Metaproteomics Service

What Is Metaproteomics

Microbial communities, as a primary driving force in biogeochemical cycles, play an undeniable role in the Earth's biosphere. Therefore, research on microorganisms has been incessant. However, due to their diversity and complexity, unraveling the functionality of these intricate microbial systems remains a formidable challenge.

In recent years, with the advancement of scientific techniques, we have gradually unveiled the "mysterious veil" surrounding microorganisms. Metaproteomics has emerged as an effective method for the spatiotemporal characterization of microbial communities. Metaproteomics, utilizing mass spectrometry technology, enables large-scale qualitative and quantitative analysis of proteins within microbial communities. This approach unveils molecular-level information about microbial phenotypes, answering questions about "what each organism in the microbial ecosystem is doing." It also allows the identification of specific microbial components associated with variations in metabolite abundance, providing unique insights into microbial community functions.

(Manuel Kleiner 2019)

However, metaproteomics faces challenges due to the complexity of sample environments, a multitude of interferences, and susceptibility to external environmental factors and spatial variations. These factors contribute to increased difficulty in protein extraction and pose challenges in data analysis. Moreover, the presence of diverse species in the sample, potentially including proteins from other animals and plants, further complicates data analysis.

Upgraded Metaproteomics Services

Creative Proteomics has introduced a groundbreaking upgrade to address the challenges in metaprotein research, resulting in a substantial increase in the number of identified proteins, easily reaching 10,000+. Concurrently, we have launched mass spectrometry-based metaproteomics, covering over ten translation-related modifications such as acetylation and dihydroxyisobutyrylation. This approach allows for a systematic and in-depth analysis of the protein translation modification panorama within microbial communities, offering a forward-looking perspective to decipher the intricate interactions among "microbiota-host-environment."

Metaproteomics Services Workflow

(Sheng Pan 2019)

Sample Preparation Key Points

• The microbial community is compositionally complex, and the system contains a large number of impurities, necessitating targeted optimization for protein extraction.
• Microbial communities within the same sample exhibit diverse properties, requiring distinct extraction and lysis protocols (Gram-negative bacteria/positive bacteria).
• Potential contamination from exogenous animal, plant, and human proteins, as well as microbial contamination from other sources during storage.
• Extracting proteins from complex samples is challenging, with a high risk of significant protein loss or extraction failure
• The wide range of protein abundances necessitates a sophisticated and effective set of separation and analysis experimental strategies.

Mass Spectrometry Detection

• The microbial community comprises a diverse array of microorganisms, leading to a wide range of protein abundances.
• High requirements for mass spectrometry scanning speed, resolution, and mass accuracy.
• Incompatibility issues may arise with some commonly used quantitative proteomic techniques.

Data Search

• Lack of a comprehensive microbial proteome database.
• Many microorganisms in the samples remain unidentified, making reliance on metaproteomics or public databases incomplete.
• Building excessively large databases can increase the error rate.
• To achieve a higher protein identification rate, massive spectral data and the use of extensive databases result in prolonged data analysis times.
• Due to the high sequence similarity among microbial populations, accurately attributing peptide sequences to completely specific species for quantification is currently not feasible. The challenges mentioned above make the integration of quantitative results more challenging.

Metaproteomic Analysis Results

After qualitatively and quantitatively analyzing peptide sequences through database searching, we further provide comprehensive analysis results, including protein species identification, functional annotations, differential protein analysis, functional composition analysis, sample comparison analysis, inter-group comparison analysis, and functional divergence analysis. These results offer a comprehensive insight into the data obtained.

Note:

• Acylation Modification Proteomics: Includes acetylation, 2-hydroxyisobutyrylation, succinylation, butyrylation, 3-hydroxybutyrylation, etc.;
• Intestinal Contents: Due to variations in microbial content among different animals' intestinal . contents, it is essential to base the extraction on the actual situation;
• Active Sludge: This category refers to sludge samples rich in microbial communities, distinct from conventional sludge, and regular sludge quantities need to be doubled for submission;
• Other Samples: Given the significant variation in the state of macro-modified proteomics samples, it might be challenging to accurately estimate some samples. Feel free to consult in advance.

Technical advantages

1. Unique and effective protein extraction method, providing solutions for protein extraction of various types of environmental samples;

2. Combined with grading technology to obtain deep coverage of metaproteome expression profiles;

3. Utilize advanced mass spectrometry platforms to obtain high-quality metaproteomics data;

4. Provide professional and comprehensive metaproteomic data analysis results.

Application

Biomedicine: Study on the relationship between intestinal flora and major diseases

Microbial field: pathogenic mechanism, drug resistance mechanism, pathogen-host interaction research, etc.;

Clinical diagnosis: biomarkers, disease mechanism, disease classification, precise treatment, etc.

Q: Which type of proteomics is suitable for microbiome-related research?

A: Metaproteomics. For host proteins, conventional proteomics is appropriate, while metaproteomics is preferable for studying microbial proteins.

Q: What is required for metaproteomics preparation?

A: You need the corresponding metagenomic data.

Q: Can PRM (Parallel Reaction Monitoring) be applied to metaproteomics?

A: Currently, it is not feasible due to the complexity of the samples.

Q: What are the current challenges in metaproteomics research?

A: Challenges in current macroproteomics research can be categorized into three main aspects: sample preparation, mass spectrometry detection, and data searching.

Sample Preparation: a) Microbial community composition is complex, requiring targeted optimization for protein extraction. b) Different microbial community properties within the same sample necessitate the use of distinct extraction and lysis strategies. c) Contamination from exogenous animal, plant, human proteins, or other microbial sources during storage. d) High difficulty in extracting proteins from complex samples. e) Large span of protein abundance.

