To maximize the resolution and protein identification of cell wall proteins, the delipidated cell wall of Mtb
was subjected to extraction based on solubility within three reagents—an anionic (SDS), a chaotrophic (GuHCl), and a nonionic (TX-114) detergent. 2DGE of all CWP fractions demonstrated the majority of proteins resolving within a range of MW 25−75 kDa (Supplementary Figure 1
). On average, 210 spots were resolved for GuHCl and SDS CWP fractions and 170 from TX-114. From the 2DGE analysis, 290 proteins were identified with 122, 131, and 37 proteins identified in the GuHCl, SDS, and TX-114 fractions, respectively. For all fractions, over half were unique to each subset (Figure A). Analysis of the data demonstrated 80% of proteins had known functions (Figure ). In contrast, the TX-114 extracted fraction was less amenable to gel separation most likely due to its increased hydrophobic content. A multidimensional chromatography approach40,41
was employed to better resolve proteins in this fraction, in order to exploit any unique or uncharacterized protein families within the TX-114 subset. While 2DGE was insufficient in identifying a significant number of proteins in the TX-114 detergent extract (Figure A), SCX chromatography combined with reverse phase chromatography of a digest of this CWP preparation allowed the resolution of 364 proteins, including an additional 294 proteins not found in any of the 2DGE preparations (Figure B).
Figure 1 (A) Identification of cell wall proteins by two-dimensional gel electrophoresis and two-dimensional liquid chromatography. Detergent extraction and separation of cell wall protein subsets via 2DGE demonstrated the majority of proteins to be resolved using (more ...)
Figure 2 The functional category distribution by biological sample. Proteins identified for each extraction method (GuHCl, SDS, TX114) were sorted by functional category as a percentage of total proteins per sample. Representation of all protein groups was identified (more ...)
Validation of a few selected proteins, by Western blot analysis of samples resolved by 2DGE (Supplementary Figure 1
) and SDS-PAGE (Supplementary Figure 2
), corroborated the identification of these proteins by mass spectrometry.
All identified proteins were grouped by functional category as defined by Institute Pasteur, which demonstrated Mtb
protein families present in each extract. Proteins in categories 3 (cell wall and cell wall processes) and 7 (intermediary metabolism) were consistently overrepresented among the CWP preparations (Figure ). Combining the data sets of both gel and non-gel based protein identifications led to the detection of 528 proteins (Supplementary Table S2
). These were further classified into functional groups as defined by the Sanger Institute (Figure A−C). One hundred and five of the 528 cell wall proteins identified were not reported in previous Mtb
including a comprehensive proteomic database http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi
) and http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi
) (Table ).
Figure 3 The functional category distribution of the 528 identified proteins. Assignments were made based on the Sanger Institute gene database. The distributions are among the major functional groups and the subgroups within functional groups I and II. The percentage (more ...)
Cell Wall Protein Identifications Unique to This Study
A majority of proteins were classified in either Category I, Small Molecule Metabolism (35%), or Category II, Macromolecule synthesis and degradation (25%) (Figure ). Subclasses of category I showed 19% classified in Small molecule metabolism-other (I.X), which included the classes: Central intermediary metabolism and Amino acid biosynthesis (Figure ; upper right); and subclasses of category II demonstrated an even distribution of proteins between synthesis/degradation of macromolecules (II.A, II.B, 12.5%) and cell envelope proteins (II.C, 12.1%) (Figure ; lower right).
The secreted proteins of Mtb
have traditionally been characterized as important antigens and immune-modulators. Prior to being exported, many of these proteins are resident within the cell wall where their function remains largely unknown. To find the putative secreted proteins identified within the cell wall proteome, all identified proteins were subject to interrogation against Neural Network (NN) and Hidden Markov model (HMM) algorithms (SignalP, http://www.cbs.dtu.dk/services/SignalP/
) which revealed 18%, 19%, 27% putative secreted proteins in GuHCl, SDS, TX-114 CWP samples, respectively, and 13% in the 2DLC resolved TX-114 CWP fraction. Cumulatively, of the 528 proteins identified in this study, 87 proteins were predicted to contain secretion signals and included the identification of 23 proteins uniquely found in this study (Table ). A majority (60%) of the CWP associated with secretion are indeed within the category of the cell wall and its processes (Figure ). Further, many of these CWP are also membrane associated, either by description22,24,25
or functional annotation. Of the 23 secreted CWP unique to this study, 11 have been described to be involved with small molecule and peptide binding. Additionally, 23 of the 87 secreted proteins are classified as hypothetical or unknown (Categories V and VI of Sanger Institute) illustrating that to a large extent, the functions of these exported proteins are poorly understood.
Secreted Cell Wall Proteins and Putative Lipoproteinsa
Figure 4 Functional classification for putative secreted proteins and lipoproteins. Eighty-seven cell wall proteins were predicted to contain secretion signals and putative lipoprotein motifs. Sixty percent of these proteins are classified within the cell wall (more ...)
Next, we interrogated the CWP to identify those with putative lipoprotein motifs. Here, a total of 16 proteins were predicted by LipoP (LipoP, http://www.cbs.dtu.dk/services/LipoP/
) to contain a signal peptidase II cleavage motif, and an additional 8 proteins were identified when compared to the 99 putative lipoproteins (2.5% of the genome) that reportedly exist within the Mtb
) A majority of the lipoproteins contain no additional functional classification; however, 4 proteins (Rv0928, Rv0934, Rv2864c, Rv3666c) are involved with substrate binding and transport within the periplasm. One of two superoxide dismutases in Mtb
, SodC (Rv0432), was also identified within the lipoprotein-enriched TX-114 CWP fraction. This protein has recently been defined as a highly glycosylated putative lipoprotein and is a defined B-cell antigen.44,45
Other putative lipoproteins identified in this study contain O-glycosylation motifs as predicted by NetOglyc and were found within the Mtb
glycoproteome (Table ).(46
) The function of these dual-modified lipo-glycoproteins, as well as the nature of their modification remain largely undefined.