Proteomics

       Extracellular vesicles are membrane-bound particles containing biomolecules such as proteins, RNA and DNA. EV proteomics can help investigate EV function and potential roles in disease. Methods include mass spectrometry and immunoassays, and can inform research into EV biogenesis, secretion, cell signaling and disease processes. Services typically include EV isolation, protein separation and mass-spectrometry analysis.

1. Label-free quantitative proteomics

       Label-free quantitative proteomics uses mass spectrometry to compare relative protein abundance without isotope labeling. Peptides produced by protein digestion are measured by liquid chromatography and mass spectrometry; ion peak areas are integrated for relative quantification.

2. TMT-labeled quantitative proteomics

       Tandem mass tags (TMT), developed by Thermo Fisher Scientific, are isobaric peptide labels used for quantitative proteomics. The source describes 6-, 10- and 16-plex labels that bind peptide amino groups, followed by tandem high-resolution mass spectrometry to compare protein abundance across up to 16 samples.

3. DIA quantitative proteomics

       In data-independent acquisition (DIA), the mass-spectrometry scan range is divided into windows, and ions within each window are cyclically selected, fragmented and measured. In contrast, data-dependent acquisition (DDA) selects a limited number of high-response peptide ions, typically the top 20 in the source example, for fragmentation. Coverage and identification depend on the assay and analysis.

4. PRM targeted proteomics

       Parallel reaction monitoring (PRM) uses high-resolution, high-accuracy mass spectrometry, such as an Orbitrap instrument, to monitor selected ions. A quadrupole selects precursor ions for target peptides; these are fragmented in a collision cell, and fragment ions are measured by the analyzer. PRM can selectively detect target proteins or peptides, including modified peptides, and support relative or absolute quantification.

5. Metaproteomics

       Metaproteomics applies proteomics to microbial communities, identifying proteins present at a given time to investigate microbial functions in an ecosystem. Analyses across environments can help identify functional genes, metabolic pathways and stress-associated proteins. Combining proteomic and metagenomic data can support studies of community composition, functional diversity and biological processes.

6. PBA single-EV surface proteomics

       The source describes proximity barcoding assay (PBA) using micrometer-sized single-stranded DNA clusters containing repeated DNA copies generated by rolling-circle amplification to label individual EVs. Antibody-DNA conjugates bind surface proteins and encode information that is amplified by PCR and read by next-generation sequencing. Detection depends on binding specificity, sequencing coverage and depth, and antibody affinity and selectivity. Comparing EV profiles may help identify surface-protein combinations for further biomarker research.