mycobacterium 
 tuberculosis is the etiological 
 agent of human tuberculosis ( tb ) . despite efforts aimed at the treatment 
 and prevention of tb ,
tb is particularly associated with individuals with compromised immune 
 function , including people co - infected with hiv and those with diabetes . 

as the diabetes epidemic expands in the coming decades , m. 
 tuberculosis poses an ongoing threat . the esx / wxg-100 
 ( wss ) protein exporters directly promote virulence 
 of both mycobacterial and gram - positive pathogens .
the genomes of pathogenic mycobacteria can encode 
 up to four or five independent esx exporters ( esx-15 ) .
the esx / wss exporters are also conserved and functional in several 
 nonpathogenic bacteria . in mycobacterial pathogens ,
the esx-1 system promotes the export 
 of several small protein substrates to both the cell surface and the 
 bacteriological media in vitro.in vivo , the esx-1 system permeabilizes the macrophage 
 phagosomal membrane and engages the human immune response to promote 
 both mycobacterial infection and host resistance .
the esx-1 system is of particular interest 
 because it is lacking 
 from the genome of the m. bovis bacillus calmette - gurin 
 ( bcg ) vaccine strain , which is the only tb vaccine currently in use . the bcg strain is not efficacious at preventing 
 adult pulmonary tb .
understanding how the esx-1 system 
 functions at the molecular level and defining secreted virulence factors 
 that are poised to interface with the human immune response may provide 
 insight into why the vaccine is not protective . in m. 

these include the esx-1 locus ( including the rd1 , region of difference-1 , deletion missing 
 from the bcg strain ) , the espacd ( esxsecretion - associated protein - a etc . ) locus , and the espr gene .
orthologous loci are conserved 
 in the genome of the mycobacterial pathogen mycobacterium 
 marinum , which is an established model for mycobacterial 
 esx-1 export . in m. marinum ,
the major esx-1 locus includes the espe ( mmar_5439 ) through mycp3_1 ( mmar_5459 ) genes ( figure 1 ) .
five protein substrates 
 are encoded within the esx-1 locus , including esxa 
 ( esat-6 ; early secreted antigenic target , 6 kda ) , esxb 
 ( cfp-10 ; culture filtrate protein , 10 kda ) , espe , espf , and espb .
proteins are 
 characterized as substrates if disruption of the esx-1-associated 
 genes results in retention in the mycobacterial cell in vitro .
accordingly , all five substrates are 
 exported across the m. marinum cytoplasmic membrane 
 either to the cell wall or cell surface or into the bacteriological 
 media in vitro in an esx-1-dependent manner ( figure 1 ) . two additional substrates , pe35 
 and ppe68_1 ( mmar_0186 ) , are encoded at another genomic 
 locus but are not known to be required for esx-1 substrate export . in m. tuberculosis , espamt and espcmt
( subscript 
  mt  indicates the proteins are from m. tuberculosis , and 
 
 indicates m. marinum ) are also exported by esx-1 , 
 but this has not been confirmed for the m. marinum espamm and espcmm proteins .
however , when the espamt and espcmt proteins were expressed in m. marinum , they were exported in an esx-1-dependent manner demonstrating that 
 the mechanisms of substrate recognition are conserved between the 
 two species .
the esx-1 region in m. marinum . the 10 kb genomic 
 region including the esx-1 secretion 
 system from m. marinum is shown .
briefly , esp 
 = esx secretion - associated proteins ; ecc = esx conserved components .
genes include letters based on the order of the genes at 
 the esx-1 locus .
transposon ( tn ) insertion strains 
 used in this study are represented with an inverted open triangle . 

