title: Trypsin-mediated ¹⁸O/¹⁶O labeling for biomarker discovery.
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.
authors.affiliation: Frederick National Laboratory for Cancer Research, Frederick, MD, USA.
authors.forename: Xiaoying
authors.initials: X
authors.lastname: Ye
authors.forename: King C
authors.initials: KC
authors.lastname: Chan
authors.forename: Darue A
authors.initials: DA
authors.lastname: Prieto
authors.forename: Brian T
authors.initials: BT
authors.lastname: Luke
authors.forename: Donald J
authors.initials: DJ
authors.lastname: Johann
authors.forename: Luke H
authors.initials: LH
authors.lastname: Stockwin
authors.forename: Dianne L
authors.initials: DL
authors.lastname: Newton
authors.forename: Josip
authors.initials: J
authors.lastname: Blonder
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002853:Chromatography, Liquid; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D008545:Melanoma / Q000378:metabolism*; D010103:Oxygen Isotopes / Q000737:chemistry*; D011506:Proteins / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods; D014357:Trypsin / Q000378:metabolism*
chemical_list: D014408:Biomarkers, Tumor; D010103:Oxygen Isotopes; D011506:Proteins; D014357:Trypsin