ERJ Express. Published on April 17, 2014 as doi: 10.1183/09031936.

00175113

ORIGINAL ARTICLE
IN PRESS | CORRECTED PROOF

The identification of tuberculosis

biomarkers in human urine samples
Brandy L. Young1, Zandile Mlamla2, Putuma P. Gqamana3, Salome Smit4,
Teri Roberts5, Jonathan Peter6, Grant Theron6, Ureshnie Govender6,
Keertan Dheda1,6,7 and Jonathan Blackburn1,2,7

Affiliations: 1Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of
Cape Town, Cape Town, 2Division of Medical Biochemistry, Dept of Clinical Laboratory Sciences, University of
Cape Town, Cape Town, 3University of the Western Cape, Cape Town, 4Proteomics Laboratory, Central
Analytical Facility, University of Stellenbosh, Cape Town, and 6Lung Infection and Immunity Unit, Division of
Pulmonology and UCT Lung Institute, Dept of Medicine, University of Cape Town, South Africa. 5Médecins Sans
Frontières, Geneva, Switzerland. 7Both authors contributed equally.

Correspondence: Jonathan Blackburn, Institute of Infectious Disease and Molecular Medicine, Faculty of
Health Sciences, University of Cape Town, Anzio Road, Observatory, Cape Town 7925, South Africa. E-mail:
[email protected]

ABSTRACT We aimed to determine whether shotgun proteomic approaches could be used to identify
tuberculosis (TB)-specific biomarkers in the urine of well-characterised patients with active TB versus
no TB.
Patients with suspected TB (n563) were classified as: definite TB (Mycobacterium tuberculosis positive
culture, n521); presumed latent-TB infection (LTBI) (M. tuberculosis negative culture, no radiological
features of active TB, a positive QuantiFERON-TB Gold In-Tube (QFT-IT) test and a positive T-SPOT.TB
test, n524); and presumed non-TB/non-LTBI (M. tuberculosis negative culture, no radiological features of
active TB, a negative QFT-IT test and a negative T-SPOT.TB test, n518). Urine proteins, in the range of
3–50 kDa, were collected, separated by a one-dimensional SDS-PAGE gel and digested using trypsin, after
which high-performance liquid chromatography-tandem mass spectrometry was used to identify the
urinary proteome.
10 mycobacterial proteins were observed exclusively in the urine of definite TB patients, while six
mycobacterial proteins were found exclusively in the urine of presumed LTBI patients. In addition, a gene
ontology enrichment analysis identified a panel of 20 human proteins that were significant discriminators
(p,0.05) for TB disease compared to no TB disease. Furthermore, seven common human proteins were
differentially over- or under-expressed in the TB versus the non-TB group.
These biomarkers hold promise for the development of new point-of-care diagnostics for TB.

@ERSpublications
The application of proteomics for the identification of novel urinary tuberculosis biomarkers
http://ow.ly/tN0Rm

This article has supplementary material available from www.erj.ersjournals.com

Received: Oct 07 2013 | Accepted after revision: Feb 13 2014

Support statement: J. Blackburn thanks the Department of Science and Technology and the National Research
Foundation for a South African Research Chair Initiative grant. B.L. Young thanks the Carnegie Corporation for a
postdoctoral research fellowship. The work was funded by the National Research Foundation (grant no. 64760) and the
TB-NEAT grant from the European and Developing Countries Clinical Trials Partnership (grant number 09.32040.009).
Conflict of interest: Disclosures can be found alongside the online version of this article at www.erj.ersjournals.com
Copyright ßERS 2014

Eur Respir J 2014; in press | DOI: 10.1183/09031936.00175113 1

Copyright 2014 by the European Respiratory Society.

TUBERCULOSIS | B.L. YOUNG ET AL.

Introduction
Tuberculosis (TB) is a major global health problem and to improve case detection the World Health
Organization recently endorsed the Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) assay as a frontline
diagnostic test in TB endemic settings [1]. Although Xpert MTB/RIF is a significant step forward for
improved diagnosis, it has several drawbacks. Xpert MTB/RIF requires patients to produce a representative
sputum sample, which excludes many HIV positive individuals and children, it has a sensitivity of ,70% in
smear-negative TB patients, it cannot directly diagnose extrapulmonary TB, and is relatively expensive [2, 3].
Other diagnostic tests such as smear microscopy have a sensitivity of only ,50%. The interferon-c release
assay (IGRA) is not useful for the diagnosis of active TB [4], and the Determine TB LAM antigen detection test
(Alere Inc., Waltham, MA, USA) is only useful in HIV-infected persons [5–9]. Hence, new TB diagnostic tests,
designed for use at the point of care in resource-poor settings, represent a major unmet global health priority.
However, the lack of suitable biomarkers that distinguish active TB disease from non-TB constitutes a major
impasse in the development of new TB diagnostic tests.
One way to address this is to use proteomics, a comprehensive protein identification methodology, to
discover novel diagnostic biomarkers for TB [10]. Shotgun proteomics, using liquid chromatography (LC)
coupled with tandem mass spectrometry (MS/MS), has gained much traction in recent years. Some of the
initial TB proteomic studies involved analysing Mycobacterium tuberculosis (MTB) proteins from different
infectious strains [11]. This provided insights into the proteome of the bacteria and served as a useful map
for protein annotation. AGRANOFF et al. [12] used proteomic fingerprinting of serum to look for diagnostic
biomarkers that could differentiate between active TB, latent tuberculosis infection (LTBI) and no TB in
HIV positive and negative patients. In another study, KASHINO et al. [13] used one-dimensional LC
combined with MS/MS to identify four M. tuberculosis-derived proteins in the urine of patients with
pulmonary TB. A more recent follow-up urinary proteomics study found that a putative molybdopterin
biosynthesis protein (Rv1681) was detected in 11 out of 21 TB patients [14].
Here, we used state-of-the-art proteomic approaches to discover urinary diagnostic biomarkers associated
with pulmonary TB [15, 16]. We report 10 M. tuberculosis and 27 human proteins that were selectively
identified in the urine of patients with active pulmonary TB disease, as well as six M. tuberculosis proteins
that were selectively identified in the urine of patients with presumed LTBI.

