Enrichment and quantitation of newly secreted proteins under serum condition by combining BONCAT and pulsed SILAC

ABSTRACT: Despite the increasing interest of secretome involved in paracrine/autocrine mechanisms, the majority of the mass spectrometric cell secretome studies have been performed using serum-free medium (SFM), due to low-abundance of secreted proteins and high-abundance of contaminating fetal bovine serum (FBS) proteins in serum-containing medium (SCM). In this study, through the combination of bioorthogonal non-canonical amino acid tagging (BONCAT) and pulsed-SILAC (pSILAC), the low-abundant secreted proteins were captured selectively and quantified. We set up the combined BONCAT-pSILAC method by optimizing capturing condition and by using a composite human-FBS database, and applied it to the analysis of differentially secreted proteins between SFM and SCM. The amount of secreted proteins for 24 h in SCM was much bigger than that from SFM for U-87MG glioblastoma cells (1.28-fold) and mesenchymal stem cells from human umbilical cord blood (hUCB-MSCs; 2.01-fold). The proteins secreted more in SCM were predominantly predicted to be truly secretory. Therefore, this study suggests the analysis of the secretome should be processed in serum-containing medium that promotes cell proliferation and secretion.

INSTRUMENT(S): LTQ, Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Jihye Shin  

LAB HEAD: Cheolju Lee

PROVIDER: PXD007529 | Pride | 2019-03-13

REPOSITORIES: Pride