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. 2020 May 14;9(5):1220. doi: 10.3390/cells9051220

Figure 1.

Figure 1

Identification of an O-glucosylated peptide from EGF2 from NOTCH1 by LC-MS/MS. NOTCH1 EGF1-18 protein was produced in the wild type HEK293T cells and purified from the culture medium, as described in the Materials and Methods. The protein was reduced, alkylated, and purified by SDS-PAGE, and subjected to in-gel trypsin digestion. The resulting peptides were analyzed by LC-MS/MS, as described in the Materials and Methods. (A) The top panel show an MS spectrum of 23.8 to 24.0 min. The bottom panel shows the MS/MS spectrum of the glycopeptide from EGF2 modified with a Xyl-Xyl-Glc trisaccharide. The red diamond denotes the parental ion. Numerous b- and y-ions confirmed the identity of the peptide 57-CQDSNPCLSTPCK-69 from NOTCH1. (B) Extracted ion chromatograms (EICs) searched for the glycopeptides modified with different glycoforms are shown. (C) Quantification was performed using the height of the EICs of the detected ions and the total amount of detected (glyco)peptides with different glycoforms set at 100% (n = 3). Error bar shows standard error of the mean. Black bar—naked peptide; blue circle—glucose; orange star—xylose.