Methods
Urine collection and clinical case definition
In this study, urine samples were collected from patients attending South African clinics who were suspected
of having active pulmonary TB (fig. 1). Informed consent was obtained from all participants and the study
was approved by the Faculty of Health Sciences Human Research Ethics Committee (University of Cape
Town, Cape Town, South Africa) (approval number HREC421/2006). Patients with HIV co-infection and
other major comorbidities (e.g. diabetes and chronic organ failure) were excluded from this study. Definite
TB patients were classified based on having clinical presentation compatible with TB infection, a positive
M. tuberculosis culture and a positive smear. Patients with presumed LTBI were defined as having: a negative
M. tuberculosis culture, a negative smear, two positive IGRA test results, the QuantiFERON-TB Gold In-
Tube (Qiagen Inc., Valencia, CA, USA) and the T-SPOT.TB (Oxford Immunotec Global PLC, Abingdon,
UK), and chest radiography that was not compatible with active TB. Finally, the presumed no TB (non-TB/
non-LTBI) group had: negative M. tuberculosis cultures, negative smears, two negative IGRAs, and chest
radiography that was not compatible with active TB. The patient demographic information is shown in
table 1.

Sample preparation
A total of 63 urine samples were collected. The urine samples were grouped according to the clinical cohort
(TB, presumed LTBI and presumed non-TB/non-LTBI). Protein readings were obtained on the individual
urine samples using a Bradford assay. Equal amounts of protein were obtained from each patient and then
pooled based on the patient group (pooled n56 patients, ,10 mL of urine in each pooled sample). For the
definite TB and non-TB/non-LTBI groups, three biological replicate pools were prepared (i.e. six patient
urine samples per group with three biological replicates; n518 patients per group). For the LTBI group four
biological replicate pools were prepared. The pooled urine sample protein concentrations were then
compared and normalised across all groups and biological replicates. In addition, a set of three individual
definite TB patient urine samples were similarly prepared and normalised.
All pooled or individual samples were filtered through a 50 kDa molecular weight cut-off (MWCO) filter to
remove abundant, higher molecular weight human proteins that would otherwise prevent identification of
lower abundance proteins. The flow-through was then concentrated on a 15 mL 3 kDa MWCO filter

2 DOI: 10.1183/09031936.00175113
 TUBERCULOSIS | B.L. YOUNG ET AL.

Patients with suspected

pulmonary TB (n=63)

Collection of 2
sputum samples
(n=63) and IGRAs (n=24)

Positive culture and Negative culture and

positive smear negative smear

LTBI (positive QFT-IT non-TB/non-LTBI

Definite TB (n=21) and positive T-SPOT.TB) (negative QFT-IT and
(n=24) negative T-SPOT.TB) (n=18)

FIGURE 1 Flow chart detailing the patient’s diagnostic outcomes stratified by final tuberculosis (TB) diagnosis category.
IGRA: interferon-c release assay; LTBI: latent TB infection; QFT-IT: QuantiFERON-TB Gold In-Tube (Qiagen Inc.,
Valencia, CA, USA).

followed by a 0.5 mL 3 kDa MWCO filter to reduce the volume. Proteins in the 3 kDa to 50 kDa range
were collected, separated on a 10% SDS-PAGE gel and digested using trypsin (fig. 2).

High-performance liquid chromatography

The chromatographic separation was conducted using a Proxeon Easy n-LC II (Proxeon Biosystems,
Odense, Denmark). A gradient chromatography separation was performed using a Proxeon Easy C18 trap
column (2 cm, ID 100 mm, 5 mm, C18) and analytical column (10 cm, ID 75 mm, 3 mm, C18).
Chromatography was performed at room temperature with a binary mobile phase system (A: water with
0.1% formic acid; and B: acetonitrile with 0.1% formic acid) at a flow rate of 300 nL?min-1. Tryptic peptides
were eluted from the column with a gradient of: 5% B at 0.00 min; 5% B at 5.00 min; and 80% B at
120 min.

