPH remains a diagnostic and therapeutic challenge. The present study investigated the disease-specific proteins that aid in the diagnosis and treatment of INPH and thus to study their role in the disease process. METHODS: A comparative proteomic analysis was used for clinical screening of cerebrospinal fluid (CSF) proteins in 15 patients with INPH and compared with 12 normal subjects. Furthermore, enzyme linked immunosorbent assay (ELISA) was performed for comparison with CSF proteins between individual INPH patients and controls. RESULTS: Seven proteins and their isoforms, including leucine-rich alpha-2-glycoprotein (LRG), alpha1-antichymotrypsin, apolipoprotein D, apolipoprotein J, haptoglobin alpha1, serum albumin, and alpha-1-microglobulin/bikunin precursor showed significant changes in CSF of INPH patients compared with controls by proteomic analysis. And significant higher CSF levels of LRG in INPH patients compared with controls were found by ELISA. CONCLUSIONS: These results indicate that there are significant differences in the expression of certain proteins in the CSF of patients with INPH and normal subjects. In particular, the CSF level assay of LRG suggests that LRG is a specific biomarker for INPH and has potential use in the diagnosis and indication for CSF shunting.

[Micro-electrophoresis of unconcentrated cerebrospinal fluid (author's transl)]. / Mikroelektrophorese des nicht eingeengten liquor cerebrospinalis.

100 divided samples of cerebrospinal fluid were examined by micro-electrophoresis and conventional paper-electrophoresis. Results of the two tests agreed well for gamma-globulin, albumin, alpha1 and beta fractions, but poorly (because of methodological differences) for pre-albumin, alpha2 and tau fractions.

[The normal range for total protein and protein fractions in the cerebrospinal fluid of children (author's transl)]. / Normalbereich des Gesamteiweisses und der Eiweissfraktionen des Liquor cerebrospinalis bei Kindern

In order to establish normal values, samples of cerebrospinal fluid from 109 healthy children between the ages of 0 and 13 years were analyzed for total protein, different protein fractions after electrophoretic separation, and IgG. Total protein was determined by the biuret method after precipitation with trichloroacetic acid. For the determination of the different protein fractions, the fluid was concentrated by high pressure filtration; the fractions were separated by microzone elektrophoresis on cellulose acetate membranes, then assayed photometrically after staining with amido black B. IgG was determined by radial immunodiffusion.

[Perinatal relation of the blood-cerebrospinal fluid barrier and its effect on protein composition of the cerebrospinal fluid]. / Die perinatalen Verhältnisse der Blut-Liquorschranke und ihr Einfluss auf die Proteinzusammensetzung des Liquors.

The lumbar spinal fluid of 70 infants aged 238-461 days after conception was examined for total protein with the Cu-Folin-differential test, and for variation of single CSF proteins as a function of developmental age using agar gel electrophoresis. In the early stages of development all CSF fractions as well as total protein are increased compared with adult persons. The absolute amounts decrease after birth more or less rapidly (partly exponentially) and reach a minimum between the 52nd and 57th week of development (prealbumin and beta 1-globulin somewhat earlier, albumin somewhat later). Except for beta 1-globulin this minimum is always lower than the normal adult values. There seems to be a linear correlation between the logarithm of the total protein (also of certain single proteins) and the gestational age over a wide range of development. The behaviour of the haptoglobin and immunoglobulin fractions demonstrates especially well that many independent factors may simultaneously influence the concentration of the CSF proteins (e.g. plasma content of proteins, permeability of the blood/brain or blood/CSF barrier) and that these factors themselves depend on both the developmental state (gestational age) and chronological age (birth). This study, in agreement with the results of other authors suggests that in human neonates the reference range of those CSF parameters which depend on barrier conditions should be related to the gestational age rather than to the chronological age.

Low molecular weight plasma proteins in the cerebrospinal fluid of children with hematological malignancies.

The concentration of beta-2-microglobulin (beta 2-m) and of post gamma globulin (P gamma G) was examined in serum and cerebrospinal fluid from children with acute lymphatic leukemia (ALL) and non-Hodgkin's lymphoma (NHL). Data were analysed in order to determine whether concentration of beta 2-m or P gamma G during remission would be of value in predicting relapse or eventual outcome. Mean serum concentration of beta 2-m was similar in good and poor prognosis patients with ALL in remission and was not significantly altered in CNS or marrow relapse. Mean CSF concentration in NHL was also similar in both prognostic groups, and in poor prognosis patients was not significantly altered in relapse. The same pattern was seen when P gamma G was measured in CSF (serum concentration of this protein being too low for accurate determination). High within patient variability of levels of beta 2-m and P gamma G appeared to relate to chemotherapy rather than the disease process. Concentration of P gamma G was persistently raised in three children with brain damage of differing etiologies. Levels of two other low molecular weight proteins, retinol binding protein and alpha 1-microglobulin, were also determined in order to establish that beta 2-m and P gamma G concentration was not influenced by alteration in permeability of the blood-brain barrier. The beta 2-m and P gamma G concentration, although higher than reported in healthy children [5] does not appear to be of value as a prognostic indicator in ALL and NHL in children.

[Cerebrospinal fluid protein electrophoresis in non-inflammatory central nervous system diseases]. / Liquirprotein-Elektrophoresen bei nicht-entzündlichen Erkrankungen des Zentralnervensystems

Cerebrospinal fluids (CSF) (N = 365) from patients with non-inflammatory diseases of the central nervous system were analyzed for protein distribution by agar gel microelectrophoresis. After subdivision into diagnostically well defined groups, these patients were compared with 79 normal controls. Most of the diseases investigated were found to follow the "plasma-type" pattern. In some of them the deviations from normal were so extensive that a CSF-protein pattern similar to the "acute-phase reaction" in the serum occurred. Moreover, the following characteristics were found: a considerable increase in total protein and the gamma-globulin fractions in neurinomas; a reproducible increase in the alpha1-globulins in metastases of the central nervous system; and a statistically significant difference of CSF protein findings between male and female patients with protrusion of lumbar intervertebral discs.

CSF circulation and blood-CSF barrier.

Results of microzone electrophoresis of non-concentrated CSF after staining with nigrosine and after evaluation on non-transparent acetate film are compared with those of isotope cisternography (111In-DTPA). We found that blood-CSF barrier disturbances begin with an increase of the absolute values of prealbumins in normal CSF circulation. When a barrier impairment occurs, by first increasing the absolute values of alpha1-globulins, we state a pathological CSF circulation. In this case, most of the globulin region is pathologic (globulin-type).

The fractionation of cerebrospinal fluid proteins by cellulose acetate electrophoresis in children with infectious diseases of the central nervous system (author's transl). / Die Celluloseacetatfolien-Elektrophoreses der Liquorproteine von Kindern mit entzündlichen Erkrankungen des Zentralnervensystems

The increased permeability of the blood-brain barrier during acute inflammation of the central nervous system leads to changes of the cerebrospinal fluid (C.S.F.) protein pattern. Initially, in the cases of bacterial meningitis, cellulos acetate electrophoresis revealed decreased prealbumin, albumin and tau-globulin fraktion whereas alpha- and gamma-globulin fractions were found increased. In later stages of purulent inflammation a hydrocephalus occurred in five children, associated with an increased amount of albumin in the C.S.F. Cases of viral meningoencephalitis had a characteristic decrease of prealbumin and increase of gamma-globulin, the lowered prealbumin values were found more often. In three cases of congenital encephalitis pathological patterns of C.S.F. proteins were still found 1--1 1/2 years postpartum. Children with acute peripheral facial palsy and febrile convulsions had a normal C.S.F. protein profile.