Bioorthogonal Labeling of Human Prostate Cancer Tissue Slice Cultures for Glycoproteomics

Abstract

Sialylated glycans are found at elevated levels in many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to the cancer cell-surface sialylation are not well characterized, specifically in bona fide human disease tissue. Metabolic and bioorthogonal labeling methods have previously enabled the enrichment and identification of sialoglycoproteins from cultured cells and model organisms. Herein, we report the first application of this glycoproteomic platform to human tissues cultured ex vivo. Both normal and cancerous prostate tissues were sliced and cultured in the presence of the azide-functionalized sialic acid biosynthetic precursor Ac4ManNAz. The compound was metabolized to the azidosialic acid and incorporated into cell surface and secreted sialoglycoproteins. Chemical biotinylation followed by enrichment and mass spectrometry led to the identification of glycoproteins that were found at elevated levels or uniquely in cancerous prostate tissue. This work therefore extends the use of bioorthogonal labeling strategies to problems of clinical relevance.

Membrane glycomics reveal heterogeneity and quantitative distribution of cell surface sialylation

“…In parallel with metabolic labeling, advanced analytical tools are emerging to enable full characterization of unnaturally glycosylated proteins using isotopic labeling or chemical ligation followed by affinity purification. 19 – 21 Here, we present a platform to comprehensively survey the structures of modified glycans on the cell membrane in addition to their associated glycoproteins and glycosphingolipids with minimal sample manipulation. In our approach, the protein-, site-, glycan-, and lipid-specific locations of azido sialic acids were probed by using the monosaccharide as a mass label, removing the need for additional conjugation and capture steps.…”

Global and site‐specific analysis of protein glycosylation in complex biological systems with Mass Spectrometry

“…The combination of metabolic labeling and click chemistry has emerged as a powerful method to study proteins and protein modifications (Mahal et al, 1997;Hang et al, 2003;Bond and Kohler, 2007;Dieterich et al, 2007;Hanson et al, 2007;Hong et al, 2009;Smeekens et al, 2015;Cheng et al, 2016;Spiciarich et al, 2017;Xiao and Wu, 2017a;Xiao et al, 2018b). Metabolic labeling of glycans with unnatural sugar analogs is an excellent way to study glycans and protein glycosylation (Kayser et al, 1992;Saxon and Bertozzi, 2000;Feng et al, 2013;Aguilar et al, 2017aAguilar et al, , 2017bPark et al, 2018;Qin et al, 2018).…”

Gewinner der Emanuel‐Merck‐Vorlesung und des Heinrich‐Emanuel‐Merck‐Preises

“…Nach einem Postdoktorat bei Steven Rosen an der UC San Francisco wechselte sie im Jahr 1996 an die UC Berkeley und von dort aus im Jahr 2015 an die Stanford University. Kürzlich hat sie in der Angewandten Chemie über die bioorthogonale Markierung von Prostatakrebszellen in Gewebeschnittkulturen berichtet . Bertozzi gehörte von 2010 bis 2015 dem International Advisory Board der Angewandten Chemie an.…”