Mass spectrometry
The shotgun proteomic analysis was performed using an LTQ-Orbitrap Velos Pro mass spectrometer
(Thermo Fisher, San Jose, CA, USA) with sample introduction via flow injection analysis from the Proxeon
high-performance liquid chromatography system. Positive ion electrospray ionisation was used with the
following conditions: an ion spray voltage of 1500 V and a source temperature of 200uC. For the collision
induced dissociation analysis, helium was used as the collision gas with a collision gas pressure of 1.2 mTorr
and variable collision energy.

Results
In this analysis a multi-algorithm database search strategy was used. The search summary can be found in
table 2, which shows the average number of peptide spectral matches, peptides and protein groups
identified across the three patient groups (presumed non-TB/non-LTBI, presumed LTBI and TB). The
average number of peptide spectral matches was higher for X! Tandem (X! TANDEM Spectrum Modeler,
www.thegpm.org/tandem/) than for Omssa (Open Mass Spectrometry Search Algorithm, ftp://ftp.ncbi.nih.
gov/pub/lewisg/omssa/CURRENT/) and accordingly X! Tandem identified 1332 peptides and 524 proteins
compared to 830 peptides and 412 proteins identified by Omssa (see online supplementary material). The
final data used in this analysis was the combined nonredundant subsumed protein list from the two
algorithms for each patient group [17]. Figure 3a shows the Venn diagrams that highlight the overlapping
and unique human and M. tuberculosis proteins identified across the three patient groups. An average of 560
proteins was found across the three cohorts. The data from each patient group showed that 319 (57%) out
of 560 of the proteins identified were seen in all three groups. With this dataset the following questions were

DOI: 10.1183/09031936.00175113 3
TUBERCULOSIS | B.L. YOUNG ET AL.

TABLE 1 Patient demographic information

Group 1 Group 2 Group 3

definite TB non-TB/LTBI non-TB/non-LTBI

Subjects 21 24 18
Age years 43 (42) 43 (41) 35 (31)
Male 15 20 14
Previous TB 10 8 3
Current Smoker 13 17 11
Mean weight kg 62 66 63
Ethnicity
Black 15 12 12
Mixed ancestry 6 12 3
White 0 0 3
Diagnostic test
Culture
Positive 21 0 0
Negative 0 24 18
Smear
Positive 17 0 0
Negative 4 24 18
T-SPOT.TB
Positive 13 24 0
Negative 2 0 18
No result 6
QFT-IT
Positive 13 24 0
Negative 1 0 18
No result 7

Data are presented as n or mean (median), unless otherwise stated. TB: tuberculosis; LTBI: latent TB
infection; QFT-IT: QuantiFERON-TB Gold In-Tube.

Sample preparation SDS-PAGE gel

Collect Collect
Urine protein
flow-through concentrate

50 KDa filter 3 KDa filter

1) Excise protein
LC-MS/MS analysis bands and
2) Digest protein

MS/MS MS LC

m/z m/z Time min

Bioinformatic analysis

Database Protein Statistical

searching identification analysis

FIGURE 2 Schematic workflow detailing the proteomic sample preparation process, liquid chromatography (LC) tandem
mass spectrometry (MS/MS) analysis and bioinformatics analysis.

4 DOI: 10.1183/09031936.00175113
 TUBERCULOSIS | B.L. YOUNG ET AL.

TABLE 2 The database search parameters used and search summary for the bioinformatics algorithms

Parameter Algorithm

X! Tandem Omssa

Precursor charge state +2 to +4 +2 to +4

Enzyme Trypsin Trypsin
Missed cleavages n 2 2
Precursor mass error ppm 25 25
Peptide mass error tolerance Da 0.5 0.5
Fixed modifications Carbamidomethylation on cysteine Carbamidomethylation on cysteine
Variable modifications Asparagine/glutamine deamidation Asparagine/glutamine deamidation
Methionine oxidation Methionine oxidation
FDR# % 1 1
Average PSMs" n 7877 6642
Average peptides" n 1332 830
Average proteins" n 524 412

FDR: false discovery rate; PSM: peptide spectral matches. #: For each algorithm the PSM were filtered based on a score threshold of 1% FDR,
which was established using a decoy database search; ": the average parameters shown are average combined lists taken from the merged X!
Tandem and merged Omssa data.

addressed. 1) Can exogenous M. tuberculosis-specific proteins be identified in urine from patients infected
with pulmonary TB? 2) Can a panel of human proteins be identified that are associated with TB infection?
3) Can significant changes in common human urinary proteins be observed that are associated with
TB disease?

