PMID- 25694144
OWN - NLM
STAT- MEDLINE
DCOM- 20160503
LR  - 20240322
IS  - 1618-2650 (Electronic)
IS  - 1618-2642 (Print)
IS  - 1618-2642 (Linking)
VI  - 407
IP  - 10
DP  - 2015 Apr
TI  - Label-free detection and identification of protein ligands captured by receptors 
      in a polymerized planar lipid bilayer using MALDI-TOF MS.
PG  - 2777-89
LID - 10.1007/s00216-015-8508-6 [doi]
AB  - 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.
FAU - Liang, Boying
AU  - Liang B
AD  - Department of Chemistry and Biochemistry, University of Arizona, 1306 East 
      University Boulevard, Tucson, AZ, 85721, USA.
FAU - Ju, Yue
AU  - Ju Y
FAU - Joubert, James R
AU  - Joubert JR
FAU - Kaleta, Erin J
AU  - Kaleta EJ
FAU - Lopez, Rodrigo
AU  - Lopez R
FAU - Jones, Ian W
AU  - Jones IW
FAU - Hall, Henry K Jr
AU  - Hall HK Jr
FAU - Ratnayaka, Saliya N
AU  - Ratnayaka SN
FAU - Wysocki, Vicki H
AU  - Wysocki VH
FAU - Saavedra, S Scott
AU  - Saavedra SS
LA  - eng
GR  - R01 EB007047/EB/NIBIB NIH HHS/United States
GR  - U54 AI065359/AI/NIAID NIH HHS/United States
GR  - R01EB007047/EB/NIBIB NIH HHS/United States
GR  - U54-AI065359/AI/NIAID NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20150219
PL  - Germany
TA  - Anal Bioanal Chem
JT  - Analytical and bioanalytical chemistry
JID - 101134327
RN  - 0 (Bacterial Toxins)
RN  - 0 (Enterotoxins)
RN  - 0 (Escherichia coli Proteins)
RN  - 0 (Ligands)
RN  - 0 (Lipid Bilayers)
RN  - 0 (Phosphatidylcholines)
RN  - 0 (Polymers)
RN  - 0 (Proteins)
RN  - 0 (Receptors, Cell Surface)
RN  - 0 (ganglioside receptor)
RN  - 0 (poly(bis-sorbyl phosphatidylcholine))
RN  - 101839-44-5 (ganglioside GD1alpha)
RN  - 37758-47-7 (G(M1) Ganglioside)
RN  - 9012-63-9 (Cholera Toxin)
RN  - D9K3SN2LNY (heat-labile enterotoxin, E coli)
RN  - EC 2.4.2.31 (Pertussis Toxin)
SB  - IM
MH  - Amino Acid Sequence
MH  - Animals
MH  - Bacterial Toxins/analysis
MH  - Cholera Toxin/analysis/metabolism
MH  - Enterotoxins/analysis
MH  - Escherichia coli Proteins/analysis
MH  - G(M1) Ganglioside/analogs & derivatives/chemistry
MH  - Ligands
MH  - Limit of Detection
MH  - Lipid Bilayers/*chemistry
MH  - Molecular Sequence Data
MH  - Pertussis Toxin/analysis
MH  - Phosphatidylcholines/chemistry
MH  - Polymerization
MH  - Polymers/chemistry
MH  - Proteins/*analysis
MH  - Receptors, Cell Surface/chemistry
MH  - Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/*methods
MH  - Tandem Mass Spectrometry
PMC - PMC4417943
MID - NIHMS665703
EDAT- 2015/02/20 06:00
MHDA- 2016/05/04 06:00
PMCR- 2016/04/01
CRDT- 2015/02/20 06:00
PHST- 2014/10/29 00:00 [received]
PHST- 2015/01/21 00:00 [accepted]
PHST- 2015/01/10 00:00 [revised]
PHST- 2015/02/20 06:00 [entrez]
PHST- 2015/02/20 06:00 [pubmed]
PHST- 2016/05/04 06:00 [medline]
PHST- 2016/04/01 00:00 [pmc-release]
AID - 10.1007/s00216-015-8508-6 [doi]
PST - ppublish
SO  - Anal Bioanal Chem. 2015 Apr;407(10):2777-89. doi: 10.1007/s00216-015-8508-6. Epub 
      2015 Feb 19.