additional proteins encoded by genes within the esx-1 locus likely form part of the esx-1 apparatus based on conservation 
 across esx systems and predicted cellular localization .
the  components  include two predicted chaperones , 
 espg1 and ecca1 ( esx conserved components ) ; two ftsk - spoiiie - like aaa atpases , eccca and 
 ecccb ; and two predicted membrane - associated proteins , eccb1 and eccd1 .
several additional proteins encoded 
 by the esx-1 locus ( espk , esph , espl , espj , and espi ) 
 are required for substrate 
 export .
it is unclear if these proteins are esx-1 components or substrates 
 ( or both ) . we have recently 
 identified a second genomic region ( mmar_16631668 ) in m. marinum that is required for esx-1 export 
 and virulence . in both mycobacterial 
 species , disruption of genes within esx-1-associated 
 loci
generally results in the loss of esx-1 substrate export in vitro and attenuation of virulence in cell culture and 
 animal models of infection .
however , recent studies have identified both attenuated m. tuberculosis strains that still secrete esx-1 substrates 
 and virulent m. tuberculosis strains that do not 
 secrete esx-1 substrates into the bacteriological media in 
 vitro .
we found that in m. marinum substrates localized 
 to the cell surface , and not necessarily the culture supernatant , 
 correlate with virulence .
therefore , 
 the complex relationship between esx-1 export and virulence requires 
 further investigation . further confounding our understanding 
 of this relationship are 
 limitations with how we measure the export of esx substrates ; in vitro esx-1
export and secretion assays measuring the 
 presence of substrates in the bacteriological media are generally 
 performed using western blot analysis .
it has been difficult to precisely 
 quantitate change in esx protein secretion using this approach in 
 part because of antibodies that are of poor quality or lacking altogether 
 for most proteins associated with the mycobacterial esx-1 system .
multiple reaction monitoring ( mrm ) has emerged as the proteomics 
 tool to target and precisely determine protein levels directly from 
 complex biological mixtures . in practical 
 terms
mrm functions as a replacement for western blot analysis , allowing 
 highly parallel stacking of targets ( proteins ) . for microbes in particular , 
 mrm data
is also largely portable , and online databases to collect 
 and curate these targets are available .
the 
 esx-1 export apparatus is a multiprotein complex , spanning 
 at least the mycobacterial cytoplasmic membrane .
it is not yet known 
 how or if the esx-1 system promotes translocation of substrates across 
 the mycolate outer membrane ( mom ) and out of the cell .
it is also not well - defined how or if individual esx-1-associated 
 proteins affect the levels and stability of other esx-1 proteins in 
 the export complex .
a lack of understanding of how each esx-1-associated 
 protein contributes to protein export makes it difficult to establish 
 how the esx-1 system works at the molecular level .
elucidating esx-1 
 protein translocation at the molecular level will advance our understanding 
 of mycobacterial virulence mechanisms and potential courses of treatment 
 and prevention of human tb .
it has been reported several times 
 that esx-1 substrates require 
 each other for export .
direct interaction between substrates or shared genetic requirements 
 for export could underlie this observation .
we hypothesized that the quantification of substrate codependency 
 could define rules for identifying novel esx-1 substrates . in a single 
 experiment
we employed targeted mass spectrometry 
 to differentially quantify changes in the levels and export of these 
 esx-1-associated proteins from both secreted and cytosolic protein 
 fractions across the 16 esx-1-deficient m. marinum strains as compared to the wild - type ( wt ) m. marinum m strain .
m. marinum strains were grown at 30 c and 
 maintained 
 on middlebrook 7h11 agar ( accumedia , lansing , mi ) or in 7h9 broth 
 ( accumedia , lansing , mi ) supplemented with 0.1% tween-80 and kanamycin 
 ( 20 g / ml ; ibi , poesta , il ) when appropriate . m. marinum strains used in this study are in supplemental 
 table s1 .
the majority of the strains in this 
 study were obtained from the laboratory of eric j. brown ; these strains 
 are esx-1-deficient and attenuated for virulence .
strains bearing transposon ( tn ) insertions in the eccb1 ( f11 ) , eccd1 ( 17 ) , and espl ( 2 ) genes were identified from a nonsaturating m. marinum tn - insertion library .
the tn - insertions were mapped by extracting and digesting genomic 
 dna from each strain and generating a plasmid library in pbluescript 
 as previously explained .
the plasmid bearing 
 the tn - insertion was selected for kanamycin resistance in e. coli , and the precise site of each insertion was mapped 
 by sequencing analysis using the 821a and 822a primers .
the loss of esx-1 secretion in vitro was confirmed using western blot analysis and nlc 
briefly , m. marinum strains were grown 
 in 7h9 broth to saturation and diluted to an od600 of 0.8 
 in 50 ml of sauton s defined broth with 0.05% tween-80 .
after 
 48 h of growth , the cells were harvested by centrifugation , and the 
 supernatant was filtered to generate culture filtrates ( cf ) .
the cell 
 lysate ( cl ) fractions were generated using a mini - bead - beater-24 ( biospec , 
 bartlesville , ok ) .
culture filtrates were concentrated 100-fold 
 using amicon ultra-4 centrifugal filter units ( emd millipore , temecula , 
 ca ) .
protein concentrations were determined using the microbca protein 
 assay according to the manufacturer s instructions ( thermo 
 scientific pierce ) .
a 10 g portion of cell lysates and cell filtrates was separated 
 using 420% mini protean tgx tris - glycine polyacrylamide gels 
 ( bio - rad ) .
nitrocellulose membranes were incubated with antibodies 
 against esat-6 ( esxa ) ( 1:3000 , abcam ab26246 ) , cfp-10 ( esxb ) ( 1:5000 , 
 pierce pa1 - 19445 ) , mpt32 ( 1:5000 , nr-13807 ; bei resources ) , or groel 
 ( 1:5000 , assay designs
immunoblots for esxa , esxb , and mpt32 were incubated with goat anti - mouse 
 igg ( 1:5000 , hrp - conjugate ) or goat anti - rabbit igg ( 1:5000 ; hrp - conjugate , 
 millipore ) .
the immunoblots were imaged using the lumiglo chemiluminescent 
 substrate kit ( kpl , gaithersburg , md ) .
the immunoblot for groel was 
 incubated with dylight anti - rabbit igg ( 1:10000 , 680 conjugate ) ( cell 
 signaling technologies , danvers , ma ) .
the immunoblot was imaged using 
 a li - cor odyssey imager and analyzed with li - cor odyssey software .
bands resulting from the western blot analysis 
 were quantified using imagej as described here : http://www.di.uq.edu.au/sparqimagejblots .
a 50 g portion 
 of protein as determined by micro bca ( pierce ) from either culture 
 filtrate or whole cell lysate was prepared and digested with trypsin 
 as previously described with the exception 
 that samples were resuspended in 0.2% trifluoroacetic acid ( tfa ) to 
 a final concentration of 500 ng/l , and 5 g aliquots 
 were desalted using a c18 zip tip ( millipore ) according to manufacturer s 
 instructions and then dried .
we utilized two 
 empirical data sources 
 to determine and develop our mrm / srm transitions : existing lc 
we maintain 
 an in - house database of identified m. marinum proteins 
 from culture filtrates and whole cell lysates and an in - house atlas 
 of mrm transitions derived from these protein identifications , many 
 of which have been previously used in other publications and validated 
 with synthetic stable - isotope analogues of the peptides .
in general , our available proteome 
 was collected by triplicate analysis of m. marinum cell lysates and culture filtrates by lc 
ms / ms as described 
 in li et al . , using nano uhplc  ms / ms .
mgf ( matrix science ) peak lists from the ltq - velos orbitrap data 
 were searched against the current m. marinum m strain 
 fasta database using the paragon algorithm in  rapid search 
 mode. fdr rates were determined using the target - decoy method 
 of elias et al . , and fdr rates were < 0.001% 
 for all proteins used in this study .
mrm transitions were determined 
 from these data using skyline software from the maccoss lab ( university 
 of washington ) and mrm pilot ( ab sciex , framingham , ma ) .
peptide and 
 protein validation was performed using a procedure nearly identical 
 to that described for empirical lc 
cross validation was made using independent 
 western blot analysis ( supplemental figure s2 ) as well as the unique aspect here of the availability of mutant 
 strains for most of the monitored proteins .
two independent mutant 
 ecca1 and eccb1 strains were analyzed for verification 
 of phenotypes , clustering , and statistical reproducibility .
individual 
 mrm transitions were tested to ensure peptide profiles degenerated 
 in the presence of strains with genes disrupted for the respective 
 protein product .
stable heavy - isotope analogues of 
 several esx-1 proteins , to validate the normalization and lysis controls , 
 were used as in kennedy et al . for further correction .
the list of endogenous transitions used in the 
 relative method are included as supplemental figure 
 s1 .
protein samples ( 5 g ) 
 were resuspended in 0.3% tfa ( 20 l ) .
then 2 l ( 500 ng ) 
 was injected onto a 100 mm  100 m c18beh column ( waters , 
 milford , ma ) and separated over a 90 min gradient from 2% to 40% b 
 ( a = 0.1% formic acid in water ) ( b = acetonitrile , 0.1% fa ) on an 
 nano ultra2d uhplc ( eksigent , dublin , ca ) running at 650 nl / min .
hplc 
 solvents were of the highest grade available ( burdick and jackson ) . 

