Document Detail

A Microfluidic Flow-Cell for Sequential Digestion of Immobilized Proteoliposomes.
MedLine Citation:
PMID:  22656064     Owner:  NLM     Status:  Publisher    
We have developed a microfluidic flow-cell where stepwise enzymatic digestion is performed on immobilized proteoliposomes and the resulting cleaved peptides are analysed with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The flow-cell channels consist of two parallel gold surfaces mounted face-to-face with a thin spacer, and feature an inlet and an outlet port. Proteoliposomes (50-150 nm in diameter) obtained from red blood cells (RBC), or Chinese hamster ovary (CHO) cells, were immobilized on the inside of the flow-cell channel, thus forming a stationary phase of proteoliposomes. The rate of proteoliposome immobilization was determined using a quartz crystal microbalance with dissipation monitoring (QCM-D) which showed that 95% of the proteoliposomes bind within 5 min. The flow-cell was found to bind a maximum of 1 µg proteoliposomes/cm², and a minimum proteoliposome concentration required for saturation of the flow-cell was determined to be 500 µg/ml. Atomic force microscopy (AFM) studies showed an even distribution of immobilized proteoliposomes on the surface. The liquid encapsulated between the surfaces has a large surface-to-volume ratio, providing rapid material transfer rates between the liquid phase and the stationary phase. We characterized the hydrodynamic properties of the flow-cell, and the forces acting on the proteoliposomes during flow-cell operation was estimated to be in the range of 0.1-1 pN, too small to cause any proteoliposome deformation or rupture. A sequential proteolytic protocol, repeatedly exposing proteoliposomes to a digestive enzyme, trypsin, was developed and compared with a single digest protocol. The sequential protocol was found to detect ∼65% more unique membrane-associated protein (p<0.001, n=6) based on peptide analysis with LC-MS/MS, compared to a single digest protocol. Thus, the flow-cell described herein is a suitable tool for shotgun proteomics on proteoliposomes, enabling more detailed characterization of complex protein samples.
Erik Tomas Jansson; Carolina Linnéa Trkulja; Jessica Bodil Olofsson; Maria Millingen; Jennie Wikström; Aldo Jesorka; Anders Karlsson; Roger Karlsson; Max Davidson; Owe Orwar
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2012-5-30
Journal Detail:
Title:  Analytical chemistry     Volume:  -     ISSN:  1520-6882     ISO Abbreviation:  -     Publication Date:  2012 May 
Date Detail:
Created Date:  2012-6-4     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0370536     Medline TA:  Anal Chem     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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