Obtained trypsin digests of IgA samples were enriched for glycopeptides by a two-step microtip cotton HILIC SPE, using cotton thread as the solid phase, as described before38 (link), with minor modifications in the used solution volumes and composition. Briefly, 15 μL of the digest was transferred to a 96 wells V-bottom plate, and adjusted to 70% ACN by addition of 35 μL ACN. The cotton thread was washed three times with 20 μL MQ, and conditioned three times with 20 μL 70% ACN, using a 12-channel micropipette (2–20 μL). The sample was then loaded on the cotton by pipetting up-and-down 20 times. Subsequently, the cotton was washed 3 times with 20 μL 70% ACN containing 1% TFA and three washes with 20 μL 70% ACN, followed by elution in 5 μL MQ. Twenty-five microliter ACN was then added to the tryptic digest to bring the sample to 80% ACN for a sequential HILIC enrichment with 80% instead of 70% ACN, to capture glycopeptides that were not detectable in the 70% HILIC eluates. The eluates from both HILIC experiments were mixed separately with 15 μL 4-chloro-α-cyanocinnamic acid matrix (Cl-CCA; 0.33 mg/mL in acetone:ethanol 1:2) and 1 μL of each eluate was spotted on a Bruker AnchorChip plate with 800 μm anchors. All glycopeptide samples were spotted in duplicate, resulting in 384 spots per 96 serum samples.
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