Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase.
An important class of carbohydrates studied within the field of glycobiology, heparin and heparan sulfate (HS) have been implicated in a diverse array of biological functions. Changes in their sulfation pattern and domain organization have been associated with different pathological situations such as viral infectivity, tumor growth, and metastasis. To obtain structural information about these biomolecules, and the modifications they may undergo during different stages of cell growth and development, a mass spectrometry-based method was developed and used to obtain unambiguous structural information on the glycosaminoglycans (GAGs) that comprise heparin/HS. The method was applied to assay for the heparin substrate specificity of a newly discovered human extracellular endosulfatase, HSulf-2, which has been implicated in tumorigenesis. This new protocol incorporates 12 known heparin disaccharides, including three sets of isomers. A unique response factor (R) is determined for each disaccharide, whereas a multiplexed and data processing method is incorporated for faster data acquisition and quantification purposes. Proof of principle was performed by using various heparin/HS samples isolated from bovine and porcine tissues.
Saad
Ola M
OM
Department of Chemistry, University of California, Berkeley, CA 94720, USA.
Ebel
Heiner
H
Uchimura
Kenji
K
Rosen
Steven D
SD
Bertozzi
Carolyn R
CR
Leary
Julie A
JA
D000818:Animals; D001681:Biological Assay; D001943:Breast Neoplasms / Q000201:enzymology; D002417:Cattle; D004187:Disaccharides / Q000032:analysis*; D006493:Heparin / Q000031:analogs & derivatives* / Q000032:analysis; D006497:Heparan Sulfate / Q000737:chemistry*; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism; D007668:Kidney / Q000378:metabolism; D008168:Lung / Q000378:metabolism; D011485:Protein Binding; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013429:Sulfatases / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D004187:Disaccharides; D006493:Heparin; D006497:Heparan Sulfate; D013429:Sulfatases; C464536:heparin disaccharide