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, Steven S Saavedra

Research output: Contribution to journalArticle

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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    Liang, B., Ju, Y., Joubert, J. R., Kaleta, E. J., Lopez, R., Jones, I. W., Hall, H. K., Ratnayaka, S. N., Wysocki, V. H., & Saavedra, S. S. (2015). Label-free detection and identification of protein ligands captured by receptors in a polymerized planar lipid bilayer using MALDI-TOF MS. Analytical and Bioanalytical Chemistry, 407(10), 2777-2789. [8508]. https://doi.org/10.1007/s00216-015-8508-6