Mesoporous Silica as Sorbents and Enzymatic Nanoreactors for Microbial Membrane Proteomics

The
membrane proteins of microbes are at the forefront of host
and parasite interactions. Having a general view of the functions
of microbial membrane proteins is vital for many biomedical studies
on microbiota. Nevertheless, due to the strong hydrophobicity and
low concentration of membrane proteins, it is hard to efficiently
enrich and digest the proteins for mass spectrometry analysis. Herein,
we design an enzymatic nanoreactor for the digestion of membrane proteins
using methylated well-ordered hexagonal mesoporous silica (Met-SBA-15).
The material can efficiently extract hydrophobic membrane proteins
and host the proteolysis in nanopores. The performance of the enzymatic
nanoreactor is first demonstrated using standard hydrophobic proteins
and then validated using membrane proteins extracted from Escherichia coli (E. coli) or a mixed bacterial
sample of eight strains. Using the nanoreactor, 431 membrane proteins
are identified from E. coli, accounting for 38.5%
of all membrane proteins of the species, which is much more than that
by the widely used in-solution digestion protocol. From the mixed
bacterial sample of eight strains, 1395 membrane proteins are identified
using the nanoreactor. On the contrary, the traditional in-solution
proteolysis workflow only leads to the identification of 477 membrane
proteins, demonstrating that the Met-SBA-15 can be offered as an excellent
tool for microbial membrane proteome research and is expected to be
used in human microbiota studies, e.g. host–microbe interactions.

微生物膜蛋白处于宿主与寄生物相互作用的前沿。全面解析微生物膜蛋白的功能，对于诸多围绕微生物群的生物医学研究而言至关重要。然而，由于膜蛋白疏水性极强且丰度较低，难以高效富集并酶解这些蛋白以开展质谱分析。在此，我们设计了一款基于甲基化有序六方介孔二氧化硅（methylated well-ordered hexagonal mesoporous silica，Met-SBA-15）的酶促纳米反应器，用于膜蛋白的酶解。该材料可高效萃取疏水性膜蛋白，并能在纳米孔内完成蛋白水解过程。该酶促纳米反应器的性能首先通过标准疏水性蛋白得到验证，随后又分别以从大肠杆菌（Escherichia coli，E. coli）中提取的膜蛋白，以及8株菌的混合细菌样本进行了验证。使用该纳米反应器，我们从大肠杆菌中鉴定出431种膜蛋白，占该物种全部膜蛋白的38.5%，这一数量远高于广泛使用的溶液内酶解方案所得结果。从8株菌的混合细菌样本中，该纳米反应器可鉴定出1395种膜蛋白；与之相反，传统的溶液内蛋白水解流程仅能鉴定出477种膜蛋白。上述结果表明，Met-SBA-15可作为一款优异工具用于微生物膜蛋白质组学研究，且有望应用于人类微生物群相关研究，例如宿主-微生物相互作用领域。