Exogenous M. tuberculosis proteins can be identified in patient urine samples

A total of 26 M. tuberculosis proteins were identified across the various patient urine samples (fig. 3b).
These M. tuberculosis proteins do not appear to have any functions in common and were primarily found to
be membrane-associated or localised to the extracellular space (table 3). Surprisingly, we identified
M. tuberculosis proteins in all the suspected TB patients. Given this result, a BLAST (Basic Local Alignment
Search Tool, http://blast.ncbi.nlm.nih.gov/Blast.cgi) analysis was carried out using DBToolkit [18] to map
the identified M. tuberculosis peptides to human or bacterial proteins in the UniProt database (http://www.
uniprot.org/). This analysis revealed that six out of 26 of the identified M. tuberculosis peptides could be
mapped to proteins from non-TB mycobacteria and/or other actinobacteria, in addition to proteins from

a) non-TB/non-LTBI b) TB

48

10
19
69

319

130 TB 4 1
LTBI
2
172 LTBI non-TB/non-LTBI

3
98 6 0

FIGURE 3 a) The total urinary protein repertoire and b) the unique and overlapping Mycobacterium tuberculosis proteins observed in suspected tuberculosis (TB)
patients classified as active TB, presumed latent TB infection (LTBI) and presumed non-TB/non-LTBI.

DOI: 10.1183/09031936.00175113 5
TUBERCULOSIS | B.L. YOUNG ET AL.

organisms within the M. tuberculosis complex (table 3). Those peptides that mapped to other species
outside of M. tuberculosis or the M. tuberculosis complex are identified as Mycobacterium-related peptides in
table 3. For the active-TB patient group (including both the triplicate biological replicate pooled samples
and the three individual samples), eight previously unreported M. tuberculosis-specific proteins and two
Mycobacterium-related proteins were identified in the urine samples. Interestingly, several isoforms of the
PE-PGRS family, which are glycan rich cell wall proteins, were identified in the TB and/or the presumed
LTBI groups (table 3).

Functional classification of proteins found in patient urine samples

To classify the human urinary proteins based on their cellular compartmentalisation (CC), molecular
function (MF) and biological processes (BP) a gene ontology (GO) enrichment analysis was performed (see
online supplementary information for details) [19, 20]. Figure 4 shows the enriched GO terms obtained
when the proteome of the presumed non-TB/non-LTBI group was set as the background gene product,
which includes 10 GO-BP terms plus three GO-CC terms for the TB group and two GO-BP terms plus two
GO-CC terms for the LTBI group. Interestingly, no GO-MF terms were significantly enriched for either
group. For the TB and LTBI groups, the most significant GO-BP terms were ‘‘Response to Stimulus’’

TABLE 3 Mycobacterium tuberculosis specific proteins and Mycobacterium-related proteins identified in human urine samples
from active TB, presumed LTBI and presumed non-TB/non-LTBI patients

Cohort Accession Protein name M. tuberculosis Mycobacterium-related"

number specific#

TB only Rv1475c Aconitate hydratase 6

Rv1977+ Conserved protein 6
Rv0014c Serine/threonine protein kinase 6
Rv2748c DNA translocase FtsK 6
Rv1664 Polyketide synthase 6
Rv1161 Nitrate reductase a-subunit 6
Rv2280 Uncharacterised FAD-linked 6
oxidoreductase
Rv2694c Conserved hypothetical protein 6
Rv2490c1 PE-PGRS family protein 6
(1661 amino acids)
Rv0578c PE-PGRS family protein 6
(1307 amino acids)
TB and LTBI Rv1450c PE-PGRS family protein 6
Rv1522c Putative membrane protein mmpL12 6
Rv2737c RecA 6
Rv2981c D-alanine– D-alanine ligase 6
TB and non-TB/non-LTBI Rv0765c Probable oxidoreductase 6
LTBI only Rv2126c PE-PGRS family protein 6
Rv1759c WAG22 antigen 6
Rv1464e Probable cysteine desulfurase 6
Rv3202c## Possible ATP-dependent DNA helicase 6
Rv0668 DNA-directed RNA polymerase subunit 6
Rv3345c PE-PGRS family protein 6
LTBI and non-TB/non-LTBI None
Non-TB/non-LTBI only Rv3775 Probable lipase LipE 6
Rv3736 Transcriptional regulator, AraC family 6
Rv2455c 2-oxoglutarate oxidoreductase subunit 6
KorA
TB, LTBI and non-TB/ Rv3885c Possible membrane protein 6
non-LTBI Rv1235 Probable sugar-binding lipoprotein LpqY 6

Online supplementary tables S1 and S2 contain detailed peptide statistics and homology matching information. TB: tuberculosis; LTBI: latent TB
infection; FAD: flavin adenine dinucleotide. #: Proteins were classified as M. tuberculosis specific if the identifying peptides only mapped to proteins
found in the M. tuberculosis complex; ": proteins were classified as Mycobacterium-related if the identifying peptides mapped to both M. tuberculosis
specific proteins as well as to other non-infectious mycobacteria and/or other proteins found in a combined UniProt human and bacteria database;
+
: this protein was identified by a peptide that mapped to both M. tuberculosis and Mycobacterium marinum; 1: this protein was identified based on
three peptides, all of which were M. tuberculosis specific; e: this protein was identified by a peptide that that mapped to both M. tuberculosis and
Mycobacterium parascrofulaceum; ##: this protein was identified by two different peptides, one of which was M. tuberculosis specific and the other
was Mycobacterium related.

