Journal of Proteome Research ARTICLE Figure 1. Reactivity of the Immunoproteome. Each bar represents one of the 3046 proteins of B.melitensis. The bar height reﬂect the number of sera reactive to each protein. Nonreactive proteins account for approximately 52% of the proteome. The raised bars of any color represent 1464 reactive proteins, which deﬁne the immunoproteome (approximately 48% of the proteome). There are 538 antigens reactive in 1 sample, 648 antigens reactive in 2 5 samples, 143 antigens reactive in 6 9 samples and 135 antigens reactive in 10 42 samples. BMEI0536 and BMEI1079 reacted in all 42 Bm culture positive samples. All serodiagnostic antigens except BMEI0503 reacted in 10 42 samples; BMEI0503 reacted in 9 samples. Figure 2. Discovery of new human serodiagnostic antigens by probing the full B. melitensis proteome arrays with a collection of B. melitensis Peruvian na€ive and culture positive human sera. The mean sera reactivity of the 3046 antigens was compared between the culture positive and Peruvian naive groups. Antigens with Benjamini Hochberg corrected p-value less than 0.05 (serodiagnostic) are organized to the left and cross-reactive (BH_p > 0.05) antigens to the right. Shown are the 33 serodiagnostic protein antigens, Bm LPS, and a subset of cross-reactive antigens. were sequenced and >99% of sequenced clones had the correct sequence. All 3046 Bm ORFs cloned in pXT7 vector (>95% of proteome) were expressed under T7 promoter in the E. coli in vitro transcription/translation system, and printed on microar- rays. Over 98% of protein spots were conﬁrmed positive for expression (Figure S1A, Supporting Information). Human Antibody Profile Bm protein arrays were probed with sera from acute brucel- losis patients in Lima, Peru obtained within 1 3 weeks of the onset of symptoms, and sera from Bm culture-positive humans (Figure S1B, Supporting Information) showed robust reactivity against a collection of antigens compared to unexposed indivi- duals. Among the 3046 antigens tested, 1464 antigens reacted with at least one culture positive individual, accounting for 48% of the proteome (Figure 1). Within this immunoproteome, 122 protein antigens were deﬁned “serodominant”, with mean re- activity greater than the mean of the no DNA controls plus 2.5 standard deviations among culture positive individuals (Figure 2, Figure S2 and Table S1, Supporting Information). Of these, 33 protein antigens were serodiagnostic, and were signiﬁcantly diﬀerentially reactive between na€ive and culture positive patients from Peru (Benjamini and Hochberg adjusted Cyber-T p-value <0.05). Most of these antigens also reacted strongly with sera from brucellosis cases that were culture negative but positive for the Rose Bengal diagnostic test. An unbiased hierarchical cluster- ing analysis segregated most of the individual specimens into two groups, with a few infected cases with weak reactivity sporadically clustered among samples diagnosed as uninfected (Figure S2, Supporting Information). Peruvian na€ive samples exhibited very low responses to this collection of antigens. We also identiﬁed 90 cross-reactive antigens that reacted similarly among all human 4816 dx.doi.org/10.1021/pr200619r |J. Proteome Res. 2011, 10, 4813–4824