The Rise of Micro and Nanoflow In Proteomics – Technology Networks

A key driver for the advent of proteomics was the realization that complexity is driven by protein variation. Contrary to expectation, the genome is thought to be predominantly invariant1; however, the proteome displays significant plasticity that is a product of protein complexation, post-translational modification (PTM), splicing, and both the spatial and temporal regulation of proteins.2 Proteomics is the concomitant and systematic study of numerous and diverse proteins. Given that the proteome is a readout of the changing state of cells, tissues, and therefore the organism, it underpins our understanding of both health and disease.

The sensitivity associated with Nanoflow-LC has contributed to its use in a variety of novel analytical settings that may have a sample limited input. Tissues are often heterogenous in nature, and the ability to interrogate cellular heterogeneity is vitally important, for example, microheterogeneity in tumor biology. Ultra-sensitive nanoflow-LC has been combined with FACS and bespoke nanodroplet sample preparation (nanoPOTS), enabling the identification of >700 proteins from a single HeLa cell. This proteome coverage (for a single cell) is more comprehensive than previously reported.17 This affords the possibility of investigating single cells and their microenvironment to help determine their contribution to disease progression.

Biomarker discovery is often complicated by methodological challenges, where low concentrations of analytes must be determined in a complex matrix. Poor ovarian response is typically difficult to predict. Biomarker discovery studies (conducted during IVF treatment) on follicular fluid were performed using a highresolution orbitrap mass spectrometer coupled to a nanoflowLC system. Numerous proteins were identified (1079), and three of these proteins (renin, pregnancy zone protein, and sushi repeat-containing protein (SRPX)) were identified as predictors of a poor response.19Imaging mass spectrometry (IMS) is an emerging technique for mapping the spatial distribution of analytes (e.g., lipids) across tissue. However, various technical challenges have limited its application to proteomics. Applying these methods would have traditionally relied on labels that require prior knowledge of protein targets. Label-free LC-nanoflow proteomics has been used to analyze tissue voxels, prepared from mouse uterus prior to blastocyst implantation. This generated quantitative cell-type-specific images for more than 2000 proteins with a spatial resolution of 100 m.20Tooth enamel is the densest, hardest, and most mineralized human tissue. Analysis of its proteome is further complicated by the meager (<1%) presence of proteinaceous material. Amelogenin is a dimorphic and abundant tooth protein and expressed from both X and Y chromosomes. Gender may, therefore, be revealed by sequencing the gender dimorphic peptide regions. The analysis of enamel is crucial in archeological or forensic specimens where no other tissue is available and DNA may be irreparably degraded. In these circumstances, the amount of sample available may also be severely restricted. Unique peptides have been identified by acid etching single teeth and peptide identification made possible using nanoflow LC-MS. This workflow has enabled the identification of major structural enamel peptides, including amelogenin isoforms, in teeth obtained from Anglo-Saxon burials (600900 AD).21Given the drive toward increasingly small sample sizes, both micro and nano-LC are expected to play larger roles in research proteomics and will therefore remain fundamental to the advancement of biomedical science.

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The Rise of Micro and Nanoflow In Proteomics - Technology Networks

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