mrm - based ms / ms acquisition was performed on a qtrap 5500 ( ab sciex ) 
 running in triple - quadrupole mode .
each sample was analyzed in - triplicate 
 with biological duplicates . in total > 200
peaks were integrated 
 using multiquant 2.0 ( ab sciex ) using intelliquan integration with 
 a 3 point gaussian smooth with no baseline correction .
the apex transition 
 was used for quantification ( quantifier ) , and the remaining transitions 
 were used as qualifiers to determine relative peptide levels and ensure 
 transition ratios .
multiple peptides ( 14 ) were averaged together 
 to arrive at the average peak area for the protein . in several cases ,
the low - molecular weight substrates yielded 
 one well - integrated peptide , which passed all other criteria . statistical 
 analysis for protein quantitation
[ ( cv % 1 ) + ( cv % 2 ) + ( cv % n ) ) ] .
the peak area of the proteotypic 
 peptides from each protein was converted to an area response / ratio 
 against the groes or groel2 cytosolic protein standards to control 
 for lysis .
area ratios of each protein were averaged and normalized 
 to the peptide response from the wt strain to allow direct comparison 
 among proteins between strains .
direct comparisons in protein levels 
 between cl ( cell lysate ) and cf ( culture filtrate ) were largely not 
 performed because the sample complexity and dynamic range vastly differ .
this approach is sensitive to low levels of 
 autolysis and can detect proteins in the secreted fraction that are 
 not necessarily secreted .
we exploited 
 coincidental detection to normalize sample amounts and correct for 
 cytosolic contamination of the cf .
further validation of the stability of differential 
 area - ratio analysis was performed by selected nuhplc 
ms injections 
 using stable heavy - isotope ( aqua ) versions of peptides corresponding 
 to esxa and groes ( new england peptide , 
 gardner , ma ) .
data from these selected absolute quantitative standards 
 are in excellent agreement with the ratios observed by the differential 
 analysis described here .
pearson s 
 correlation coefficients of the proteins were calculated independently 
 from normalized area ratios of each protein compared to the peptide 
 response found in wild - type m. marinum for the cell 
 pellet and cell lysate data sets , and their p values 
 were computed by fitting a linear model using r , an open source statistical 
 analysis environment .
the r code and 
 the raw data files used to calculate the correlation and its significance 
 are included in the supporting information ( supplemental figure s6 and supplemental files ) .
hierarchical clustering 
 was performed using hce 3.5 software ( schneiderman lab ) using normalized 
 peak area mrm data as cell input with peptide ( column ) and mutant / strain 
 ( row ) for consistency .
data were normalized by row ( protein ) across 
 strains using ( x  m)/ 
 to normalize the response ratio between high and low abundance proteins 
 relative to the internal standards .
hierarchical aggregate clustering 
 was employed to cluster the data for both m. marinum strains and esx-1-associated proteins as described in seo et al .
first , we determined calculated 
 protein ratios from the mrm - peptide measurements , their respective 
 % cv , and then the propagated error to determine the analytical robustness . 

a paired two - tailed student s t test was used 
 to compare the protein ratios for each protein between the wt m. marinum and esx-1-deficient strains ( microsoft excel ) .
a 50 g portion 
 of protein as determined by micro bca ( pierce ) from either culture 
 filtrate or whole cell lysate was prepared and digested with trypsin 
 as previously described with the exception 
 that samples were resuspended in 0.2% trifluoroacetic acid ( tfa ) to 
 a final concentration of 500 ng/l , and 5 g aliquots 
 were desalted using a c18 zip tip ( millipore ) according to manufacturer s 
 instructions and then dried .
we utilized two 
 empirical data sources 
 to determine and develop our mrm / srm transitions : existing lc 
we maintain 
 an in - house database of identified m. marinum proteins 
 from culture filtrates and whole cell lysates and an in - house atlas 
 of mrm transitions derived from these protein identifications , many 
 of which have been previously used in other publications and validated 
 with synthetic stable - isotope analogues of the peptides .
in general , our available proteome 
 was collected by triplicate analysis of m. marinum cell lysates and culture filtrates by lc 
mgf ( matrix science ) peak lists from the ltq - velos orbitrap data 
 were searched against the current m. marinum m strain 
 fasta database using the paragon algorithm in  rapid search 
 mode. fdr rates were determined using the target - decoy method 
 of elias et al . , and fdr rates were < 0.001% 
 for all proteins used in this study .
mrm transitions were determined 
 from these data using skyline software from the maccoss lab ( university 
 of washington ) and mrm pilot ( ab sciex , framingham , ma ) .
peptide and 
 protein validation was performed using a procedure nearly identical 
 to that described for empirical lc 
cross validation was made using independent 
 western blot analysis ( supplemental figure s2 ) as well as the unique aspect here of the availability of mutant 
 strains for most of the monitored proteins .
two independent mutant 
 ecca1 and eccb1 strains were analyzed for verification 
 of phenotypes , clustering , and statistical reproducibility .
individual 
 mrm transitions were tested to ensure peptide profiles degenerated 
 in the presence of strains with genes disrupted for the respective 
 protein product .
stable heavy - isotope analogues of 
 several esx-1 proteins , to validate the normalization and lysis controls , 
 were used as in kennedy et al . for further correction .
the list of endogenous transitions used in the 
 relative method are included as supplemental figure 
 s1 .
protein samples ( 5 g ) 
 were resuspended in 0.3% tfa ( 20 l ) .
then 2 l ( 500 ng ) 
 was injected onto a 100 mm  100 m c18beh column ( waters , 
 milford , ma ) and separated over a 90 min gradient from 2% to 40% b 
 ( a = 0.1% formic acid in water ) ( b = acetonitrile , 0.1% fa ) on an 
 nano ultra2d uhplc ( eksigent , dublin , ca ) running at 650 nl / min .
hplc 
 solvents were of the highest grade available ( burdick and jackson ) . 

mrm - based ms / ms acquisition was performed on a qtrap 5500 ( ab sciex ) 
 running in triple - quadrupole mode .
peaks were integrated 
 using multiquant 2.0 ( ab sciex ) using intelliquan integration with 
 a 3 point gaussian smooth with no baseline correction .
the apex transition 
 was used for quantification ( quantifier ) , and the remaining transitions 
 were used as qualifiers to determine relative peptide levels and ensure 
 transition ratios .
multiple peptides ( 14 ) were averaged together 
 to arrive at the average peak area for the protein . in several cases ,
the low - molecular weight substrates yielded 
 one well - integrated peptide , which passed all other criteria .
[ ( cv % 1 ) + ( cv % 2 ) + ( cv % n ) ) ] .
the peak area of the proteotypic 
 peptides from each protein was converted to an area response / ratio 
 against the groes or groel2 cytosolic protein standards to control 
 for lysis .
area ratios of each protein were averaged and normalized 
 to the peptide response from the wt strain to allow direct comparison 
 among proteins between strains .
direct comparisons in protein levels 
 between cl ( cell lysate ) and cf ( culture filtrate ) were largely not 
 performed because the sample complexity and dynamic range vastly differ .
this approach is sensitive to low levels of 
 autolysis and can detect proteins in the secreted fraction that are 
 not necessarily secreted .
we exploited 
 coincidental detection to normalize sample amounts and correct for 
 cytosolic contamination of the cf .
further validation of the stability of differential 
 area - ratio analysis was performed by selected nuhplc 
ms injections 
 using stable heavy - isotope ( aqua ) versions of peptides corresponding 
 to esxa and groes ( new england peptide , 
 gardner , ma ) .
data from these selected absolute quantitative standards 
 are in excellent agreement with the ratios observed by the differential 
 analysis described here .
pearson s 
 correlation coefficients of the proteins were calculated independently 
 from normalized area ratios of each protein compared to the peptide 
 response found in wild - type m. marinum for the cell 
 pellet and cell lysate data sets , and their p values 
 were computed by fitting a linear model using r , an open source statistical 
 analysis environment .
the r code and 
 the raw data files used to calculate the correlation and its significance 
 are included in the supporting information ( supplemental figure s6 and supplemental files ) .
hierarchical clustering 
 was performed using hce 3.5 software ( schneiderman lab ) using normalized 
 peak area mrm data as cell input with peptide ( column ) and mutant / strain 
 ( row ) for consistency .
data were normalized by row ( protein ) across 
 strains using ( x  m)/ 
 to normalize the response ratio between high and low abundance proteins 
 relative to the internal standards .
hierarchical aggregate clustering 
 was employed to cluster the data for both m. marinum strains and esx-1-associated proteins as described in seo et al .
first , we determined calculated 
 protein ratios from the mrm - peptide measurements , their respective 
 % cv , and then the propagated error to determine the analytical robustness . 

