96 well vacuum manifold

N-Glycosidase F (PNGase F) was purchased from New England Biolabs (Ipswich, MA). Neuraminidase, sodium hydroxide, methyl iodide, β-mercaptoethanol, chloroform, dimethyl sulfoxide (DMSO), HPLC-grade acetonitrile (ACN), and water and the 96-well vacuum manifold were purchased from Sigma-Aldrich (St. Louis, MO). The MALDI matrix, 2,5-dihydroxybenzoic acid (2,5-DHB), the UltraLink hydrazide resin, the Zeba Spin Desalting column, and the SPE 96-well plate packed with 100% porous graphitic carbon (PGC) were purchased from Thermo Scientific (Rockford, IL). The 96-well SpinColumn plate was purchased from Harvard Apparatus (Holliston, MA). Antihuman haptoglobin antibody (CatLog No. ab13429) and a human haptoglobin standard were purchased from Abcam (Cambridge, MA). The empty PEEK column was purchased from MicroSolv Technology Corp. (Eatontown, NJ).

Subsequently the N-glycans were permethylated using a solid-phase permethylation method^{10 (link)} on a 96-well SpinColumn plate^{6 (link)} with 5 μm frit (Harvard Apparatus, Holliston, MA). In brief, NaOH beads (20–40 mesh) that were prepared in ACN were packed into the 96-well SpinColumn plate to ~1 cm depth.^{10 (link)} The well was then washed three times with 200 μL of DMSO and kept in DMSO prior to applying the samples. The sample was suspended in 70.8 μL of DMSO, 26.4 μL of iodomethane (CH₃I), and 2.8 μL of water, where the ratios were optimized according to the procedure of Kang.^{10 (link)} After discarding the DMSO from the NaOH packed 96-well SpinColumns, the sample was applied through the NaOH beads and recycled for at least 8 times to minimize the sample loss. Subsequently, the permethylated sample was eluted into a 96-well collection plate by adding 100 μL of ACN. The eluent was collected by suction using a 96-well vacuum manifold (Sigma) and then transferred to an eppendorf tube. The permethylated glycans were extracted with 200 μL of chloroform and washed with 200 μL of water for four times. Finally, the permethylated N-glycans were dried under a SpeedVac concentrator (Labconco).