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Figure 1 | Clinical Proteomics

Figure 1

From: Identification of sialylated glycoproteins from metabolically oligosaccharide engineered pancreatic cells

Figure 1

Strategy for analyzing azide-modified sialoglycoproteins. The strategy used to analyze the samples includes multiple steps as follows: 1) Cells were metabolically labeled using 1,3,4-O-Bu3ManNAz. 2) Proteins were extracted using RIPA buffer at which point samples were divided with one set of aliquots used for steps 4 and 5 and another set of aliquots used for steps 6 through 9. 3) Azide-labeled proteins were biotinylated using through the Staudinger reaction using biotin-PEG3-phosphine and excess reagent was removed by protein precipitation. 4) Biotinlabeled, azide-modified proteins were purified using monomeric avidin agarose. 5) Glycan profiles of biotin-labeled, azide-modified proteins were determined by lectin microarray analysis. 6) Proteins were trypsin digested after biotinylation. 7) Biotin-labeled peptides were coupled to streptavidin agarose. 8) PNGase F was used to release the formerly N-glycosylated peptide from the agarose beads. 9) The released peptides were analyzed by LC-MS.

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