%0 Journal Article
%A Jansson, Erik T.
%A Trkulja, Carolina
L.
%A Olofsson, Jessica
%A Millingen, Maria
%A Wikström, Jennie
%A Jesorka, Aldo
%A Karlsson, Anders
%A Karlsson, Roger
%A Davidson, Max
%A Orwar, Owe
%D 2012
%T Microfluidic Flow Cell
for Sequential Digestion of
Immobilized Proteoliposomes
%U https://acs.figshare.com/articles/journal_contribution/Microfluidic_Flow_Cell_for_Sequential_Digestion_of_Immobilized_Proteoliposomes/2508610
%R 10.1021/ac300519q.s001
%2 https://acs.figshare.com/ndownloader/files/4151539
%K CHO
%K proteoliposome
%K flow cell channel
%K flow cell channels
%K AFM
%K quartz crystal microbalance
%K Microfluidic Flow Cell
%K flow cell operation
%K microfluidic flow cell
%K Chinese hamster ovary
%K material transfer rates
%K RBC
%K LC
%K flow cell
%X We have developed a microfluidic flow cell where stepwise
enzymatic
digestion is performed on immobilized proteoliposomes and the resulting
cleaved peptides are analyzed 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 force 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.
%I ACS Publications