Deae sepharose fast flow resin

An enoxaparin reference standard was purchased from SANDOZ. 2-Aminoacridone (AMAC) and sodium cyanoborohydride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Three ¹³C-labeled 8-mer calibrants were synthesized using the chemoenzymatic synthesis method.^{10 (link)} Recombinant heparan lyase I, II, and III were expressed in E. coli and purified by a Ni-agarose column. DEAE-Sepharose Fast Flow resin was purchased from GE Healthcare (Chicago, IL, USA). All reagents and chemicals were high-performance LC (HPLC) grade or LC-mass spectrum (MS) grade.

Chemicals, biochemicals, and buffers were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO), Fisher Scientific, Inc. (Pittsburgh, PA), EMD Millipore, Inc. (Billerica, MA) or Bio-Rad Laboratories, Inc. (Hercules, CA), unless otherwise noted below. Phenylenolpyruvate (PP) was purchased from Fluka Chemical Corp. (Milwaukee, WI) as the crystalline free acid, which exists in the enol form. 2-Oxo-3-pentynoate (2-OP) was synthesized, as described elsewhere.^{21 (link)} The compounds in the substrate library were obtained from sources reported elsewhere.²³ The DEAE-Sepharose fast flow resin and the PD-10 Sephadex G-25M buffer-exchange columns were obtained from GE Healthcare Bio-Sciences (Pittsburgh, PA). The Econo-Column chromatography columns (used for the DEAE-Sepharose columns) were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). The Amicon stirred cell concentrators and the ultrafiltration membranes (10000 Da, MW cutoff) were purchased from EMD Millipore Inc. (Billerica, MA). InstantBlue for staining sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels was obtained from C.B.S. Scientific Company, Inc. (Del Mar, CA).

2-Aminoacridone (AMAC) and sodium cyanoborohydride were purchased from Sigma-Aldrich (St. Louis, MO, USA). Eight ¹³C-labeled heparan sulfate disaccharide internal standards and ¹³C-labeled polysaccharide (NSK5P) were synthesized as described previously.^{8 (link)} Eight unlabeled native disaccharide standards were purchased from Iduron (Manchester, UK). Recombinant heparan lyase I, II, and III were expressed in E. coli and purified by a Ni–agarose column. DEAE-Sepharose Fast Flow resin was obtained from GE Healthcare (Chicago, IL, USA). Formalin-fixed paraffin-embedded (FFPE) mice tissue slides sectioned in the coronal plane at 5 μm thickness were prepared at the core facility of the University of North Carolina at Chapel Hill (Chapel Hill, NC, USA). Each slide contains 2–3 sections. A Pierce BCA Protein Assay Kit was purchased from Thermo Fisher Scientific (Waltham, MA USA). All reagents and chemicals were LC grade or LC-MS grade.