a paired two - tailed student s t test was used 
 to compare the protein ratios for each protein between the wt m. marinum and esx-1-deficient strains ( microsoft excel ) .
targeted proteomics ( mrm)-based assays are well - suited 
 for the analysis of proteins from microbial systems and particularly 
 well - suited for the analysis of mycobacterium spp .
we previously used targeted proteomics 
 on nano - hplc - separated peptide digests of bacterial proteins ( nlc  mrm - ms ) 
 to detect the secretion of individual esx-1 substrates , including 
 those that were largely undetectable by western blot analysis .
mrm has recently been used to determine the 
 genetic requirements for export of the esxh and esxg substrates of 
 the esx-3 system in mycobacterium smegmatis .
mrm - based analysis is suited to generate relative 
 detection of numerous substrates simultaneously .
mrm - based approaches 
 can also be made specific to domains of proteins and facilitate a 
 substantially larger potential set of controls .
contamination , cellular 
 lysis , gene duplications , actual changes in secretion , and protein loading can greatly affect the denominator used to 
 normalize secreted / compartmented proteomics .
targeted multiplexed mrm allows development of appropriate normalization 
 proteins and schemes simultaneously , including the use of multiple 
 normalization proteins such as mmar_2929 , groel2 , groes , mpt64 , and 
 mpt32 used here and in numerous mycobacterial studies .
we hypothesized 
 that by using nlc  mrm - ms on a larger scale to measure esx-1-associated 
 proteins in a collection of isogenic m. marinum strains 
 bearing tn insertions or deletions in the esx-1 locus , 
 we could both identify esx-1 substrates and gain a deeper insight 
 into the molecular mechanisms of esx-1 export . to address this hypothesis
, 
 we induced esx-1 export in m. marinum by growth in 
 sauton s defined medium and generated cell lysate ( cl ) and 
 culture filtrate ( cf ) protein fractions from each strain ( supplemental table s1 ) .
the protein fractions 
 were digested with trypsin to generate proteotypic peptides for detection 
 and quantitation .
we quantified changes in esx-1 protein levels within 
 and secreted from each esx-1-deficient m. marinum strain normalized to levels in the wt m. marinum using nlc 
mrm in a single experiment ( see supplemental figure s1 for mrm transitions monitored in this 
 study ) .
the peak area for each peptide was normalized for total quantity 
 and autolysis ( false - secretion ) to stable cytoplasmic proteins , chiefly 
 groel2 and groes .
the area ratio for each protein - derived peptide 
 was then compared directly with the exact same peptide response from 
 the wt m. marinum to allow direct comparison among 
 strains and proteins ( see experimental procedures ) . to test our approach , we selected the proteomics data describing 
 the relative levels of the best - characterized and most abundant esx-1 
 substrates , esxa and esxb .
the data from this study are presented 
 as figure 2a and b ( log2 transformed 
 data in supplemental figure s4 ) . in parallel , 
 we also separately conducted western blot analysis followed by quantitation 
 by densitometry , which is presented as supplemental 
 figure s2 . for esxa and esxb , the m. marinum rd1 , esxa , and esxba strains , which lack the esxa and/or the esxb genes , served as controls for specificity for both 
 nlc  mrm and western blot analysis . in these strains , esxa and 
 esxb
were not detected in the cl and cf fractions , indicating that 
 both detection methods were specific for the esxa and esxb proteins 
 ( figure 2a and b and supplemental 
 figure s2 ) .
the western blot analysis using the same samples 
 ( supplemental figure s2 ) contained 10 g 
 of protein from m. marinum fractions to detect 12 
 protein bands per analysis with virtually no dynamic range or quantification . 
 here , each proteomic analysis utilized 20-fold less material , with 
 multiplexing of target proteins and controls simultaneously .
further 
 dilution experiments and the inherent linearity of triple - quadrupole 
 instrumentation gave at least 3.5 orders of magnitude in linear dynamic 
 range for all substrates and sensitivities < 100 amol on column 
 per protein ( confirmed by selected aqua peptides , data not shown ) . 

the dynamic range of western blots for esxb and esxa for which we 
 have reliable antibodies was < 22.5 orders of magnitude 
 ( supplemental figure s2 ) .
absolute peak 
 intensities for esxb often exceed 5  10 cps , and 
 we can typically detect these peptides down to ca . 300 cps ( s / n > 5:1 ) .
this is nearly 5-orders 
 of 
 linear dynamic range and represents a potential reduction in starting 
 material to detect esxb from less than 0.5 ng of trypsin - digested 
 cf ( < 0.005 % of what was loaded for western blot analysis ) .
the 
 reproducibility of the measurements here ( cv  10% ) further 
 demonstrates the superior precision of our approach .
relative esxa levels within the 
 cell lysate ( cl , blue bars ) or in the culture filtrate ( cf , red bars ) 
 generated from esx-1-deficient strains as compared to wild - type m. marinum .
for esxa 
 and esxb , the rd1 , esxa , and esxba strains served as negative controls for esxa and esxb 
 detection and demonstrated the specificity of the approach .
mrm 
 analysis of the levels of the espb substrate in the cl and cf relative 
 to the levels of espb in the wild - type strain .
for espb , the espb::tn strain served as a negative control for espb detection 
 and demonstrated the specificity of the approach .
mrm 
 analysis of the levels of the espf substrate in the cl and cf .
error bars represent the average propagated standard error 
 and were calculated as described in the experimental 
 procedures . in all graphs changes in the levels of protein 
 in each of the esx-1-deficient strains as compared to the wild - type 
 strain were considered significantly different from each other if 
 the p value from a two - tailed student s t test was 0.05 .
changes in protein levels between 
 the wild - type and each esx-1-deficient strain that were not statistically 
 significant ( p  0.05 ) were as follows : ( a ) 
 the levels of esxa in the cell lysates from the espi , eccd1 , espj , and espk strains were not statistically different from the levels 
 in the wild - type strain .
( b ) the levels of esxb in the cell lysates 
 from the eccd1 , espj , 
 and espk strains were not statistically different 
 from the levels in the wild - type strain .
( c ) the levels of espb in 
 the cell lysates from the eccb1 - 2 , ecccb , esxba , esxa , eccd1 , espk , and espl were not significantly different from the levels in the wild - type 
 strain .
( f ) the levels of esxn in the lysates from the rd1 , espg1 , eccb1 - 2 , espi , eccd1 , espj , espk , espl , and espb strains were not statistically different from the wild - type strain . 

