Secreted proteins of Mycobacterium tuberculosis are implicated in its disease pathogenesis and so are considered as potential diagnostic and vaccine candidates. The search for these has been slow, even though the entire genome sequence of M. tuberculosis is now available; of the 620 protein spots resolved by 2-D gel electrophoresis, 114 secreted proteins have been identified, but for only 13 has the primary structure been partly characterized. For comparison, in this top down mass spectrometry (MS) approach the secreted proteins were precipitated from cell culture filtrate, resuspended, and examined directly by electrospray ionization (ESI) Fourier transform MS. The ESI spectra of three precipitates showed 93, 535, and 369 molecular weight (M(r)) values, for a total of 689 different values. However, only approximately 10% of these values matched (+/-1 Da) the DNA predicted M(r) values, but these identifications were unreliable. Of nine molecular ions characterized by MS/MS, only one protein match was confirmed, and its isotopic molecular ions were overlapped by those of another protein. MS/MS identified a total of ten proteins by sequence tag search, of which three were unidentified previously. The low success of M(r) matching was due to unusually extensive posttranslational modifications, including loss of a signal sequence, loss of the N-terminal residue, proteolytic degradation, oxidation, and glycosylation. Although in eubacteria the latter is relatively rare, a 9 kDa protein showed 7 hexose attachments and two 20 kDa proteins each had 20 attachments. For MS/MS, electron capture dissociation was especially effective.

中文翻译：

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通过电子捕获解离质谱从上到下表征结核分枝杆菌分泌的蛋白质。

结核分枝杆菌的分泌蛋白与疾病的发病机理有关，因此被认为是潜在的诊断和疫苗候选者。尽管现在可以找到结核分枝杆菌的整个基因组序列，但是对它们的搜索一直很缓慢。通过2-D凝胶电泳可分辨出620个蛋白质斑点，其中114个分泌蛋白质被鉴定出来，但是只有13个蛋白质的一级结构得到了部分表征。为了进行比较，在这种自上而下的质谱（MS）方法中，分泌的蛋白质从细胞培养滤液中沉淀出来，重悬并通过电喷雾电离（ESI）傅里叶变换MS直接检查。三个沉淀物的ESI光谱显示93、535和369分子量（M（r））值，总共689个不同值。然而，这些值中只有约10％与DNA预测的M（r）值相匹配（+/- 1 Da），但这些鉴定是不可靠的。在MS / MS表征的9个分子离子中，只有一种蛋白质被确认，其同位素分子离子被另一种蛋白质的分子离子重叠。MS / MS通过序列标签搜索共鉴定了十种蛋白质，其中三种以前未被鉴定。M（r）匹配的成功率低是由于异常广泛的翻译后修饰，包括信号序列丢失，N末端残基丢失，蛋白水解降解，氧化和糖基化。尽管在真细菌中后者相对较少，但一个9 kDa的蛋白质显示7个己糖附着，两个20 kDa的蛋白质每个都有20个附着。对于MS / MS，电子捕获解离特别有效。