Mass Spectrometry Detection: a) Microbial communities comprise a diverse range of species, resulting in a wide span of protein abundances. b) High requirements for mass spectrometry scan speed, resolution, and mass accuracy. c) Incompatibility with some commonly used quantitative proteomic techniques.

Data Searching: a) Lack of protein databases specific to microbial communities. b) Many microorganisms in samples remain unidentified. c) Building excessively large databases increases error rates. d) Prolonged data analysis times. e) Difficulty in accurately assigning peptide segments to specific species due to high sequence similarity among microbial populations.

Q: Which genes are annotated in the KEGG pathway maps?

A: KEGG Pathway Maps provide annotation results for all genes, allowing users to explore all annotated pathways. The pathway maps display gene information for all annotations and allow users to perform pathway analysis on specific areas of interest selected from the annotation results.

Q: Can protein samples be submitted in batches for detection?

A: It is not recommended to submit samples in batches. Different batch effects may arise due to instrument stability, variations in experimental operators, and maintenance/cleaning of mass spectrometers. Batch effects are challenging to eliminate, and sometimes differences between batches can be more significant than differences between samples.

Q: What are the common factors contributing to unsuccessful protein identification in mass spectrometry?

A: Unsuccessful protein identification can be categorized into two main factors: poor mass spectrometry performance and the absence of a reference protein database.

1. Poor mass spectrometry performance may result from factors such as faint protein spots (indicating low protein content), errors in protein digestion or mass spectrometry operations. To avoid this, it is recommended to select darker protein spots with appropriately larger spot areas. For small but distinct protein spots, choosing a larger nozzle for sampling and including blank areas around the spot can help prevent contamination.

2. If poor identification results are due to the lack of a suitable database, improving the identification can be achieved by using a larger database, databases of closely related species, or adopting databases of larger taxonomic groups.

Q: Can macroproteomics be performed in species without metagenomic/metatranscriptomic sequencing?

A: Many species have undergone whole-genome sequencing, predicting corresponding genes. For species without sequencing, one can use gene sequences from closely related species as a database for proteomic studies. If suitable closely related species are not available, databases from larger taxonomic groups, such as genera or families, can be used for searching.

Q: Why do some differentially expressed genes not have corresponding differentially expressed proteins at the proteome level?

A: Differentially expressed genes are studied at the transcriptome level, while differentially expressed proteins are studied at the proteome level. Inconsistencies between RNA expression levels and protein expression levels can arise due to post-transcriptional, translational, and post-translational regulation.

Q: What are pros and cons of common quantitative proteomics techniques?

A: TMT/iTRAQ: Pros: Strong separation capability, broad applicability, high throughput, reliable results, high automation. Cons: Expensive, limited sample numbers.

Label-free: Pros: No labeling required, simple operation, no restriction on the number of compared samples, suitable for high-throughput testing, minimal preprocessing, higher coverage for low-abundance peptides. Cons: Requires high stability and repeatability in experimental operations, slightly lower accuracy compared to labeled quantification.

DIA: Pros: Good repeatability, high sensitivity, high quantification accuracy, high throughput without isotope labeling, suitable for large sample sizes. Cons: Requires advanced analysis algorithms, some difficulty in analyzing mass spectra from complex samples.

Case 1: Investigating the Regulation Mechanisms of the Gut Microbiota in Inflammatory Bowel Disease

Inflammatory bowel diseases, including Crohn's disease and ulcerative colitis, are chronic and complex disorders influenced by clinical, immunological, molecular, genetic, and microbial factors. The etiology and pathogenesis of these diseases remain incompletely understood. The gut microbiota has long been considered closely associated with the pathogenesis of inflammatory bowel diseases, and some studies have even suggested that microbiota transplantation could serve as a novel intervention for the treatment of these conditions.

Through the integration of metaproteomics and metaproteomic PTM analysis, researchers have conducted an in-depth analysis of the gut microbiota in patients with Crohn's disease. This study unveiled alterations in the acetylation modification profile under disease conditions. The investigation revealed that acetylation modifications are widely present in crucial metabolic pathways of the human gut microbiota, particularly in the production of short-chain fatty acids within the Firmicutes phylum. This research underscores the significance of post-translational modifications (PTMs), such as acetylation, in the study of functional microbial communities.

Case 2 Environmental Microbiology and Ecological Adaptation

Post-translational modifications are enriched within protein functional groupsimportant to bacterial adaptation within adeep-sea hydrothermal vent environment

Protein Post-Translational Modifications (PTMs) exhibit variances among orthologous proteins in different microbial taxa and are unevenly distributed across microbial taxonomy and functional categories.

Researchers employed a multi-omics approach integrating metagenomics and metaproteomics to comprehensively investigate PTMs, including acetylation, deamination, hydroxylation, methylation, nitrosylation, and phosphorylation, within the microbial communities of hydrothermal vent ecosystems. The study revealed non-uniform distributions of various modification types in both microbial taxonomy and functional classifications, indicating associations between specific modifications and distinct functionalities. This highlights the high diversity and distinctiveness of PTMs in deep-sea microbes, showcasing their crucial role in adapting to the extreme conditions of hydrothermal vents. The application of metaproteomics in exploring the PTM landscape of deep-sea microbes emerges as a meaningful research avenue for understanding how bacteria adapt to their environments.

References

1. Xu Zhang, et al., 2020. Widespread protein lysine acetylation in gut microbiomeand its alterations in patients with Crohn's disease. Nature Communications.
2. Xu Zhang, et al., 2021, Exploring the Microbiome-Wide Lysine Acetylation, Succinylation,and Propionylation in Human Gut Microbiota. Anal. Chem.
3. Weipeng Zhang, et al., 2016, Post-translational modifications are enriched within protein functional groups important to bacterial adaptation within a deep-sea hydrothermal vent environment. Microbiome.