6 DOI: 10.1183/09031936.00175113
 TUBERCULOSIS | B.L. YOUNG ET AL.

Response to stimulus
Regulation of biological quality
Immune response
Macromolecule localization
Tissue development
Response to external stimulus
Ectoderm development GO-BP
Response to stress
Epidermis development
Homeostatic process
FIGURE 4 Gene ontology (GO) enrich- Immune response
ment analysis of the proteins identified Defense response
from the suspected tuberculosis (TB)
patients with the presumed non-TB/non-
Extracellular region
latent TB infection (LTBI) patient group TB
set as the background gene product. The Extracellular region part
following terms were obtained via David Extracellular space GO-CC LTBI
Pathway Analysis with a cut-off of pf0.05. Extracellular region
GO-BP: terms that represent biological Extracellular space
operations or sets of molecular events;
GO-CC: terms that represent part of the 0.00 0.02 0.04 0.06 0.08
cell or extracellular space. p-value

(GO:0050896) and ‘‘Immune Response’’ (GO:0006955), respectively. While the most significant GO-CC
term for both groups was ‘‘Extracellular Region’’ (GO:0005576).
In total, these 17 enriched GO terms encompassed a total of 1230 redundant proteins. To reduce this list of
potential biomarkers towards a smaller panel of proteins that could potentially predict TB disease, we did
the following. First, the proteins that comprised the most significant GO-BP term for the TB group,
‘‘response to stimulus’’ (GO:0050896), were clustered. Secondly, the proteins that comprised the two GO-
BP terms for the LTBI group, ‘‘immune response’’ (GO:0006955) and ‘‘defense response’’ (GO:0006952),
were clustered. Finally, all the GO-CC terms and the corresponding proteins from both the TB and LTBI
groups were clustered together since there was significant overlap in the GO-CC terms and proteins here.
Figure 5 shows a Venn diagram of these clustered proteins and reveals that 20 of the proteins in the
‘‘response to stimulus’’ (GO:0050896) group were unique to this term in the TB group (table 4). The
complete list of GO terms and proteins can be found in online supplementary table S3.

Immune-related proteins are differentially expressed in urine samples of patients with active-TB
The normalised spectral abundance factor is a label-free spectral counting method that enables the relative
quantitation of protein abundances across samples. The spectral abundance is enumerated by summing the
number of identified peptide spectra for each protein, normalising this sum to the protein length and then
further normalising to the sum of all protein abundances in the given sample. Here, we used normalised
spectral abundance factor values to determine the level of differential protein expression in the common
human urinary proteins observed in the TB-suspected patients’ urine by comparing the active TB and,
separately, the LTBI patients to the presumed non-TB/non-LTBI group. An ANOVA test showed that when
using either of the two bioinformatics algorithms, the TB group had upregulated levels of immunoglobulin
kappa chain C (IGKC; accession number: P01834), retinol binding protein 4 (RBP4; accession number:
P02753), a-1-acid glycoprotein 1 (ORM1; accession number: P02763) and immunoglobulin lambda-2
chain C (IGLC2; P0CG05, Omssa database only) that were statically significant (pf0.05) compared to the
non-TB/non-LTBI group (fig. 6). A statistically significant difference (pf0.05) was also observed between
the LTBI group compared to the non-TB/non-LTBI group for IGKC and ORM1 (X! Tandem database
only). Although the difference in ORM1 abundance between the LTBI group and the non-TB/non-LTBI
group found when using the Omssa database failed to reach statistical significance, we note that the trend is
similar to that observed when using the X! Tandem database.
By contrast, in the X! Tandem dataset, the proteins prostaglandin-H2 D-isomerase (PTGDS; accession
number: P41222), secreted and transmembrane protein 1 (SECTM1; accession number: Q8WVN6) and
a-1-microglobulin/bikunin precursor (AMBP; accession number: P02760) were significantly (pf0.05)
downregulated in the TB group compared to the non-TB/non-LTBI group (fig. 6a). A similar pattern of
relative expression values was observed for PTGDS in the Omssa dataset (fig. 6b).

DOI: 10.1183/09031936.00175113 7
TUBERCULOSIS | B.L. YOUNG ET AL.

20
35

FIGURE 5 Gene ontology (GO) enrich-

ment analysis where A: is the clustered
17 GO-biological processes (GO-BP) pro-
62
teins from the GO term ‘‘response to
stimulus’’ (GO:0050896) enriched in the
tuberculosis (TB) group; B: is the clustered
B GO-BP proteins from the GO terms ‘‘im-
92
mune response’’ (GO:0006955) and
C 6 ‘‘defense response’’ (GO:0006952) enriched
in the presumed latent TB infection
4
(LTBI) group; and C: is the clustered
GO-cellular compartmentalisation (GO-
CC) proteins from the terms in the TB
and presumed LTBI group.

