Updated project metadata. The O-glycosyltransferase LARGE2 attaches a laminin-binding matriglycan structure on alpha-Dystroglycan (α-DG) to mediate the interaction of different cell types with their laminin-containing basement membrane. We used HT-29 CRC cells, characterized by low endogenous LARGE2 gene expression, to ectopically express LARGE2 and to drive O glycosylation of its protein substrates. Immunoblot analyses had revealed that the addition of matriglycan-structures on LARGE2 substrate proteins substantially increased their molecular weight (MW). In order to take into account this shift in MW of candidate proteins, glycoprotein-enriched fractions from HT-29 cells with or without ectopic LARGE2 expression were separated by gel electrophoresis and subsequently fractionated in 6 different molecular weight windows (kDa: 0-25, 25-55, 55-75, 75-110, 110-160, 160<). Subsequently, samples were prepared for quantitative mass spectrometry analysis (qLC-MS/MS) analysis according to standard procedures. qLC-MS-MS analysis identified α-DG–related peptides within higher MW protein fractions only when LARGE2 was overexpressed. HT-29 cell secreted Laminin-2 was exclusively detected together with these high MW forms of α-DG, which demonstrated the affinity of Laminin towards the LARGE2-synthesized matriglycan structure on α-DG. Additional candidate proteins identified in this study need further experimental confirmation. Overall, we show that size fractionation after gel electrophoresis followed by quantitative mass spectrometry, performed separately on different MW windows, represents an elegant approach to reveal potential substrates of the O-glycosyltransferase LARGE2.