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Isolation of secreted glycoproteins using sugar‐azide labeling and alkyne‐bead capture
Author(s) -
Drake Richard R,
Roper Stephen,
Jones Ellen,
Kraft Andrew S,
Zemskova Marina
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.lb231
Subject(s) - glycoprotein , chemistry , biochemistry , sialic acid , secretory protein , cytoskeleton , secretion , extracellular matrix , cytosol , cell culture , microbiology and biotechnology , biology , cell , enzyme , genetics
Proteomic methods used to characterize proteins present in the secretome of cultured cells frequently identify many non‐glycosylated cytosolic and cytoskeletal proteins. To address this, metabolic labeling was done in a prostate stroma cell line YPMI‐1 with an azide‐sugar analog of sialic acid, N‐acetylamannosamineazide (ManNAcAz), that is incorporated into secreted glycoproteins. Serum‐free media from cells with and without ManNAcAz labeling was collected after 24 hrs. The ManNAcAz‐treated media was incubated with alkyne beads and copper catalyst for crosslinking to glycoproteins. Bead‐bound proteins and conditioned secretome media were digested with trypsin, and proteins identified using electrospray tandem mass spectrometry. Over 400 proteins were detected in the secretome media, while 75 proteins were captured in ManNAcAz‐labeled media. The majority (>80%) of the identified proteins in the secretome media were cytosolic or cytoskeletal in origin and non‐glycosylated, while >90% of the ManNAcAz proteins were associated with secretion, extracellular matrix, adhesion or ligand/receptor functions. One protein detected was TGF‐beta2, which by ELISA was determined to be present at 5–10 ng/ml. This illustrates the depth and selectivity of detection provided by the azide‐alkyne capture method, and the method can readily be applied to many cell systems.