The gut microbiota is a dynamic community that is pivotal for human health and fitness, the studies have been widely extended by the recent advances in metagenomics levels and post-genomic research. It was in 2004 when Rodriguez and Velera first brought up the concept of metaproteomics, researchers started to study into this field which looks into the presence and abundance of various proteins identified from gut microbial communities. And with a well-curated protein sequence database in hand, we can assign these proteins to individual species or higher taxa and understand the functional roles and interactions of individual members in the community. Two-dimensional gel electrophoresis (or 2D-PAGE), as the most classic method that provides excellent separation of protein and allows for the visualization of thousands of proteins in a given sample on a single gel, plays a significant role in the progress of metaproteomic research.
Figure 1. Overview of two-dimensional gel electrophoresis
Coupling two-dimensional gel electrophoresis to mass spectrometry largely improves the separation of proteins, which is widely performed in rapid whole microbial protein analysis.
Creative Proteomics, as a preeminent corporation that has many years of experience in proteomics, provides leading two-dimensional gel electrophoresis and mass spectrometry services for gut metaproteomic research, the workflow generally falls into the following steps:
Sample preparation is a key step in proteomics research, with the quality of the sample weighing heavily on the subsequent steps. We provide delicate protein extraction techniques according to the state of the raw material and research purpose, to guarantee an outcome with high fidelity and reliability.
The purification of proteins is performed via two-dimensional gel electrophoresis – isoelectric focusing and SDS-PAGE, which separate the proteins by Ip value and molecular mass respectively.
Tryptic digestion breaks down proteins into peptides for the following runs, and the digestion can be done within excised gel slices.
The digestion is processed by analysis and peptide extraction using mass spectrometry and many other technologies including peptide mass fingerprinting and MALDI-TOF or alternatively direct analysis using nano-spray.
We perform data analysis referencing not only public repositories but in-house databases to guarantee the comprehensiveness and accuracy of the results. And aligned with optimized algorithms and powerful computing platforms, we are able to handle the large datasets for in-deep biological interpretation including protein identification, metaproteomic profiling, and comparison to bring insights into dynamics and functions of the intestinal microbiota.
We dedicate to providing the highest level of service in gut microbiota research. To find out more about two-dimensional gel electrophoresis services, please feel free to contact us.
For Research Use Only. Not for use in diagnostic procedures.
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