This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Introduction: Heparan sulfate (HS) glycosaminoglycans have been implicated in a host of biological functions. To better understand their biological roles, it is necessary to gain understanding about the structure of HS. Methods involving enzymatic digestion have been developed to determine the type and location of sulfation/acetylation modifications. However, the generated disaccharides have ?-unsaturation at the non-reducing end, thus, erasing the original epimeric nature of the hexuronic acid. Hydrazinolysis and nitrous acid treatment is a classic method for GAG depolymerization that retains uronic acid epimerization. To date, it has not been combined with mass spectrometric detection. This work shows combined LC-MS and LC-MS/MS for analysis of HONO generated disaccharides to obtain information about the sulfation pattern and uronic acid epimerization. Methods: 15 ?g of intact heparosan (Hen) and HS were deacetylated by adding 70% hydrazine containing 1% hydrazine sulfate. The mixture was heated, cooled, evaporated to dryness, and desalted. For the Hen, pH 4 HONO reagent was added and pH was adjusted to 8.5 after 15 minutes. For the HS, cold pH 1.5 HONO reagent was added, pH adjusted to 4 after 10 minutes, followed by nitrous acid (HONO) treatment at pH 4. The heparin (Hep) sample was subjected to HONO treatment at pH 1.5. The resulting disaccharides were reduced with NaBH4 and then analyzed using size exclusion chromatography (SEC) LC-MS and LC-MS/MS. Preliminary Data: In most studies, disaccharide analysis of HS was performed by means of enzymatic digestion with heparin lyases. The lyase catalyzed cleavage generates a ?-4,5-HexA residue at the nonreducing terminus, resulting in planarization of the C5 carbon of the uronic acid. An alternative is the use of hydrazinolysis with nitrous acid (HONO) treatment at pH 1.5 and 4 to generate disaccharide units composed of uronic acid (IdoA or GlcA) and 2,5-anhydromannose (AnMan) bearing an aldehyde group. The first set of experiments involved determining the optimum conditions for hydrazinolysis and nitrous acid treatment. Various amounts of hydrazine solution and heating times were tested. We found that hydrazinolysis with 20 ?L of hydrazine solution at 96 oC for 4 hours yielded almost complete deacetylation. After HONO treatment, the disaccharides were reduced with 0.5 M NaBH4 for 1 hour at 37 oC followed by SEC-MS analysis. The compositions of the disaccharides of Hep and HS from porcine intestinal mucosa, and Hen from E.coli K5 bacterial culture obtained using the chemical method were found to be comparable to those obtained by the enzymatic method, showing that the hydrazinolysis and HONO treatment provide reliable information about disaccharide composition. We also found that Hen and Hep contain at least 95% GlcA and 70% IdoA, respectively. Tandem MS of disaccharides obtained from these two samples generated cross-ring cleavages as well as loss of sulfate groups. However, the differences observed in the tandem MS spectra were due to the sulfate position rather than the identity of the hexuronic acid. These results demonstrate the efficacy of LC/MS for analysis of HONO generated GAG disaccharides. It is likely that use of higher resolution chromatography mode with the LC/MS system will allow direct separation of disaccharide epimers to facilitate their identification.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR010888-15
Application #
8365497
Study Section
Special Emphasis Panel (ZRG1-BCMB-H (40))
Project Start
2011-06-01
Project End
2012-08-09
Budget Start
2011-06-01
Budget End
2012-08-31
Support Year
15
Fiscal Year
2011
Total Cost
$27,680
Indirect Cost
Name
Boston University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
604483045
City
Boston
State
MA
Country
United States
Zip Code
02118
Hu, Han; Khatri, Kshitij; Zaia, Joseph (2017) Algorithms and design strategies towards automated glycoproteomics analysis. Mass Spectrom Rev 36:475-498
Sethi, Manveen K; Zaia, Joseph (2017) Extracellular matrix proteomics in schizophrenia and Alzheimer's disease. Anal Bioanal Chem 409:379-394
Hu, Han; Khatri, Kshitij; Klein, Joshua et al. (2016) A review of methods for interpretation of glycopeptide tandem mass spectral data. Glycoconj J 33:285-96
Ji, Yuhuan; Bachschmid, Markus M; Costello, Catherine E et al. (2016) S- to N-Palmitoyl Transfer During Proteomic Sample Preparation. J Am Soc Mass Spectrom 27:677-85
Pu, Yi; Ridgeway, Mark E; Glaskin, Rebecca S et al. (2016) Separation and Identification of Isomeric Glycans by Selected Accumulation-Trapped Ion Mobility Spectrometry-Electron Activated Dissociation Tandem Mass Spectrometry. Anal Chem 88:3440-3
Wang, Yun Hwa Walter; Meyer, Rosana D; Bondzie, Philip A et al. (2016) IGPR-1 Is Required for Endothelial Cell-Cell Adhesion and Barrier Function. J Mol Biol 428:5019-5033
Srinivasan, Srimathi; Chitalia, Vipul; Meyer, Rosana D et al. (2015) Hypoxia-induced expression of phosducin-like 3 regulates expression of VEGFR-2 and promotes angiogenesis. Angiogenesis 18:449-62
Yu, Xiang; Sargaeva, Nadezda P; Thompson, Christopher J et al. (2015) In-Source Decay Characterization of Isoaspartate and ?-Peptides. Int J Mass Spectrom 390:101-109
Steinhorn, Benjamin S; Loscalzo, Joseph; Michel, Thomas (2015) Nitroglycerin and Nitric Oxide--A Rondo of Themes in Cardiovascular Therapeutics. N Engl J Med 373:277-80
Walsh, Erica M; Niu, MengMeng; Bergholz, Johann et al. (2015) Nutlin-3 down-regulates retinoblastoma protein expression and inhibits muscle cell differentiation. Biochem Biophys Res Commun 461:293-9

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