the levels of esxn in the culture filtrate generated from the ecccb , eccd1 , and espj strains were not statistically different from the wt strain .
the 
 actual p values are listed in the supporting information ( table s2 ) .
log2 transformed 
 versions of the cf data are available in supplemental 
 figure s4 .
relative to wt m. marinum , the levels of esxa 
 and esxb in the cl were significantly reduced in the majority of esx-1-deficient m. marinum strains .
the levels of esxa were comparable to 
 wt m. marinum in strains bearing transposon insertions 
 in the eccd1 , espj , and espk genes
. the decreased level of esxa in the espi::tn strain was not significantly different from that in wt m. marinum ( p = 0.0777 ) .
the levels of 
 esxb in the ecccb::tn , espj::tn , 
 and espk::tn strains were not significantly different 
 from that in wt m. marinum .
esxb , but not esxa , significantly 
 accumulated in the eccd1::tn strain relative 
 to the wild - type strain ( p = 0.0014 and 0.9782 for 
 esxb and esxa , respectively , as compared to wt m. marinum ) .
we conclude from this subset of the data that for detecting relative 
 levels of esxa and esxb in the cl , the nlc  mrm - ms analysis 
 was more robust at measuring changes in protein levels than western 
 blot analysis .
moreover , to our knowledge , this is the first report 
 of accumulation of the esxb substrate in the absence of esx-1 export .
esx-1 substrates are defined as proteins that are present in the 
 cf in an esx-1-dependent manner .
we expected 
 to measure decreased levels of substrates in the cf fractions generated 
 from the esx-1-deficient strains .
consistent with previous reports , 
 we found that the levels of esxa and esxb from m. marinum were significantly decreased in the cf protein fractions from the 
 esx-1-deficient strains as compared to those generated from wt m. marinum ( figure 2a and b ; esxb p  0.0007 , esxa p  0.0277 ; supplemental table s2 ) .
specific disruption of 
 the eccd1 gene by transposon insertion 
 abrogated the export of both esxb and esxa .
we conclude from these 
 data that , as in m. tuberculosis , the eccd1 gene in m. marinum is required for 
 esx-1 secretion .
it was previously reported by western blot 
 analysis that disruption 
 of espk did not affect the secretion of esxb .
the levels of esxa and esxb secretion from the espj and espk tn - insertion strains were 
 intermediate and significantly different from the levels of esxa and 
 esxb in the cfs generated from both wt m. marinum and the eccd1::tn strains ( for esxb , eccd1 vs espkp = 0.0011 ; eccd1 vs espjp = 0.0045 ) .
note that transposon insertions in 
 the espk and espj genes resulted 
 in a loss of espk and espj proteins , respectively ( figure 4 ) .
the intermediate secretion phenotype can not be 
 attributed to intermediate or residual levels of functional espk or 
 espj proteins in the m. marinum strains . to our knowledge 

this is the first report of an intermediate secretion phenotype for 
 the export of esxa and esxb into the bacteriological media during 
 mycobacterial growth in vitro .
however , all of 
 the esx-1-deficient strains used in this study , including the espk strain , are attenuated 
 for virulence .
thus , it is interesting to consider that 2-fold 
 reductions in the levels of known esx-1 substrates in the cf may reflect 
 attenuation of m. marinum strains .
two additional esx-1 substrates encoded by the esx-1 locus in m. marinum include espb and espf .
we conducted a similar analysis of the relative levels across esx-1-deficient 
 strains and wt m. marinum for the espb and espf substrates 
 ( figure 2c and d , supplemental 
 figure s4 ) .
espb was absent from the cl and cf fractions generated 
 from the espb::tn strain , strengthening the specificity 
 of our approach .
the levels of espb in the cl generated from the esx-1-deficient 
 strains were generally comparable to the wt m. marinum , with the exception of the strains with disruptions in espg1 , ecca1 , and espi , and the rd1 strain ( figure 2c ; for 
 these strains , p  0.0203 ; supplemental table s2 ) .
we found that 
 disruption of eccb1 , eccd1 , and espl also prevented the export 
 of espb into the cf , expanding the requirements for espb export .
for 
 the strains with insertions in espg1 , espj , and espi , intermediate levels of 
 espb were detected in the cf .
interestingly , in previous reports , 
 espb was not detectable in the cl or cf generated from the ecca1::tn strains using western blot analysis .
here we observed reduced but detectable levels 
 of espb in the cl generated from the ecca1::tn strains , reflecting the sensitivity of this approach .
finally , 
 we found that deletions in the esxa and esxb genes resulted in a loss of espb export into the cf .
, which indicate that the 
 export of esxba is required for the export of espb .
the levels of espf were significantly reduced 
 in the cl and cf fractions 
 generated from every esx-1-deficient strain as compared to the wt m. marinum ( figure 2d , p  0.0093 for cf and p  0.0024 ; supplemental table s2 ) .
these data indicate that 
 espf is distinct from the other known substrates in that it is decreased 
 in the absence of a functional esx-1 system .
these results were consistent 
 with our original finding of reduced espf stability in multiple esx-1-deficient 
 strains . to control the above experiment 
 for changes in general secretion , 
 we next compared the relative levels of mmar_2929 , a secreted protein 
 with a sec signal sequence , and esxn , a substrate of the esx-5 export 
 system , across the wt and esx-1-deficient m. marinum strains .
the sec secretion system and the esx-5 system are thought 
 to be independent of the esx-1 secretion system .
the data from mmar_2929 and esxn are shown 
 in figure 2e and f , respectively .
we measured 
 significant changes ( both increases and decreases ) in the levels of 
 mmar_2929 in the cl and cf fractions generated across the esx-1-deficient 
 strains as compared to the wt strain ( figure 2e , supplemental figure s4 ) .
however , compared 
 to esxa and esxb ( figure 2a and b ) , the levels 
 of mmar_2929 in the cf were decreased only in strains where the levels 
 in the cl were also decreased
. significantly lower levels of esxn 
 were secreted into the cf when transposon insertion or deletion disrupted 
 the esx-1 in several cases ( figure 2d , supplemental figure s4 ) .
however , 
 as compared to esxa and esxb , none of the disruptions within the esx-1 locus completely abrogated esxn secretion into the 
 cf .
one possible interpretation is that the esx-1 and esx-5 systems 
 share genes not included in this study . if the two systems were linked 
 by as of yet - undefined genes , disruption of esx-1 secretion could 
 lead to decreased esxn secretion
alternatively , these results may 
 be statistically significant due to the sensitive nature of our approach , 
 but not biologically relevant . in support of this idea ,
the export 
 of mmar_2929 and esxn do not correlate with the known and additional 
 esx-1 substrates presented here , as we illustrate below . from 
 these data , we conclude that we have established a novel assay 
 that accurately and sensitively measures esx-1-mediated substrate 
 secretion into the bacteriological media in vitro .
mrm assay recapitulates most of the 
 published observations about esx-1 substrate stability , as well as 
 the independence of esx-1 on the general sec pathway . in doing so
, 
 we identified several intermediate esx-1 secretion defects as a result 
 of disruption of the esx-1 locus .
the intermediate 
 secretion defects were widely missed using standard western blot analysis 
 but commonly observed using the nlc  mrm - ms approach .
importantly , 
 this assay is scalable based on the design of the normalization controls . 

