Proteomics of Macroproteins

In 1994, Wilkins and Williams first proposed the concept of proteome, which refers to all proteins expressed by cells, tissues, or organisms. In 1997, Peter James further introduced the concept of proteomics, which is the scientific field for studying the proteomes in cells, tissues, or organisms. Proteomics has a wide range of applications, including interpreting genomes, expressing and functioning proteins, and studying protein-protein interactions, etc.

 

Metaproteomics is a branch of proteomics. Different from traditional proteomics research, metaproteomics focuses on analyzing the protein composition of microbial communities and how they interact and respond to environmental changes. So far, metaproteomics research has covered various environmental samples, including fermented food, biofilms of acidic mine wastewater, wastewater, marine samples, soil systems, permafrost, human microbiomes, and various environmental ecology and gut flora. Since the first large-scale metaproteomics data was generated in 2005, metaproteomics has rapidly improved in scale, depth, speed, and quality.

 

Analysis Workflow

The technical process of metaproteomics mainly includes: sample preparation, protein separation, and protein identification.

 

1. Sample Preparation

The preparation of the sample directly affects the identification results, and different research objects take different preparation methods. Researchers need to collect microbial communities in specific environmental or biological samples, such as intestines, soil, water bodies, etc. After the sample is collected, non-biological components are removed by physical methods such as filtration, and then the microbial cells are lysed by chemical or physical methods to release and enrich the proteins.

 

Since there are many types of microorganisms and the abundance of proteins varies greatly, the difficulty of protein extraction is high, and it is very easy to fail to extract or lose a large amount of protein. Therefore, it is very necessary to properly optimize the extraction and lysis scheme.

 

2. Protein Separation

Protein separation technology mainly includes two categories: gel separation technology and chromatography technology.

 

Among them, the methods of two-dimensional gel electrophoresis (2-DE), two-dimensional fluorescence difference gel electrophoresis (2D-DIGE), and capillary electrophoresis (CE) are widely used in proteomics research. However, gel electrophoresis technology is limited by various conditions in practice and cannot completely and effectively separate proteins in complex samples. Therefore, chromatography technology is currently more widely used.

 

Chromatography technology can significantly increase the type and number of proteins identified, effectively making up for certain shortcomings of electrophoresis technology. Liquid chromatography (LC) as one of the commonly used chromatography technologies has a wide range of applications and is often used in combination with 2-DE to achieve automation of the experimental process. High-performance liquid chromatography (HPLC) has developed on the basis of LC. With its high specificity and high sensitivity, it is especially suitable for the separation of small molecular weight proteins, membrane proteins, and low-abundance proteins.

 

3. Protein Identification

Mass spectrometry (MS) technology identifies proteins by analyzing peptide mixtures obtained after specific proteinase (such as trypsin) hydrolysis, which is currently the most commonly used protein identification technology. Using mass spectrometry technology can quickly identify the proteome in microorganisms, and accurately measure the relative molecular weight, amino acid sequence, and post-translational modifications of peptides and proteins. Because mass spectrometry is as efficient and accurate as chromatography, the two are used together as an important means for metaproteomics research. The more frequently used mass spectrometry is Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS) and Electrospray Ionization Mass Spectrometry (ESI-MS).

 

The identification of microbial proteins also poses certain challenges: on the one hand, due to the diverse types of proteins in the microbiome and large differences in abundance, it requires higher resolution and sensitivity for mass spectrometry; on the other hand, many microorganisms have not been identified to date, the protein database of the microbiome is not perfect, and there is a high sequence similarity between microbial species, so whether it depends on a specific database (such as the metaproteomics database) or a public database, the difficulty of data analysis is quite high.

 

MtoZ Biolabs uses Thermo's latest Obitrap Fusion Lumos mass spectrometer in conjunction with Nano-LC nanoscale chromatography technology to provide efficient and accurate metaproteomics services. We offer free customized solutions according to different project needs, which can achieve efficient extraction of microbial proteins at the omics level and high-throughput identification analysis of protein samples from various environmental microbes, contributing to various basic scientific research. Feel free to consult.