Journal of Proteome Research Table 1. Enrichment Analysis for B. abortus Homolog Proteins Expressed during in vitro Growth As Detected by Mass Spectrometrya B. abortus homologs expression Expressed and detected with at least 1 peptide Expressed and detected with at least 2 peptides Expressed and detected with at least 5 peptides Expressed and detected with at least 7 peptides Expressed and detected with at least 9 peptides Not expressed Total ORFs proteins on chip 356 175 40 16 9 2690 3046 hits 41 23 8 4 2 81 122 serodominant serodiagnostic cross-reactive FoldEnrich p-value hits FoldEnrich p-value hits FoldEnrich p-value 2.9 3.3 5.0 6.2 5.5 0.8 5.28E-11 2.04E-07 1.38E-04 3.07E-03 4.76E-02 5.28E-11 20 16 7 4 2 13 33 5.2 8.4 16.2 23.1 20.5 0.4 1.87E-11 3.56E-12 1.30E-07 1.90E-05 3.91E-03 1.87E-11 21 7 1 0 0 68 89 2.0 1.4 0.9 0.0 0.0 0.9 ARTICLE 0.001 0.353 1.000 1.000 1.000 0.001 a Signiﬁcant enrichment or underrepresentation for certain features are underlined and bold. determine whether sera from blood culture-positive, Rose Bengal positive brucellosis patients (gold standard diagnosis) vs blood culture-negative, Rose Bengal positive (TAT not done) subjects (presumptive positive, unconﬁrmed) recognized diﬀerent pro- teins on the protein microarrays. Sera from blood culture-negative, Rose Bengal positive subjects reacted with 10 B. melitensis antigens much more strongly than blood culture-positive, Rose Bengal positive individuals (Figure S3, Supporting Information). This unique set of 10 antigens was distinct from the 33 antigens that distinguish culture positive cases from Peruvian na€ives. (Table S2, Supporting Information). These results indicate that protein microarray analysis was able to delineate distinct anti- body proﬁles in blood culture positive and negative patients, which has the potential to identify antigens capable of diﬀer- entiating active acute/subacute brucellosis from previously ex- posed patients. These ﬁndings may suggest novel biological interactions between bacteria and host that need to be further explored because culture negativity/Rose Bengal seropositivity might be associated with some biological diﬀerence between human hosts, perhaps an immune response that might lower bacterial loads to levels diﬃcult to culture from blood. Alterna- tively, it is possible that antibiotic use might lead to an altered host immune response to killed bacteria that no longer would subvert the host immune responses (typical of intracellular pathogens such as Brucella).52 54 Enrichment Analysis To better understand the determinants of antigenicity in bacteria, we performed a functional enrichment analysis of the serodominant and serodiagnostic antigens identiﬁed in this study. Bm proteins are annotated with NCBI Clustered Ortho- logous Group (COG) functional category. A total of 543 proteins are unassociated with COGs, and 39 proteins are associated with COGs but have not been assigned to a category, shown as “Other COGs”. Some proteins have multiple COG assignments so there are 3,291 COGs in total for 3046 proteins on chip. Proteins with predicted COG- U function, involved in intra- cellular traﬃcking and secretion, were 6.2- fold enriched in serodiagnostic antigens and 4.3- fold enriched in serodominant antigens. The enrichment was signiﬁcant with p value <0.05. Proteins in the COG-M category, cell envelope biogenesis and outer membrane, were 2-fold enriched in serodominant antigens (p-value 0.01). Proteins in COG-N category, cell motility and secretion, were 3.5-fold enriched in serodominant antigens (p = 0.027). Proteins with predicted COG-O function, posttranslational modiﬁcation, protein turnover, chaperones, were also signiﬁcantly enriched at 3.0-fold in serodominant antigens. There was only 1 serodominant antigen out of 176 COG-K category proteins, predicted to be involved in transcription and identiﬁed as sero- dominant, which was 0.1- fold enrichment and signiﬁcantly underrepresented with p value of 0.014 (Figure 5A, Table S3A, Supporting Information). We also looked at enrichment of antigens using computation- ally predicted features. As shown in Figure 5B and Table S3B (Supporting Information), proteins with 1 transmembrane do- main were signiﬁcantly enriched in serodominant, serodiagnostic and cross-reactive antigens groups, with enrichment fold at 4.3, 5.0, and 4.0, respectively. Proteins lacking transmembrane domains were signiﬁcantly underrepresented. Interestingly, proteins with more than 1 transmembrane domain were also signiﬁcantly under- represented among the serodominant and cross-reactive anti- gens. Proteins with predicted signal peptides (SignalP score > 0.7) were signiﬁcantly enriched in all three groups at a range of 2.8- to 3.6-fold. Conversely, proteins without signal peptides (SignalP score < 0.7) were signiﬁcantly underrepresented. pSortb pre- dicted 4 serodiagnostic and 5 serodominant outermembrane antigens, resulting in 11.5- and 3.9-fold enrichment, respectively. pSortb periplasmic proteins were also signiﬁcantly enriched, from 3.1- to 4.2-fold. However, pSortb cytoplasmic proteins were 0.5- to 0.7-fold underrepresented in serodominant and serodiagnostic antigens groups. We also found that proteins with isoelectric points lower than 5 were 1.7- to 2.0-fold enriched in serodominant and cross-reactive antigen groups, whereas, pro- teins with pI = 9 to 14 were 0.6- and 0.5-fold underrepresented in these two groups. Another potential feature that could account for protein antigenicity is the level of in vivo expression; proteins expressed at a high level in vivo would be expected more likely to be seen by the immune system than proteins expressed at a low level. To test this hypothesis, we compared the group of antigenic B. melitensis proteins with an existing data set of expressed protein peptide data from a very closely related species, B. abortus.23,35,36 In this study, cytosolic proteins and cell envelope proteins from B. abortus in the log phase of in vitro growth, were extracted and subjected to LC Mass spectrometry (MS) analysis as described in Materials and Methods. The number of diﬀerent peptides identiﬁed for each protein was listed in Table S4 (Supporting Information). Two-hundred forty proteins were exclusively ex- pressed in the cytosol of B. abortus with 1 to12 detected peptides, and 53 proteins exclusively expressed in the cell envelope of B. abortus with 1 to 15 detected peptides, and 80 overlapping proteins expressed in both conditions. Of these, 356 proteins 4819 dx.doi.org/10.1021/pr200619r |J. Proteome Res. 2011, 10, 4813–4824