sepharose and azidohomoalanine

Nascent proteome presents dynamic changes in response to a certain stimulus. Thus, monitoring nascent proteome is critical to uncovering the involved biological mechanism. But the low-abundance of nascent proteome against an overwhelming pre-existing proteome limits its identification and quantification. Herein, we present a novel strategy to enrich nascent proteome from whole cell lysate for further analysis by mass spectrometry. We employed a terminal alkyne and disulfide functionalized agarose resin to capture nascent proteome which had been labeled by L-azidohomoalanine. Results from the western blot, silver staining and pulse metabolic labeling suggested that the nascent proteome could be enriched efficiently. Applied to Hela cells, the method identified about 700 nascent proteins with good correlation with previous reports. The above indicates that our strategy can be used to reveal the proteome dynamics of biological processes.

Topics: Alanine; Alkynes; Chromatography, Liquid; Click Chemistry; Disulfides; HeLa Cells; Humans; Proteins; Proteome; Sepharose; Tandem Mass Spectrometry