Unraveling the Complexity of the Proteome
PolyLC: Your Partner in Proteomic Discoveries
Proteomics, the large-scale study of proteins, has revolutionized our understanding of biological processes and disease mechanisms. PolyLC offers a comprehensive suite of chromatography products designed to support your proteomics research, from protein separation and purification to peptide mapping and analysis. Our innovative solutions empower you to delve deeper into the proteome and uncover valuable insights.
Navigating the Proteomics Landscape
No single analytical method can effectively identify every protein within extensive biological samples. That’s where PolyLC comes in. We’re here to guide you through the diverse landscape of proteomics approaches and provide the tools you need to succeed.
Bottom-Up Proteomics
This approach involves breaking down proteins into smaller peptides, typically using enzymes like trypsin. These peptides are then analyzed using techniques like tandem mass spectrometry (MS/MS) to identify the original proteins by matching peptide sequences. Bottom-up proteomics is a widely used, high-throughput method for large-scale protein identification and quantification. However, it’s important to note that this method may have limitations in capturing the complete protein context and certain post-translational modifications (PTMs).
BOTTOM-UP EXAMPLE:
ERLIC of Phosphopeptides & Acidic Analogues
To achieve optimal peptide resolution in bottom-up proteomics, consider employing ERLIC or HILIC separation techniques. These methods excel at separating polar peptides, which can be particularly challenging for traditional reversed-phase chromatography.
This method demonstrates exceptional separation capabilities, effectively resolving even the least well-retained phosphopeptide (S2) from an analogue with 4 Asp- residues. This level of resolution is particularly important in complex tryptic digests, where approximately 40% of the peptides have 3 or more Asp- and Glu- residues. By effectively separating these closely related peptides, you can increase the confidence and accuracy of your protein identification and characterization.
Middle-Down Proteomics
This hybrid approach involves partially digesting proteins into larger peptide fragments before analysis. By striking a balance between studying intact proteins (as in top-down) and smaller peptides (as in bottom-up), middle-down proteomics offers valuable insights into protein structure and modifications. However, it’s essential to be aware of the potential challenges associated with the intermediate complexity of analysis and the possible loss of some PTMs.
MIDDLE-DOWN EXAMPLE:
Histone Modification Analysis with PolyCAT A™
To achieve optimal peptide resolution in bottom-up proteomics, consider employing ERLIC or HILIC separation techniques. These methods excel at separating polar peptides, which can be particularly challenging for traditional reversed-phase chromatography.
This method demonstrates exceptional separation capabilities, effectively resolving even the least well-retained phosphopeptide (S2) from an analogue with 4 Asp- residues. This level of resolution is particularly important in complex tryptic digests, where approximately 40% of the peptides have 3 or more Asp- and Glu- residues. By effectively separating these closely related peptides, you can increase the confidence and accuracy of your protein identification and characterization.
Top-Down Proteomics
Top-down proteomics involves analyzing intact, full-length proteins directly, without any prior digestion. This approach allows for the study of post-translational modifications (PTMs) and protein isoforms in their native forms, providing a comprehensive view of protein diversity and function. However, it’s important to consider the challenges associated with top-down proteomics, such as the requirement for high-resolution mass spectrometry and the complexity arising from the size and heterogeneity of proteins.
TOP-DOWN EXAMPLE:
Optimizing Top-Down Proteomics with HIC
PolyLC’s HIC columns excel in the analysis of antibodies, enabling the separation and characterization of glycoform variants with high resolution and sensitivity.
Further, the conditions achievable with PolyLC HIC columns allow for direct coupling with mass spectrometry, facilitating in-depth characterization of antibody structure and modifications.
The Importance of Ligand Length on Retention in HIC
PolyLC offers a specialized line of extended ligand HIC stationary phases specifically designed for top-down proteomics. These phases enable efficient separation of intact proteins while minimizing the salt concentration required, making them highly compatible with mass spectrometry.
FEATURED APPLICATION NOTE:
High-Resolution Analysis of Antibodies
PolyLC’s HIC columns excel in the analysis of antibodies, enabling the separation and characterization of glycoform variants with high resolution and sensitivity.
Further, the conditions achievable with PolyLC HIC columns allow for direct coupling with mass spectrometry, facilitating in-depth characterization of antibody structure and modifications.
Ready to Experience the PolyLC Difference?
Contact us to discuss your chromatography needs and let us help you find the optimal solution for your specific application.