Discussion
Our initial urinary proteomics efforts showed that the abundant, high molecular weight human proteins,
such as uromodulin and albumin (reported to be 60% and 20% of the total urinary proteome, respectively
[21]), masked the signal of the lower abundant proteins; resulting in ,50 total protein identifications (data
not shown). However, noting that the average molecular weight of M. tuberculosis proteins is only ,20 kDa
(online supplementary fig. S1), we found that simple use of MWCO filters to remove the abundant, higher
molecular weight human proteins enabled us to identify and quantify on average 560 proteins across the
three patient groups. This figure compares well to the benchmark of ,600 protein identifications set by
NAGARAJ et al. [22] for label-free, quantitative urinary proteomic analyses, although it is some way short of a
nonquantitative proteomic analysis that reported ,1500 proteins in a human urine sample [23].
Using this experimental approach we identified 26 M. tuberculosis proteins across the various patient urine
samples, of which 10 were unique to the TB group, six were unique to the LTBI group and three were
unique to the non-TB/non-LTBI group (table 3). Given the continuing uncertainty about the metabolic
status of M. tuberculosis bacilli within infected macrophages during a latent infection [24], the observation
of unique M. tuberculosis peptides in urine from LTBI patients is not unreasonable. However, we note that
the number of such peptides uniquely identified in LTBI patients urine is lower than that found uniquely in
active-TB patients’ urine, in accord with expectation. In addition, different isoforms of the PE-PGRS
protein family were uniquely identified in the TB and LTBI patient groups, with one isoform shared
between the two groups. Importantly, since several peptides in this protein family have been identified in
both active-TB and LTBI patient urine samples in the present work, they may represent further interesting
biomarker candidates, in addition to the 10 M. tuberculosis proteins that are unique to the active TB group.
The identification of M. tuberculosis proteins in the presumed non-TB/non-LTBI group was unexpected,
but it is important to note that there is currently no reliable test for predicting LTBI. For example, RANGAKA
et al. [4] reported that IGRAs are not good at predicting active TB disease let alone LTBI. It is, therefore,
possible that some patients in the non-TB/non-LTBI group did in fact have LTBI; further work will be
needed to evaluate this possibility. Nonetheless, the M. tuberculosis proteins identified selectively in the
active TB and presumed LTBI patient urine samples represent important observations and warrant further
investigation as candidate urinary biomarkers for active TB infection. For example, it is striking that we
observed the same peptide derived from the M. tuberculosis protein Rv2981c in all four LTBI pools, as well
as in two out of the six TB samples, but in none of the non-TB/non-LTBI samples, suggesting that this
peptide might represent a more direct measure for LTBI than IGRAs (online supplementary table S1).
In all cases, further validation studies are clearly needed to determine the frequency with which the 26
M. tuberculosis proteins identified here are also observed in individual urine samples from a larger cohort of
patients with suspected TB. However, it is encouraging that our data already hints strongly at high
frequencies for at least three of these proteins: Rv0765c, Rv1235 and Rv2981c.
A GO enrichment analysis [19, 20] aims to cluster proteins based on three defined ontology terms: GO-BP,
GO-CC and GO-MF. Here, we observed 17 significantly enriched GO terms that together contained 1230

8 DOI: 10.1183/09031936.00175113
 TUBERCULOSIS | B.L. YOUNG ET AL.

TABLE 4 The 20 unique human proteins identified from the enriched gene ontology (GO)-
biological processes term ‘‘response to stimulus’’ (GO:0050896)

Accession number Protein Name

P08758 Annexin A5
P10523 S-arrestin
P12830 Cadherin-1
P05937 Calbindin
Q9UBG3 Cornulin
O60494 Cubilin
P15924 Desmoplakin
P54764 Ephrin type-A receptor 4
P05413 Fatty acid-binding protein
P07359 Platelet glycoprotein Ib a chain
Q9HCN6 Platelet glycoprotein VI
P07476 Involucrin
Q9Y5Y7 Lymphatic vessel endothelial hyaluronic acid receptor
P04156 Major prion protein
P17900 Ganglioside GM2 activator
P18827 Syndecan-1
Q8TF72 Protein Shroom 3
Q9UBC9 Small proline-rich protein
P08138 Tumour necrosis factor
P30530 Tyrosine-protein kinase receptor

redundant proteins in our dataset (fig. 5). With this dataset, a set of 20 human proteins was mined from the
most significant GO enrichment term ‘‘response to stimulus’’ (GO:0050896) (table 4). It is plausible that
discrimination of TB disease status might be achieved using this panel of 20 human proteins; however,
further studies will be required to determine the true discriminatory power of this panel.
Our analysis further identified seven immune-related human proteins (IGKC, RBP4, PTGDS, ORM1,
IGCL2, AMBP and SECTM1) of which six were differentially expressed in active TB urine samples (fig. 6).
A number of these have been previously observed to be associated with TB infection. For example, two
related studies showed that RBP4 was a marker for TB infection, with elevated levels of RBP4 being found in
the serum of cattle infected with Mycobacterium bovis [25] and with differential expression of RBP4 being

