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Phospho-peptide enrichment was performed using TiO 2 (Titansphere Phos-TiO Bulk 10 um, GL Science), ratio of beads to sample 10:1, mixed for 30 minutes at room temperature in 80% acetonitrile, 6% trifluoroacetic acid, 5 mM monopotassium phosphate, 20 mg/mL 2.5-dihydroxybenzoic acid

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Elevated methylmalonic acid above 0.4 micromoles per liter confirms functional B12 deficiency even when serum levels appear adequate