Xiao Hua's research group and collaborators of Shanghai Jiaotong University proposed a new strategy for macroproteomics research

Recently, Research, a comprehensive scientific and technological journal co-founded by the Chinese Association for Science and Technology and the American Association for the Promotion of Science, published online the research paper "In-Depth Metaproteomics Analysis of Oral Microbiome for Lung Cancer"
by Xiao Hua's research group and collaborators at the School of Life Science and Technology of Shanghai Jiao Tong University 。 This paper creates a new strategy for metaproteomics research, which deeply explores the microbiome from the level of protein, the executor of microbial function, realizes the deep metaproteomic analysis of the oral microbiome, and provides new clues
for discovering the relationship between lung cancer and microbiome disorders 。 Shanghai Jiao Tong University is the first research paper, Jiang Xiaoteng, a doctoral graduate of the School of Life Science and Technology of Shanghai Jiao Tong University, and Associate Professor Zhang Yan of the School of Pharmacy are the co-first authors of the paper, and Professor Xiao Hua from the School of Life Science and Technology of Shanghai Jiao Tong University, Professor Cao Chengxi from the School of Electronic Information and Electrical Engineering, and Professor Hu Shen from the UCLA School of Dentistry are the co-corresponding authors
of the paper.

Figure 1.
New strategies for metaproteomics research on the oral microbiome

The human microbiome plays an important role
in maintaining homeostasis in vivo.
As the second largest microbial microbiome in the human body, the oral microbiome contains nearly 800 microorganisms and up to 20 million
non-redundant genes.
As the first hub connecting the external environment with the human digestive tract and respiratory tract, the composition and function of the oral microbiome are crucial to the composition of commensal microorganisms in the human body, and its imbalance can lead to a variety of systemic diseases
in the human body, including lung cancer.
Therefore, it is important to conduct in-depth research on the oral microbiome
.
Currently, most microbiome studies associated with lung cancer are conducted through sequencing methods, and analyses based on changes in species taxonomic often do not accurately reflect changes in their function
.
Characterizing the functional properties of the microbiome is a key factor
in understanding its role in disease.
Functional analysis relies on protein detection, which is an advantage of
mass spectrometry-based metaproteomics.

However, the metaproteomics research of oral microorganisms is still in its infancy, and the challenges mainly include: (1) oral microbiome samples contain a large number of host proteins, cells and cellular debris, and the background interference is serious; (2) The composition and structure of oral microorganisms are complex, and the lack of preparative microbial separation technology makes it easy for the separation and detection of liquid chromatography-mass spectrometry to be saturated with massive microbial peptides; (3) The abundance dynamic range of oral microorganisms is wide, and the low-abundance flora is easy to be masked, which greatly limits the characterization of low-abundance species, and often needs to rely on microbial culture, which is not conducive to the analysis of non-cultured microorganisms; (4) Lack of efficient oral microbial metaproteome data analysis process
.
These factors severely hinder the in-depth analysis
of the oral microbiome.
In order to break through the above bottlenecks, researchers developed a new strategy for preprocessing microbial samples, constructed high-resolution free-flow isoelectric focusing electrophoresis, realized the isolation, enrichment and preparation of complex microorganisms, significantly improved the protein elucidation and identification ability of microbiome by liquid chromatography-mass spectrometry, and established an integrated macroproteomics analysis module, which provided a comprehensive analysis strategy
for in-depth exploration of the species composition and executive function of the microbiome.

Figure 2.
Macroproteomic identification results based on free-flow isoelectric focusing electrophoresis

Figure 3.
Metaproteomics analyzes species classification and function of the microbiome

First, differential centrifugation and double membrane filtration were used to effectively separate the host cells and proteins from microorganisms in the sample preparation stage, which significantly reduced the interference of host proteins on microbial identification, and improved the number of protein identification and experimental reproducibility
of microorganisms.
Secondly, the researchers constructed an electrophoresis system suitable for the isolation and preparation of complex microorganisms, and at the same time realized the enrichment of low-abundance microorganisms, further improved the identification efficiency of microbial proteins, and raised the number of oral microbial proteins to a new level
.
Third, through the integrated metaproteomics data processing tools, the researchers not only explored the protein function of the oral microbiome, but also jointly analyzed
the species composition of microorganisms and the functions performed by each species.
Finally, the researchers systematically compared the differences between the oral microbiome of lung cancer patients and the normal control group, and found and verified two oral microorganisms
that perform dysfunctional functions in lung cancer.
The new macroproteomics strategy created by this study will facilitate comprehensive analysis of the functions performed by complex microbiomes and provide new tools
for in-depth exploration of the microbiome's relationship with disease.

Fig.
4.
Analysis of oral microorganisms in lung cancer group and normal control group

The research was supported
by the National Key R&D Program, the National Natural Science Foundation of China, the National Major Scientific Research Instrument Development Project of the National Foundation of China, and the Shanghai Natural Science Foundation.

Links to papers: https://spj.
sciencemag.
org/journals/research/2022/9781578/