***
a) b) ***
***
5 5 ***
TB
Normalised spectral abundance factor

Normalised spectral abundance factor

LTBI
4 4
Non-TB/non-LTBI

3 *** 3
*** ***

*** *
2 *** 2 *
* *
***
1 1

0 0
IGKC RPB4 ORM1 PTGDS SECTM1 IGKC RPB4 ORM1 IGLC2 AMBP PTGDS

FIGURE 6 The relative quantitative analysis of the common human urine proteins from tuberculosis (TB) patients with
active TB, presumed latent TB infection (LTBI) and presumed non-TB/non-LTBI observed from a) X! Tandem and b)
Omssa. Significance was evaluated via two-way ANOVA with a Bonferroni correction by comparing the presumed LTBI
and TB groups to the presumed non-TB/non-LTBI group. IGKC: immunoglobulin kappa chain C; RBP4: retinol binding
protein 4; ORM1: a-1-acid glycoprotein 1; PTGDS: prostaglandin-H2 D-isomerase; SECTM1: secreted and
transmembrane protein 1; IGLC2: immunoglobulin lambda-2 chain C; AMBP: a-1-microglobulin/bikunin precursor.
*: p,0.05; ***: p,0.001.

DOI: 10.1183/09031936.00175113 9
TUBERCULOSIS | B.L. YOUNG ET AL.

observed in human whole blood supernatant [26]. In addition, SECTM1 has been shown to be a cognate of
CD7, a protein found on T-cells, natural killer cells and pre-B cells and, while the function of SECTM1 is
not well established [27], the SECTM1 cognate has been reported to stimulate the upregulation of CD25,
CD54 and CD69 on human natural killer cells in vitro in TB infection. Furthermore, serum levels of AMBP
have also been observed to be elevated levels in cattle with sub-clinical M. bovis infections [25]. Reduced
levels of PTGDS have been reported in cerebrospinal fluid samples from patients with bacterial meningitis
[28]; however, to our knowledge there has been no previous correlation between PTGDS and TB.
Based on our analyses, we hypothesise that the immune-related proteins IGKC, RBP4, PTGDS, AMBP,
ORM1, IGCL2 and SECTM1 might be collectively used to diagnose TB disease in patient urine samples. For
example, our observation of significantly increased abundance of RBP4 in active TB patients compared to
the other two patient groups may provide the basis for a novel ‘‘rule-in’’ test for TB, whilst the significantly
decreased abundance of PTGDS in active TB patients compared to LTBI patients may provide the basis for a
‘‘rule-out’’ test for TB (fig. 5). However, we note that it will be important to first determine whether such
biomarkers can discriminate between TB and other respiratory diseases such as asthma, emphysema,
pneumonia or sarcoidosis.
Proteomics offers tremendous opportunities for the discovery of diagnostic and/or prognostic biomarkers
associated with localised or systemic disease, thus facilitating global policy for TB elimination [29]. Urine is
a particularly good biological fluid for this type of analysis because, as an ultra-filtrate of blood, it is
potentially representative of molecules from all organs of the body, including the lungs. Through the
proteomic analysis of pooled and single urine samples collected from patients with definite pulmonary TB,
presumed LTBI and presumed non-TB/non-LTBI, we have identified 10 exogenous M. tuberculosis proteins,
as well as various panels of human proteins and seven common human proteins that appear to be indicative
of TB infection. Validation studies are underway to confirm the frequency of these candidate biomarkers of
TB disease.

Acknowledgements
We are grateful to the patients who agreed to take part in this study. Also, we would like to thank the research nursing
staff involved in the recruitment of patients and collection of urine samples.

