Trypsin-Mediated 18O/16O Labeling for Biomarker Discovery View Full Text


Ontology type: schema:Chapter     


Chapter Info

DATE

2013

AUTHORS

Xiaoying Ye , C. Chan , DaRue A. Prieto , Brian T. Luke , Donald J. Johann , Luke H. Stockwin , Dianne L. Newton , Josip Blonder

ABSTRACT

Differential (18)O/(16)O stable isotopic labeling that relies on post-digestion (18)O exchange is a simple and efficient method for the relative quantitation of proteins in complex mixtures. This method incorporates two (18)O atoms onto the C-termini of proteolytic peptides resulting in a 4 Da mass-tag difference between (18)O- and (16)O-labeled peptides. This allows for wide-range relative quantitation of proteins in complex mixtures using shotgun proteomics. Because of minimal sample consumption and unrestricted peptide tagging, the post-digestion (18)O exchange is suitable for labeling of low-abundance membrane proteins enriched from cancer cell lines or clinical specimens, including tissues and body fluids. This chapter describes a protocol that applies post-digestion (18)O labeling to elucidate putative endogenous tumor hypoxia markers in the plasma membrane fraction enriched from a hypoxia-adapted malignant melanoma cell line. Plasma membrane proteins from hypoxic and normoxic cells were differentially tagged using (18)O/(16)O stable isotopic labeling. The initial tryptic digestion and solubilization of membrane proteins were carried out in a buffer containing 60 % methanol followed by post-digestion (18)O exchange/labeling in buffered 20 % methanol. The differentially labeled peptides were mixed in a 1:1 ratio and fractionated using off-line strong cation exchange (SCX) liquid chromatography followed by on-line reversed-phase nano-flow RPLC-MS identification and quantitation of peptides/proteins in respective SCX fractions. The present protocol illustrates the utility of (18)O/(16)O stable isotope labeling in the context of quantitative shotgun proteomics that provides a basis for the discovery of hypoxia-induced membrane protein markers in malignant melanoma cell lines. More... »

PAGES

133-49

Identifiers

URI

http://scigraph.springernature.com/pub.10.1007/978-1-62703-360-2_12

DOI

http://dx.doi.org/10.1007/978-1-62703-360-2_12

DIMENSIONS

https://app.dimensions.ai/details/publication/pub.1016259937

PUBMED

https://www.ncbi.nlm.nih.gov/pubmed/23625401


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