as new esx-1-deficient strains are identified , the levels of the proteins 
 of the existing target list can be measured and compared to the data 
 presented here without regenerating the entire data set .
finally , 
 we contribute the first single - data - set quantitative assessment of 
 the changes in both the levels and secretion of the esx-1 substrates 
 into the bacteriological media in vitro .
we postulated that the levels of esx-1 substrates 
 should statistically correlate across cl and cf fractions generated 
 from the strain collection in this study because they all require 
 the esx-1 system for export . to test this idea , we calculated the 
 pearson s correlation and measured the significance by fitting 
 the data to a linear model to determine if the levels of esx-1-associated 
 proteins in the cl and cf fractions significantly correlated across 
 the collection of m. marinum strains .
accordingly , 
 we found that the known substrates , espf , espb , esxa , and esxb , all 
 significantly correlated with each other in the cf ( table 1 , r  0.9275 ) .
conversely , 
 proteins in the cf as a result of lysis or the active secretion by 
 a non - esx-1 system did not significantly correlate with esx-1 substrates . 

rather , proteins secreted independently of esx-1 , including mmar_2929 
 and esxn , correlated with the presence of known cytoplasmic or membrane 
 components of the esx-1 system ( for example , ecca1 ) in 
 the cf , which were detected due to a combination of cell lysis and 
 the high sensitivity of this approach .
pearson s 
 correlation 
 coefficients ( r ) were calculated using the levels 
 of esx-1-associated and control ( esxn and mmar_2929 ) proteins in the 
 culture filtrate protein fractions .
the significance of the correlation 
 coefficients was measured on the basis of p values 
 computed by fitting a linear model .
( * p  
 0.05 ; * * p  0.01 , colored grey ) .
5 and s superscripts refer to proteins secreted 
 by the esx-5 and sec secretion systems , respectively .
the control proteins 
 ( mmar_2929 and esxn ) did not significantly correlate with esx-1 substrates 
 in the cf .
we further reasoned 
 that statistical correlation with known substrates 
 in cf could identify esx-1 substrates based on correlation - clustering . 
 in the cf , three additional esx-1-associated proteins , espj , espk 
 and ppe68 ,
significantly correlated with the known esx-1 substrates 
 but not with the esx-1-independent controls , mmar_2929 and esxn . to confirm that espk , espj , and ppe68 were esx-1 substrates , we 
 mined the nlc  mrm - ms experiment to determine the relative levels 
 of espk , espj and ppe68 in and secreted from wt m. marinum as compared to the esx-1-deficient strains .
the results of this 
 analysis are shown in figure 3 . the rd1 strain and the espk::tn 

on the basis of the absence of detectable espk levels in these two 
 strains , we validated the espk measurements .
espk was also absent 
 from the strain bearing the transposon insertion in espl , the gene transcribed immediately upstream of espk , indicating the two could be operonic ( figure 1 ) .
espk was clearly detected in the cl and cf fractions generated 
 from wt m. marinum ( figure 3a and supplemental figure s4 ) .
espk levels 
 were significantly reduced in cl generated from all esx-1-deficient 
 strains except the espg1::tn strain ( for 
 all strains except espg1::tn , p  0.0005 in the cl ; for espg1::tn , p = 0.6519 ) .
espk levels in the cf 
 were significantly reduced in all of the strains tested as compared 
 to wt m. marinum ( p  0.0001 ) . 
 from these results
we conclude that the presence of espk in the cf 
 fraction was dependent upon esx-1-associated genes , indicating that 
 espk is indeed an esx-1 substrate .
these findings expand upon sani 
 et al . , who demonstrated that espk was found in the capsule of m. marinum in an esx-1-dependent manner .
the addition of n- and c - terminal protein tags to espk abrogated 
 secretion by esx-1 .
our findings support 
 the necessity of analyzing espk and other esx components without epitope 
 tags or indirect reporters .
mrm 
 analysis of the levels of espk in cell lysate ( cl , blue bars ) and 
 culture filtrate ( cf , red bars ) relative to the levels of espk in 
 the wt m strain . the rd1 and espk::tn strains 
 served as a negative control for espk detection and demonstrate specificity 
 of the approach .
( b ) nlc  mrm analysis of the levels of the 
 espj substrate in the cl and cf .
the rd1 strain and espj::tn strain serve as a negative control for espj detection . 

we report a weak false positive signal indicating extremely low levels 
 of espj peptides in the espj::tn strain .
one espj 
 peptide was observed at < 0.5% of the levels in the espb::tn strain and < 0.2% of the espj levels observed in the wt m 
 strain .
this low false - positive signal was due to the large espj signal 
 observed in the espb::tn strain , which was the previous 
 nlc injection on the mass spectrometer .
for example , the espj tryptic 
 peptide tssmstaadiyak was present at 1ecps2 in 
 the m strain and was not detected in rd1 or the espj::tn strains .
aeplavdpar is a high - intensity proteotypic peptide 
 for espj that was measured at > 2ecps2 in the wt strain 
 and 1.3ecps2 in the espb::tn strain , 
 which was injected immediately prior to espj::tn 
 analysis .
further evidence of this is the gradual reduction in carryover 
 signal with each successive analysis of the espj::tn 
 strain .
( c ) nlc  mrm analysis of the levels of ppe68 in the 
 cl and cf .
error bars represent the average propagated standard 
 error and were calculated as described in the experimental 
 procedures .
the differences between the levels of each protein 
 in the wt and each esx-1-deficient strain were considered statistically 
 significant if p values were less than 0.05 based 
 on a two - tailed student s t test .
the levels 
 of the indicated protein in each esx-1-deficient strain were significantly 
 different from the wild - type levels with the following exceptions : 
 the levels of espk in the cell lysates of the espg strain were not different from the levels of espk in the wild - type 
 lysate .
the levels of ppe68 in the cell lysate from the espj strain were not significantly different from the wild - type strain . 

the levels of ppe68 in the culture filtrate generated from the espk strain were not significantly different from the levels 
 of ppe68 in the culture filtrate from the wild - type strain .
we readily detected espj in both 
 cls and cfs generated from wt m. marinum ( figure 3b ) .
as such , espj 
 was not detectable in the cl from the rd1 strain , indicating 
 that we were specifically detecting espj by nlc  mrm .
the espj peptide was observed at < 0.5% of the espj 
 levels in the espb::tn and < 0.2% of the espj levels 
 in the wt strain .
this low false - positive signal was inconsistent 
 with the genetics and was found due to the large espj signal observed 
 specifically in the espb::tn strain , which arbitrarily 
 was the prior nlc injection on the mass spectrometer .
this was the 
 only case of measurable carryover we observed in the entire experiment 
 ( see figure 3 legend for more detail ) .
the 
 levels of espj were otherwise significantly altered in all of the 
 cl fractions for all esx-1-deficient strains tested ( p  0.0114 ) even considering this level of background .
we interestingly 
 observed a significant increase in the levels of espj in several strains 
 indicating accumulation of espj in the mycobacterial cell ( ecccb::tn , esxba , esxa and eccd1::tn ) .
for all 
 mutant strains tested , the levels of espj were significantly decreased 
 in the cf ( p  0.0001 ) .
we have experimentally 
 confirmed that espj is a substrate of the esx-1 system , as predicted 
 by daleke et al . on the basis of the yxxxd / e motif .
ppe68 was detected in the cl and cf from the m. marinum m strain ( figure 3c ) . 