References
1 World Health Organization. Rapid Implementation of the Xpert MTB/RIF Diagnostic Test: Technical and
Operational ‘How-to’ Practical Considerations. Geneva, WHO Press, 2011.
2 Theron G, Peter J, van Zyl-Smit R, et al. Evaluation of the Xpert MTB/RIF assay for the diagnosis of pulmonary
tuberculosis in a high HIV prevalence setting. Am J Respir Crit Care Med 2011; 184: 132–140.
3 Weyer K, Mirzayev F, Migliori GB, et al. Rapid molecular TB diagnosis: evidence, policy making and global
implementation of Xpert MTB/RIF. Eur Respir J 2013; 42: 252–271.
4 Rangaka MX, Wilkinson KA, Glynn JR, et al. Predictive value of interferon-c release assays for incident active
tuberculosis: a systematic review and meta-analysis. Lancet Infect Dis 2012; 12: 45–55.
5 Peter J, Green C, Hoelscher M, et al. Urine for the diagnosis of tuberculosis: current approaches, clinical
applicability, and new developments. Curr Opin Pulm Med 2010; 16: 262–270.
6 Pai M, Minion J, Steingart K, et al. New and improved tuberculosis diagnostics: evidence, policy, practice, and
impact. Curr Opin Pulm Med 2010; 16: 271–284.
7 Dheda K, Ruhwald M, Theron G, et al. Point-of-care diagnosis of tuberculosis: past, present and future. Respirology
2013; 18: 217–232.
8 Minion J, Leung E, Talbot E, et al. Diagnosing tuberculosis with urine lipoarabinomannan: systematic review and
meta-analysis. Eur Respir J 2011; 38: 1398–1405.
9 Peter JG, Theron G, van Zyl-Smit R, et al. Diagnostic accuracy of a urine lipoarabinomannan strip-test for TB
detection in HIV-infected hospitalised patients. Eur Respir J 2012; 40: 1211–1220.
10 Wheelock CE, Goss VM, Balgoma D, et al. Application of ‘omics technologies to biomarker discovery in
inflammatory lung diseases. Eur Respir J 2013; 42: 802–825.
11 Rosenkrands I, Weldingh K, Jacobsen S, et al. Mapping and identification of Mycobacterium tuberculosis proteins by
two-dimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis 2000; 21: 935–948.
12 Agranoff D, Fernandez-Reyes D, Papadopoulos MC, et al. Identification of diagnostic markers for tuberculosis by
proteomic fingerprinting of serum. Lancet 2006; 368: 1012–1021.
13 Kashino SS, Pollock N, Napolitano DR, et al. Identification and characterization of Mycobacterium tuberculosis
antigens in urine of patients with active pulmonary tuberculosis: an innovative and alternative approach of antigen
discovery of useful microbial molecules. Clin Exp Immunol 2008; 153: 56–62.
14 Pollock NR, Macovei L, Kanunfre K, et al. Validation of Mycobacterium tuberculosis Rv1681 protein as a diagnostic
marker of active pulmonary tuberculosis. J Clin Microbiol 2013; 5: 1367–1373.
15 Mehaffy MC, Kruh-Garcia NA, Dobos KM. Prospective on Mycobacterium tuberculosis proteomics. J Proteome Res
2012; 11: 17–25.
16 Zhang L, Lowrie D, Zhou H. Proteomic progress in studying tuberculosis from 2010 to 2011. J Biophys Chem 2011;
2: 395–400.
17 Nesvizhskii AI, Aebersold R. Interpretation of shotgun proteomic data: the protein inference problem. Mol Cell
Proteomics 2005; 4: 1419–1440.

10 DOI: 10.1183/09031936.00175113
 TUBERCULOSIS | B.L. YOUNG ET AL.

18 Martens L, Vandekerckhove J, Gevaert K. DBToolkit: processing protein databases for peptide-centric proteomics.
Bioinformatics 2005; 21: 3584–3585.
19 Huang DW, Sherman BT, Lempicki RA. Bioinformatics enrichment tools: paths toward the comprehensive
functional analysis of large gene lists. Nucleic Acids Res 2009; 37: 1–13.
20 Huang DW, Sherman BT, Lempicki RA. Systematic and integrative analysis of large gene lists using DAVID
bioinformatics resources. Nat Protoc 2009; 4: 44–57.
21 Klahr S, ed. Atlas of Diseases of the Kidney Volume Four. Systemic Diseases and the Kidney. Hoboken, Wiley, 1999.
22 Nagaraj N, Mann M. Quantitative analysis of the intra- and inter-individual variability of the normal urinary
proteome. J Proteome Res 2011; 10: 637–645.
23 Adachi J, Kumar C, Zhang Y, et al. The human urinary proteome contains more than 1500 proteins, including a
large proportion of membrane proteins. Genome Biol 2006; 7: R80.
24 Baena A, Porcelli SA. Evasion and subversion of antigen presentation by Mycobacterium tuberculosis. Tissue Antigens
2009; 74: 189–204.
25 Seth M, Lamont EA, Janagama HK, et al. Biomarker discovery in subclinical mycobacterial infections of cattle. PLoS
One 2009; 4: e5478.
26 Tanaka T, Sakurada S, Kano K, et al. Identification of tuberculosis-associated proteins in whole blood supernatant.
BMC Infect Dis 2011; 11: 71.
27 Lyman SD, Escobar S, Rousseau a M, et al. Identification of CD7 as a cognate of the human K12 (SECTM1)
protein. J Biol Chem 2000; 275: 3431–3437.
28 Tumani H, Reiber H, Nau R, et al. Beta-trace protein concentration in cerebrospinal fluid is decreased in patients
with bacterial meningitis. Neurosci Lett 1998; 242: 5–8.
29 Diel R, Loddenkemper R, Zellweger JP, et al. Old ideas to innovate tuberculosis control: preventive treatment to
achieve elimination. Eur Respir J 2013; 42: 785–801.

DOI: 10.1183/09031936.00175113 11