Ge Ying, El-Naggar Mariam, Sze Siu Kwan, Oh Han Bin, Begley Tadhg P, McLafferty Fred W, Boshoff Helena, Barry Clifton E
Department of Chemistry and Chemical Biology, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301, USA.
J Am Soc Mass Spectrom. 2003 Mar;14(3):253-61. doi: 10.1016/s1044-0305(02)00913-3.
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.
结核分枝杆菌的分泌蛋白与其疾病发病机制有关，因此被视为潜在的诊断和疫苗候选物。尽管结核分枝杆菌的全基因组序列现已可得，但对这些分泌蛋白的寻找进展缓慢；在通过二维凝胶电泳分离出的620个蛋白点中，已鉴定出114种分泌蛋白，但只有13种的一级结构得到了部分表征。作为比较，在这种自上而下的质谱（MS）方法中，分泌蛋白从细胞培养滤液中沉淀出来，重新悬浮，然后直接通过电喷雾电离（ESI）傅里叶变换质谱进行检测。三种沉淀物的ESI光谱显示出93、535和369个分子量（M(r)）值，总共689个不同的值。然而，这些值中只有约10%与DNA预测的M(r)值匹配（±1 Da），但这些鉴定结果并不可靠。在通过串联质谱（MS/MS）表征的九个分子离子中，仅确认了一个蛋白匹配，并且其同位素分子离子与另一个蛋白的同位素分子离子重叠。MS/MS通过序列标签搜索总共鉴定出十种蛋白质，其中三种是以前未鉴定出的。M(r)匹配成功率低的原因是存在异常广泛的翻译后修饰，包括信号序列的丢失、N端残基的丢失、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见，但一种9 kDa的蛋白显示有7个己糖附着，两种20 kDa的蛋白每种都有20个附着。对于MS/MS，电子捕获解离特别有效。
