SLAS Discovery - Special Issue on Advances in MALDI Mass Spectrometry for Drug Discovery - Volume 22 Issue 10 - December 2017 - 1232

1232

SLAS Discovery 22(10)

Table 2. InterProScan Analysis of Identified Proteins of Unknown Functions.
ORF No.
Rv1860
Rv1636
Rv1980c
Rv2140c

Rv1926c

Protein Identity

Prediction of
Conserved Domain

Alanine- and proline- Conserved fibronectin
rich secreted
attachment domain
protein
Universal stress
Universal stress protein A
protein
(USP-A) domain
Immunogenic
Conserved domain of
protein MPT64
unknown function
(DUF3298)
UPF0098
Conserved
hypothetical
phosphatidylethanolamine
protein
binding protein (PEBP)
domain
Immunogenic
Conserved domain of
protein MPT63
unknown function
(DUF1942)

Accession No. of
Conserved Domain

Amino Acid Residues
of Conserved Domain

Probable Function
of the Proteins

PF07174

1-297

Unknown

PF00582

4-144

Unknown

PF11738

147-221

Unknown

SSF49777

17-174

Unknown

PF09167

33-142

Unknown

ORF, open reading frame.

3D Modeling and Docking
Molecular docking analysis of selected 3D models (showing less than 2% discrepancy from Ramachandran plot) of
hypothetical proteins was performed to find their binding
sites with SM. Parameters used for selection of 3D models
and molecular docking are represented in Table 3. Docking
of Rv1860 (Fig. 2A) showed the interaction of SM with the
amino acid residues of the conserved fibronectin attachment domain of alanine- and proline-rich secreted protein.
Docking with Rv1636 (Fig. 2B) showed that SM bound to
the residues of the universal stress protein A (USP-A)
domain of the uncharacterized universal stress protein.
Docking with Rv1980c (Fig. 2C) showed that SM bound to
the interacting residues of the conserved domain of
unknown function (DUF3298) of the immunogenic protein
MPT64. Docking with Rv2140c (Fig. 2D) and Rv1926c
(Fig. 2E) showed that SM interacted with amino acid residues of the conserved phosphatidylethanolamine binding
protein (PEBP) domain of the hypothetical UPF0098 protein and domain of unknown function (DUF1942) of the
immunogenic protein MPT63, respectively.

Prediction of Pupylation Sites
Using the default threshold (medium) with a cutoff of 2.452,
GPS-PUP predicted pupylation sites in 13 proteins, which
were tabulated in Table 4.

STRING Analysis
We analyzed the overexpressed proteins using STRING-10
with a medium confidence score threshold of 0.4 and built
an interactome network (Fig. 3) for these proteins to find

the protein-protein interaction (PPIs) and predict functional
associations. We found that proteins involved in intermediary metabolism and respiration, lipid metabolism, information pathway/regulatory and virulence, and detoxification
and adaptation interacted with each other as well as their
partners, except the hypothetical proteins.

Discussion
In this study, we have analyzed the secretory proteome of
the SM-resistant isolate by 2DE-MALDI-TOF/MS and bioinformatic tools. Overexpressed proteins in the resistant
clinical isolate were identified, which may be used as diagnostic markers or drug targets. 2DE/MS is the direct
approach that is used for the identification of a large number
of unknown proteins as well as for the separation of protein
and its species, and it has an advantage over the traditional
methods (SDS-PAGE, chromatography, and sequencing).
Several reports on drug resistance and identification of
diagnostics/drug targets employing proteomic approach
exist.8-13,27 However, to our knowledge, no study on secretory proteome analysis with SM-resistant M. tuberculosis
isolate has been reported.
Our study revealed 16 spots that were overexpressed in
SM-resistant compared with total susceptible isolates.
However, two spots were identified as one protein (isoforms of
a protein or proteins of its species), and therefore a total of 15
proteins were found. Among them, Rv1860, Rv1980c,
Rv2140c, Rv1636, and Rv1926c were proteins of an unknown
function or undefined role. Of the 15, Rv0350, Rv0440,
Rv3846, Rv1636, and Rv3418c belonged to virulence/detoxification/adaptation; Rv3248c, Rv0896, Rv0815c, and Rv2334
belonged to intermediary metabolism and respiration; Rv1860,

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