accordingly , we did not detect 
 the ppe68 protein in the 
 cl or cf generated from the rd1 strain , indicating that we 
 were indeed measuring ppe68 .
ppe68 levels were significantly reduced 
 in the cl derived from all of the esx-1-deficient strains tested ( p  0.0059 ) except the espj::tn 
 strain ( p = 0.3475 ) .
interestingly , the levels of 
 ppe68 in the cf were significantly reduced from all esx-1-deficient 
 strains ( p  0.0393 ) as compared to the wt 
 strain , except for the espk::tn strain ( p = 0.7889 ) .
two reports demonstrated that a protein related 
 to ppe68 ( mmar_0186 , 
 ppe68_1 ) encoded outside of the esx-1 locus was an 
 esx-1 substrate .
although ppe68 and ppe68_1 are 
 related proteins , the peptides used in this study to measure ppe68 
 are not conserved in ppe68_1 , allowing us to distinguish between the 
 two proteins .
here we demonstrated for the first time that the ppe68 
 protein encoded by the esx-1 locus was secreted or 
 shed into the cf in an esx-1-dependent manner .
overall , we demonstrate 
 the development of a proteomics - based workflow 
 that successfully identified three additional esx-1 substrates in m. marinum , whose genes are conserved in the human pathogen m. tuberculosis .
we demonstrate that identifying proteins 
 that significantly correlate with known esx-1 substrates in the culture 
 filtrate can identify novel esx-1 substrates , which are key virulence 
 factors in disease caused by mycobacterial and gram - positive pathogens . 

this approach , while applied here to the esx-1 system , is broadly 
 applicable to any protein secretion system , many of which play direct 
 roles in bacterial pathogenesis . to further interpret the data we generated by this 
 approach and to elucidate relationships between substrates , substrate 
 codependency , and differences between esx-1-deficient strains
first , we plotted the relative 
 levels of the esx-1 substrates in the cf from each strain to compare 
 the genetic requirements for esx-1 substrate export ( figure 4 ) .
second , we performed 
 a hierarchical clustering array of esx-1-associated proteins across 
 the esx-1-deficient strains ( figure 5 ) .
the 
 clustering of individual peptides ( columns ) vs strain ( rows ) as compared 
 to clustering the average for each protein was performed to highlight 
 potential differences in peptide clustering .
most esx-1-associated 
 peptides cluster together as a protein , such as esxn , but some such 
 as esph and espj exhibit differential clustering depending on where 
 in the protein ( n- or c - terminus ) the tryptic peptide was derived . 

one explanation for this is that the dynamic range of esx-1 export 
 magnifies the apparent clustering differences ; however , there is some 
 evidence of proteoforms of esx-1 that may be biological or sample - handling - associated .
preserving the peptide - centric clustering of these substrates presents 
 the possibility of teasing apart what , if any , contribution proteoform 
 fragments play in the underlying biology .
the levels of secreted proteins present 
 in the cf of the esx-1-deficient strains relative to the wt strain 
 illustrate divergent genetic requirements for substrate export .
brackets 
 with asterisks highlight strains deficient for export of all of the 
 substrates tested , similar to the rd1 strain .
hierarchical clustering was performed on the quantitative peak area 
 response of esx-1-derived peptides vs wt and the esx-1-deficient m. marinum strain library .
red intensity indicates higher 
 expression , and green indicates lower expression vs the mean value 
 for each gene product .
mrm 
 data from ( a ) culture filtrate ( cf ) and ( b ) cell lysate ( cl ) peak 
 response .
displayed 
 dynamic range for cf ( log2 1.874.96 green  red ) 
 and cl ( log2 2.144.32 green  red ) . 

novel esx-1 substrates ppe68 , espj , and espk are readily apparent 
 from examination of the cf data , and loss of substrate stability in 
 esx-1 mutants is also well represented in cl fractions . white boxes 
 indicate peptides that are absent due to interruption or loss of the 
 gene that encodes the protein .
for example , in the esxa deletion strains , the peptides corresponding to esxa are boxed in 
 white .
columns are listed for each protein for simplicity but represent 
 individual peptide responses as in supplemental 
 figure s1 .
all of the substrates 
 tested had an absolute requirement for ecccb , 
 eccb1 , esxa , esxb , and espl for export ( figure 4 , marked with an asterisk ; see supplemental figure s5 for a model ) .
the cf levels of esx-1 
 substrates generated from these strains all resembled the cf levels 
 of esx-1 substrates generated from the rd1 strain , which is 
 deficient for the production of several esx-1-associated proteins . 

cluster analysis performed of the cf fraction at the protein and strain 
 level indicated a relationship among all of these strains with the 
 exception of the ecccb::tn strain ( figure 5a ) . in the espl - deficient strain , 

all of the esx-1-associated proteins measured in this study were also 
 decreased in the cl as compared to wt m. marinum ( figure 5b ) .
these proteins , including esxa and esxb , may 
 form a core part of the esx-1 apparatus that actively exports the 
 known esx-1 substrates . alternatively , 
 if there is a temporal order to esx-1 substrate export , esxa and esxb 
 may be required for the export of the other substrates because they 
 are exported prior to other substrates .
our findings indicate , 
 consistent with previous findings , that 
 esxa and esxb share export requirements .
the levels of esxa and esxb significantly and substantially 
 correlated across all m. marinum strains in the cf 
 ( r = 0.9865 , p = 2.132  10 ) .
both proteins were absent from the cf if any gene in the esx-1 region was disrupted 
 or deleted ( figure 4 , figure 5a ) with the exceptions of the espk and espj genes .
likewise , both proteins were exported to intermediate 
 levels in strains with disruptions in the espk or espj genes ( figure 4 , figure 5a ) . this highly significant correlation likely reflects 
 the molecular relationship between esxa and esxb ; the two are encoded 
 in an operon , directly interact and form a 1:1 dimer , and require 
 each other for stability .
interestingly , the espj and espk substrates shared genetic 
 requirements 
 for export into the cf .
both espj and espk were absent from cfs generated 
 from the majority of the strains in this study ( figures 4 and 5a ) .
disruption of the espg1 , espb , and espi genes resulted in intermediate secretion phenotypes for both proteins . 

