conserved throughout the Bcc [10, 11] and has been shown to
interact with the AHL-dependent circuitries to regulateB. cenocepa-
ciavirulence [12]. Our group has been a pioneer in the utilization of
comparative proteome analysis to identify QS-regulated proteins
[13, 14]. In 2003, an approach including pre-fractionation (intracel-
lular, extracellular, and surface proteins) followed by a two-dimen-
sional gel electrophoresis (2-D PAGE) was used to identify 55
proteins with differential expression in acepImutant strain [13].
Later, gel-free proteomics usingIsobaric Tags forRelative and
Absolute Quantitation (iTRAQ) analysis was utilized to detect
1257 proteins inB. cenocepacia, 82 of which were differentially
expressed in acepRmutant [15]. More recently, we employed a
highly sensitive GeLC-MS/MS shotgun proteomics analysis to elu-
cidate the BDSF stimulon and detected close to 2700B. cenocepacia
proteins which account for 35% of the proteins annotated forB.
cenocepaciastrain H111 [16]. Among the detected proteins, 116
displayed significant regulation in therpfFmutant [12].
In this review, we present a detailed practical procedure that our
lab routinely uses to carry out GeLC-MS/MS shotgun proteomics
experiments (Fig.1), usingB. cenocepaciastrain H111 as a model
organism. First, a subcellular fractionation is performed (extracellu-
lar, intracellular, and membrane fractions) to reduce the complexity
of the sample and to determine the predominant subcellular locali-
zation of a protein of interest, which may provide important infor-
mation about its function [17, 18]. Subsequently, protein samples
are separated by 1D SDS-PAGE [19, 20], which has the advantageFig. 1The overall workflow of the GeLC-MS/MS shotgun proteomics experiment
194 Yilei Liu et al.