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We developed a method that uses lectin affinity chromatography to enrich glycosylated proteins where the modification terminates with N-acetyl-D-glucosamine (GlcNAc), and used this method in conjunction with ETD MS/MS to identify Arabidopsis proteins with this type of modification. We found forty-eight proteins with N-linked modification consisting of single GlcNAc attached by an N-linkage to the Asn of the Asn-X-Ser/Thr sequon that is commonly modified with larger N-linked oligosaccharides. An enzymatic and chemical labeling method that changes the mobility of GlcNAc modified protein during PAGE through addition of PEG was employed to investigate the origin N-GlcNAc modifications on the β-thioglucoside glucohydrolases (TGG1 and TGG2), which are both modified with multiple N-linked oligosaccharides. It has been hypothesized that N-GlcNAc modifications are produced when ENGase remove N-linked oligosaccharides. Arabidopsis has two ENGases (AtENGase85A and B). Through the analysis of AtENGase85A, AtENGase85B single and AtENGase85A/B double mutants, we discovered that the N-GlcNAc modification of TGG2 was greatly reduced by AtENGase85A, but TGG1 was not affected in any of the mutants. These results support that ENGase producing N-GlcNAc modifications by hydrolyze N-linked oligosaccharides but also suggest that some Arabidopsis N-GlcNAc modifications may be generated by another mechanism. Since N-GlcNAc modification was detected at only one site on each myrosinase, the production of the N-GlcNAc modification may be regulated.