consistent with the shared export requirements , the levels of espj 
 in the cf highly and significantly correlated with espk ( r = 0.9854 , p = 3.704  10 ; table 1 , supplemental 
 figure s3 ) .
the correlation between espj and espk was comparable 
 to those measured for the esxa and esxb pair , indicating that there 
 is likely a molecular relationship between espj and espk that is yet 
 undefined .
the export requirements for the espj and espk proteins 
 differed from the esxa and esxb proteins , indicating that these substrates 
 are not absolutely dependent upon each other for export . espj and 
 espk
are not encoded by genes that can form an operon , so the shared 
 requirements for export can not simply be due to co - transcription ( figure 1 ) .
espj and espk are both proteins of unknown function ; 
 espk is classified as a proline- and alanine - rich protein .
interestingly , 
 bioinformatics analysis revealed that while m. tuberculosis h37rv has a single gene encoding espk and espj , the m. marinum genome contains 3 
 additional espj paralogs and 5 additional espk paralogs ( supplemental table s3 ) .
the paralogs vary with respect to conservation with each other 
 and with the m. tuberculosis orthologs .
moreover , 
 for all of the espj paralogs there are espk paralogs either adjacent or nearby .
possible explanations for both of the correlations 
 we observed and for the genomic organization that require further 
 testing include that espk and espj interact or are secreted simultaneously 
 ( supplemental figure s5 ) .
the espb , 
 espf , and ppe68 substrates had distinct genetic requirements 
 for export from the other esx-1 substrates measured here ( figures 4 and 5a ) .
based on these 
 comparisons , the ppe68 substrate was clearly divergent as compared 
 to the other monitored substrates .
for example , ppe68 did not require 
 the espk , espj , or espb substrates for export ( figure 4 ) . instead
, ppe68 levels were significantly increased in the 
 cf protein fraction generated from the espj::tn strain 
 ( p = 0.0123 ) .
ppe68 levels correlated significantly 
 with a subset of esx-1 substrates in the cf , including esxb , esxa , 
 and espf ( table 1 ) .
the relative levels of 
 espf and ppe68 in the esx-1-deficient strains were very similar in 
 the cl across the strains tested in this study ( r = 0.9456 , p = 9.48  10 ) , which likely represents a molecular relationship between the espf 
 and ppe68 proteins .
consistent with this idea , both espf and ppe68 
 directly interact with the component ecca1 ( supplementary figure s5 ) .
our findings indicate that the codependency in both secretion 
 and 
 stability of esx-1 substrates are not absolute .
indeed , several reports 
 have indicated exceptions to codependent export . for example , espfmt is not required for the secretion of esxamt and esxbmt .
one possibility is that the different groups 
 of substrates represent components that are not strictly necessary 
 for secretion in vitro or are needed at later points 
 in apparatus assembly or function .
as the number of esx-1 substrates 
 grows , it is likely that the dependency on other substrates will be 
 resolved . across all of our analyses ,
we uncovered only two examples 
 of significant substrate accumulation ( esxb in the cl of the eccd1::tn strain and espj in the cl of the ecccb::tn , esxba and eccd1::tn strains ) .
in a recent report using nlc  mrm to study the 
 esx-3 substrates , esxg and esxh , these proteins failed to accumulate 
 in the cl fractions generated from esx-3-deficient m. smegmatis strains .
these findings indicate that 
 the levels of esx substrates are likely regulated , and stability in 
 the absence of export is rare .
although the substrate secretion 
 profile generated from some esx-1-deficient 
 strains mirrored that from the rd1 strain ( ecccb::tn , eccb1::tn , esxba , and espl::tn , discussed above ) , tn - insertions 
 in other genes within the deletion in the rd1 strain ( espi , eccd1 , espj , and espk ) and others in the esx-1 locus resulted in different substrate profiles in the cf ( figures 4 and 5a ) .
these findings 
 demonstrate that all genetic disruptions of the esx-1 apparatus are 
 not equal ; disruption of a single gene or operon is not the same as 
 deleting several esx-1-associated genes .
one possible interpretation 
 of these data is that the tn - insertions in some esx-1-deficient strains result in an esx-1 system that is largely 
 intact , while others result in a lack of stability or assembly of 
 the apparatus .
note that the espj , espk , and eccd1 strains cluster closest to 
 the wt strain when comparing levels of esx-1-associated proteins in 
 the cell lysate ( figure 5b ) .
moreover , the 
 majority of the esx-1-associated proteins measured from these strains 
 were present at levels comparable to those of wt m. marinum ( figure 5b ) . in contrast , disruption of the 
 several other esx-1-associated genes resulted in a decrease in the 
 relative levels in esx-1 proteins in the cell ( figure 5b ) .
the decreased levels of esx-1 proteins were linked to 
 disruption of several esx-1 components ( ecca1 , espl , espg1 , espi , and eccb1 ) as well as the espb substrate . 

one possibility 
 is that in the absence of espl and eccb1 , the esx-1 membrane 
 complex does not form or is destabilized . this has not been reported 
 previously , as there has never before been an approach that could 
 simultaneously quantitate changes to esx-1 proteins at this scale .
espg1 , ecca1 , espi , and espb were required 
 for the export of some but not all of the substrates .
indeed , disruption of espg1 led to decreased levels of all of the esx-1 proteins measured 
 in this study . by this analogy
, several esx-1 proteins may function 
 as chaperones ( supplementary figure s5 ) . 

for example , ecca1 directly interacts with at least three 
 known esx-1 substrates , espc , espf , and ppe68 , and shares aaa+ domains with chaperones .
ecca1 may function to keep esx-1 substrates in the properly 
 folded or unfolded state for export . because espb is secreted
. it may be that in the absence 
 of espb the apparatus is unstable or that espb is a secreted part 
 of the apparatus .
overall , the present study and subsequent 
 analysis allowed us to 
 expand the genetic requirements for export of four known esx-1 substrates 
 and identified three additional substrates encoded by the esx-1 locus .
these findings set the stage for a future work 
 aimed at understanding how these additional substrates promote virulence 
 in m. marinum and m. tuberculosis .
we defined rules of assigning new substrates to the esx-1 system 
 using this approach , which can be expanded as new esx-1-deficient 
 strains are isolated .
surprisingly , we identified several intermediate 
 phenotypes of esx-1 secretion that correlated with attenuation of 
 virulence .
importantly , while the application of this approach was 
 directed at esx-1 export in m. marinum , these findings 
 can directly be tested in the human pathogen m. tuberculosis .
this method creates a framework that can be readily applied to studying 
 protein secretion or any complex molecular pathway .
 the 
 esx / wxg-100 ( esat-6/wss ) exporters are multiprotein complexes 
 that promote protein translocation across the cytoplasmic membrane 
 in a diverse range of pathogenic and nonpathogenic bacterial species . 
 
 the esx-1 ( esat-6 system-1 ) system mediates virulence factor translocation 
 in mycobacterial pathogens , including the human pathogen mycobacterium 
 tuberculosis . 
 although several genes have been associated 
 with esx-1-mediated transport and virulence , the contribution of individual 
 esx-1 genes to export is largely undefined . 
 a unique aspect of esx-1 
 export is that several substrates require each other for export / stability . 
 
 we exploited substrate  
 codependency  to identify esx-1 
 substrates . 
 we simultaneously quantified changes in the levels of 
 13 esx-1 proteins from both secreted and cytosolic protein fractions 
 generated from 16 esx-1-deficient mycobacterium marinum strains in a single experiment using mrm / srm targeted mass spectrometry . 
 
 this expansion of measurable esx-1 proteins allowed us to define statistical 
 rules for assigning novel substrates using phenotypic profiles of 
 known esx-1 substrates . using this approach , we identified three additional 
 esx-1 substrates encoded by the esx-1 region . 
 our 
 studies begin to address how disruption of specific genes affects 
 several proteins in the esx-1 complex . 
 overall , our findings illuminate 
 relationships between esx-1 proteins and create a framework for the 
 identification of secreted substrates applicable to other protein 
 exporters and pathways . 
Introduction
Experimental Procedures
Proteomics
Results and Discussion