Mass Spectral Profiling of Glycosaminoglycans from Histological Tissue Surfaces

Glycosaminoglycans (GAGs) are found in intracellular granules, cell surfaces, and extracellular matrices in a spatially and temporally regulated fashion, constituting the environment for cells to interact, migrate, and proliferate. Through binding with a great number of proteins, GAGs regulate many facets of biological processes from embryonic development to normal physiological functions. GAGs have been shown to be involved in pathologic changes and immunological responses including cancer metastasis and inflammation. Past analyses of GAGs have focused on cell lines, body fluids, and relatively large tissue samples. Structures determined from such samples reflect the heterogeneity of the cell types present. To gain an understanding of the roles played by GAG expression during pathogenesis, it is very important to be able to detect and profile GAGs at the histological scale so as to minimize cell heterogeneity to potentially inform diagnosis and prognosis. Heparan sulfate (HS) belongs to one major class of GAGs, characterized by dramatic structural heterogeneity and complexity. To demonstrate feasibility of analysis of HS, 15 μm frozen bovine brain stem, cortex, and cerebellum tissue sections were washed with a series of solvent solutions to remove lipids before applying heparin lyases I, II, and III on the tissue surfaces within 5 mm × 5 mm digestion spots. The digested HS disaccharides were extracted from tissue surfaces and then analyzed by using size exclusion chromatography/mass spectrometry (SEC-MS). The results from bovine brain stem, cortex, and cerebellum demonstrated the reproducibility and reliability of our profiling method. We applied our method to detect HS from human astrocytoma (WHO grade II) and glioblastoma (GBM, WHO grade IV) frozen slides. Higher HS abundances and lower average sulfation level of HS were detected in glioblastoma (GBM, WHO grade IV) slides compared to astrocytoma (WHO grade II) slides

Mass Spectrometric Method for Determining the Uronic Acid Epimerization in Heparan Sulfate Disaccharides Generated Using Nitrous Acid

Heparan sulfate (HS) glycosaminoglycans (GAGs) regulate a host of biological functions. To better understand their biological roles, it is necessary to gain understanding about the structure of HS, which requires identification of the sulfation pattern as well as the uronic acid epimerization. In order to model HS structure, it is necessary to quantitatively profile depolymerization products. To date, liquid chromatography–mass spectrometry (LC-MS) methods for profiling heparin lyase decomposition products have been shown. These enzymes, however, destroy information about uronic acid epimerization. Deaminative cleavage using nitrous acid (HONO) is a classic method for GAG depolymerization that retains uronic acid epimerization. Several chromatographic methods have been used for analysis of deaminative cleavage products. The chromatographic methods have the disadvantage that there is no direct readout on the structures producing the observed peaks. This report demonstrates a porous graphitized carbon (PGC)-MS method for the quantification of HONO generated disaccharides to obtain information about the sulfation pattern and uronic acid epimerization. Here, we demonstrate the separation and identification of uronic acid epimers as well as geometric sulfation isomers. The results are comparable to those expected for benchmark HS and heparin samples. The data demonstrate the utility of PGC-MS for quantification of HS nitrous acid depolymerization products for structural analysis of HS and heparin

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Characterization of the Complete Mitochondrial Genome Sequence of the Globose Head Whiptail <i>Cetonurus globiceps</i> (Gadiformes: Macrouridae) and Its Phylogenetic Analysis

<div><p>The particular environmental characteristics of deep water such as its immense scale and high pressure systems, presents technological problems that have prevented research to broaden our knowledge of deep-sea fish. Here, we described the mitogenome sequence of a deep-sea fish, <i>Cetonurus globiceps</i>. The genome is 17,137 bp in length, with a standard set of 22 transfer RNA genes (tRNAs), two ribosomal RNA genes, 13 protein-coding genes, and two typical non-coding control regions. Additionally, a 70bp tRNA^Thr-tRNA^Pro intergenic spacer is present. The <i>C</i>. <i>globiceps</i> mitogenome exhibited strand-specific asymmetry in nucleotide composition. The AT-skew and GC-skew values in the whole genome of <i>C</i>. <i>globiceps</i> were 0 and -0.2877, respectively, revealing that the H-strand had equal amounts of A and T and that the overall nucleotide composition was C skewed. All of the tRNA genes could be folded into cloverleaf secondary structures, while the secondary structure of tRNA^Ser(AGY) lacked a discernible dihydrouridine stem. By comparing this genome sequence with the recognition sites in teleost species, several conserved sequence blocks were identified in the control region. However, the GTGGG-box, the typical characteristic of conserved sequence block E (CSB-E), was absent. Notably, tandem repeats were identified in the 3' portion of the control region. No similar repetitive motifs are present in most of other gadiform species. Phylogenetic analysis based on 12 protein coding genes provided strong support that <i>C</i>. <i>globiceps</i> was the most derived in the clade. Some relationships however, are in contrast with those presented in previous studies. This study enriches our knowledge of mitogenomes of the genus <i>Cetonurus</i> and provides valuable information on the evolution of Macrouridae mtDNA and deep-sea fish.</p></div

Phylogenetic tree of Gadiform species reconstructed from concatenated DNA sequences of mitochondrial protein-coding genes.

<p>Twelve mitochondrial protein-coding genes (with the exception of <i>ND6</i>) were used for the phylogenetic tree, which was produced by Bayesian inferences (BI). <i>Sardinops melanostictus</i> was used as the outgroup. Bayesian posterior probability are shown orderly on the nodes. The asterisk indicates the sequence generated in this study.</p

Overall mean p-genetic distance of six Macrouridae species for each of 13 protein genes.

<p>They were calculated based on the first and second nucleotide positions and on the third nucleotide position of amino acid codons, and on the full sequence among six Macrouridae species, respectively.</p

Characteristic constituents of the mitochondrial genome of <i>C</i>. <i>globiceps</i>.

<p>Characteristic constituents of the mitochondrial genome of <i>C</i>. <i>globiceps</i>.</p

Intergenic T-P spacer sequence of five Gadiform species.

<p>Gm: <i>Gadus morhua</i>, Tc: <i>Theragra chalcogramma</i>, Bs: <i>Boreogadus saida</i>, Cg: <i>Cetonurus globiceps</i>, Vg: <i>Ventrifossa garmani</i>. Dots indicate identical positions and dashes indicate deletions. Conserved regions and tRNAs are marked by boxes.</p

Evolutionary rates of <i>C</i>. <i>globiceps</i> mitogenome.

<p>The rate of non-synonymous substitutions (Ka), the rate of synonymous substitutions (Ks) and the ratio of the rate of non-synonymous substitutions to the rate of synonymous substitutions (Ka/Ks) for each protein-coding gene.</p

Secondary structure prediction for the T-P spacer DNA sequence of <i>C</i>. <i>globiceps</i>.

<p>Secondary structure prediction for the T-P spacer DNA sequence of <i>C</i>. <i>globiceps</i>.</p