Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS

Boying Liang, Yue Ju, James R. Joubert, Erin J. Kaleta, Rodrigo Lopez, Ian W. Jones, Henry K. Hall, Saliya N. Ratnayaka, Vicki H. Wysocki, S. Scott Saavedra

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

Abstract Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) coupled with affinity capture is a well-established method to extract biological analytes from complex samples followed by label-free detection and identification. Many bioanalytes of interest bind to membrane-associated receptors; however, the matrices and high-vacuum conditions inherent to MALDI-TOF MS make it largely incompatible with the use of artificial lipid membranes with incorporated receptors as platforms for detection of captured proteins and peptides. Here we show that cross-linking polymerization of a planar supported lipid bilayer (PSLB) provides the stability needed for MALDI-TOF MS analysis of proteins captured by receptors embedded in the membrane. PSLBs composed of poly(bis-sorbylphosphatidylcholine) (poly(bis-SorbPC)) and doped with the ganglioside receptors GM1 and GD1a were used for affinity capture of the B subunits of cholera toxin, heat-labile enterotoxin, and pertussis toxin. The three toxins were captured simultaneously, then detected and identified by MS on the basis of differences in their molecular weights. Poly(bis-SorbPC) PSLBs are inherently resistant to nonspecific protein adsorption, which allowed selective toxin detection to be achieved in complex matrices (bovine serum and shrimp extract). Using GM1-cholera toxin subunit B as a model receptor-ligand pair, we estimated the minimal detectable concentration of toxin to be 4-nM. On-plate tryptic digestion of bound cholera toxin subunit B followed by MS/MS analysis of digested peptides was performed successfully, demonstrating the feasibility of using the PSLB-based affinity capture platform for identification of unknown, membrane-associated proteins. Overall, this work demonstrates that combining a poly(lipid) affinity capture platform with MALDI-TOF MS detection is a viable approach for capture and proteomic characterization of membrane-associated proteins in a label-free manner. [Figure not available: see fulltext.]

Original languageEnglish (US)
Article number8508
Pages (from-to)2777-2789
Number of pages13
JournalAnalytical and bioanalytical chemistry
Volume407
Issue number10
DOIs
StatePublished - Mar 22 2015

Keywords

  • Bacterial toxin
  • Ganglioside receptor
  • Matrix-assisted laser desorption/ionization
  • Planar lipid bilayer
  • Polymerizable lipid

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biochemistry

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