SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1230

1230

SLAS Discovery 22(10)

enzymatic inactivation of antibiotic molecules, over expression of novel efflux pumps, and porin alterations in the cell
wall, and trapping of drugs. As proteins manifest most of the
biological processes, these are attractive targets for developing new drugs and diagnostics.8-13 Two-dimensional gel electrophoresis (2-DE) coupled with matrix assisted laser
desorption/ionization-time-of-flight mass spectrometry
(MALDI-TOF/MS) is the direct approach for separation,
identification, and characterization of proteins and their species.14 Comparative expression proteomic studies of aminoglycoside-resistant isolates have been reported.8-11 However,
the secretory proteome of SM-resistant M. tuberculosis isolates has not been explored. To address this issue, we analyzed the secretory proteome of SM-resistant M. tuberculosis
by direct proteomic and bioinformatics approaches. Such
information could be helpful for the development of newer
diagnostics and drug targets against SM resistance.

identification. Student t test was used for the statistical analysis by PDQuest Advanced software. The system picks up
the spots with differential intensity of significant levels
built in the system. An equal amount of protein was loaded
in all gels, and experiments were repeated in biological and
technical replicates at least three times.

Materials and Methods

Bioinformatic Analysis

M. tuberculosis Isolates Collection and Drug
Susceptibility Testing
Total susceptible (rifampicin, isoniazid, ethambutol, pyrazinamide, streptomycin, kanamycin, and amikacin) and
SM-resistant (sensitive to other first-line and second-line
drugs) M. tuberculosis isolates were procured from the
Mycobacterial Repository Centre of the National JALMA
Institute for Leprosy and Other Mycobacterial Diseases,
Agra, India. Drugs susceptibility testing (DST) for all drugs
was performed by the Löwenstein-Jensen (LJ) proportion15
and resazurin microplate assay (REMA) methods.16
Cultures were grown in Sauton's liquid medium at 37 °C up
to 4 weeks for proteomic analysis.

Protein sequences of hypothetical proteins were retrieved
from a Tuberculist server (http://tuberculist.epfl.ch). The
probable conserved domain, interaction with drugs, and
interacting partners were predicted using InterProScan,
molecular docking, GPS-PUP,21-24 and STRING analysis
(Search Tool for the Retrieval of Interacting Genes/Proteins;
http://string.embl.de/).25,26 The STRING-10 server was
used to predict the interacting partners of protein-protein
interactions. The STRING database uses a combination of
prediction approaches and an integration of other information (neighborhood, transferred neighborhood, gene fusion,
co-occurrence, coexpression, experiments, databases, text
mining). The network was made at a medium confidence
level (0.400), allowing all active prediction methods.

Secretory Protein Isolation and Precipitation

Results

After 4 weeks, cells were removed by centrifugation; supernatant was sequentially filtered by 0.45- and 0.22-µm filters
and precipitated using the sodium dodecyl sulfate (SDS)-
trichloroacetic acid (TCA) precipitation method with the
published protocol.17 Protein concentrations were estimated
by the Bradford method.18 Protein extractions were performed in biological and technical replicas.

In this study, we have compared and analyzed the secretory
protein profiles of SM-resistant and total sensitive isolates.
Comparison of 2D gels (Fig. 1) by PDQuest Advanced software revealed 16 protein spots that were consistently overexpressed in resistant compared with sensitive isolates (cut
limit ≥1.5-fold changes in spot intensity). Student t test was
used for statistical analysis with PDQuest software.
Overexpressed proteins spots were identified by MALDITOF/MS and tabulated in Table 1. The level of difference
in protein spot intensity has been represented as a densitometric ratio in Table 1.

2D Gel Electrophoresis
Isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) were carried
out using the published protocol of "in-gel rehydration"
with slight modifications.8-11,19 Gel images were analyzed
using PDQuest Advanced software version 8.0.0 (Bio-Rad,
Hercules, CA). Protein spots that showed consistently
increased intensities more than 1.5-fold were selected for

In-Gel Digestion and Mass Spectrometry
In-gel digestion of proteins and MALDI-TOF/MS were
carried out using a published protocol.10,11,14,20 Mass spectra
of digested proteins were acquired using the Autoflex II
TOF/TOF 50 (Bruker Daltonik GmbH, Leipzig, Germany).
Peptide mass tolerance was set to 50 to 125 ppm with
carbamidomethyl-cysteine as the fixed modification. The
oxidation of methionine was set as a variable modification,
and one missed cleavage site was allowed.

InterProScan Analysis
InterProScan analysis of proteins of undefined roles
(Rv1860, Rv1636, Rv1980c, Rv2140c, and Rv1926c)
showed the presence of their conserved domains (Table 2).

http://tuberculist.epfl.ch http://string.embl.de/

Table of Contents for the Digital Edition of SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017