Cobalt loaded resin

HRG was isolated from human serum using immobilized metal affinity chromatography (IMAC) using a cobalt-loaded resin (Thermo Scientific). Briefly, cobalt slurries were washed with 3 × binding buffer (supplemental Table S1). A volume of 50 μl of beads was incubated on a tube revolver with 100 μl serum diluted with 1000 μl binding buffer for 3 h at 4 °C. Then the beads were subsequently washed with washing buffer 1 to 7 (supplemental Table S1). The purified HRG protein was eluted with eluting buffer twice (supplemental Table S1). The supernatant was combined and vacuum dried. Imidazole, which was used for the buffer, was purchased from Sigma-Aldrich.

Human plasma containing EDTA from a single healthy individual was obtained from Zen-Bio. One recombinant HRG variant, containing the mutations Pro204 and Asn493, was produced in CHO cell lines by PeproTech. Four other HRG variants, with pairwise combinations of targeted mutations for positions 204 and 493, were produced in HEK293E-253 cells (HEK293) by ImmunoPrecise. HRGs were expressed with sequences matching allelic variants on position 204 or 493 in human population: Pro204/Asn493, Pro204/Ile493, Ser204/Ile493, and Ser204/Asn493.
Cobalt-loaded resin was acquired from Thermo Scientific. Imidazole was purchased from Sigma–Aldrich. Tris–HCl, Tris(2-carboxyethyl)phosphine, chloroacetamide, sodium deoxycholate (SDC), and TFA were purchased from Sigma–Aldrich. Oasis HLB 96-well μElution Plates were purchased from Waters. Sequencing-grade trypsin was obtained from Promega. SialEXO and OpeRATOR were obtained from Genovis. Plasmin was purchased from Abcam. Formic acid (FA) and acetonitrile were acquired from Biosolve Chimie. The SDS-PAGE standards and Imperial Protein Stain were purchased from Bio-Rad and Thermo Scientific, respectively.

Sodium deoxycholate (SDC), Tris(2-carboxyethyl)phosphine (TCEP), Tris(hydroxymethyl)aminomethane, chloroacetamide (CAA), sodium hydroxide, and TFA were purchased from Sigma–Aldrich. Formic acid (FA) was purchased from Merck. Acetonitrile (ACN) was purchased from Biosolve. Oasis μElution HLB and PRiME HLB plates were purchased from Waters. Milli-Q was produced by an in-house system (Millipore). Both phosphoSTOP and cOmplete Mini EDTA-free were purchased from Roche. GluC was obtained from Roche. Recombinant tissue nonspecific alkaline phosphatase (TNAP) was a gift from Copenhagen Centre of Glycomics. Histidine-rich glycoprotein (HRG) was purified from human plasma with a cobalt-loaded resin (Thermo Scientific) using immobilized metal affinity chromatography–based enrichment (34 (link)). Commercial sialylglycopeptide (α2,6-SGP) and asialo-SGP were purchased from Fushimi Pharmaceutical Co, Ltd. Commercial pooled human plasma was purchased from Affinity Biologicals. Purified human neutrophils, prepared as described previously (35 (link)), were a kind gift from the Department of Molecular and Cellular Homeostasis, Sanquin Research.