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Proteomic analysis involves the comprehensive study of proteins within a given sample, which is essential for understanding biological processes and disease mechanisms. However, preparing proteomic samples, particularly from complex specimens such as human tissues, cell lines, or biological fluids with wide dynamic ranges in protein abundance, poses a significant challenge. Especially when applying liquid-chromatography (LC) mass spectrometry (MS)-based analysis, this may result in inadequate data depth. Peptide fractionation can mitigate these challenges, facilitating more reliable results with increased proteomic depth and, ultimately, more protein identifications.
Why Is Peptide Fractionation Important for Proteomic Analysis?
1. Complexity Reduction
Biological samples contain thousands of different proteins, leading to ion suppression and low sensitivity for less abundant proteins during MS-based analysis. Protein fractionation simplifies these complex mixtures into manageable subsets, reducing sample complexity and improving the detection of low-abundance proteins.
2. Increased Dynamic Range
Proteins in biological samples vary widely in concentration, with high-abundance proteins often masking low-abundance ones in MS analysis. Fractionation separates high-abundance from low-abundance proteins, expanding the dynamic range and allowing the identification of low-abundance proteins.
3. Improved Identification and Coverage
Fractionation increases the probability of identifying proteins and obtaining better sequence coverage. By dividing the proteome into smaller fractions, each with fewer proteins, the mass spectrometer can more effectively analyze peptides, leading to more confident identifications and greater sequence coverage.
4. Enhanced Sensitivity
Separating proteins or peptides into distinct fractions minimizes the complexity of each MS run, enhancing the instrument’s sensitivity. This is crucial for detecting proteins present in low amounts, which might otherwise be missed in more complex mixtures.
5. Facilitating Post-Translational Modification Analysis
Fractionation helps isolate proteins or peptides with specific post-translational modifications (PTMs), such as phosphorylation or glycosylation. This targeted approach allows for more in-depth PTM analysis, critical for understanding protein function and regulation.
6. Resolving Proteins with Similar Mass or Properties
Some proteins or peptides have similar masses or properties, making them difficult to distinguish in complex mixtures. Fractionation techniques that separate based on characteristics like isoelectric point, hydrophobicity, or size help resolve these closely related species, facilitating their identification.
Common Methodologies Used for Peptide Fractionation
Common fractionation techniques include several approaches: (1) Gel electrophoresis, which separates proteins based on size and/or isoelectric point. (2) Liquid chromatography (LC) separates proteins or peptides based on different chemical properties. There are different modalities of LC, including ion exchange, reverse phase, and size exclusion. LC is often combined with mass spectrometry (LC-MS), offering a powerful analytical tool that delivers superior separation, identification, and quantification of a wide range of chemical compounds. (3) Affinity chromatography utilizes specific interactions, such as antibody-antigen or metal affinity, to isolate subsets of proteins. (4) Subcellular fractionation separates proteins based on their localization within the cell, such as nuclear, cytoplasmic, or membrane-bound.
By employing these fractionation techniques, researchers can reduce sample complexity, improve detection limits, and achieve more comprehensive proteomic analyses. This ultimately leads to better protein identification, more detailed protein characterization, and a deeper understanding of the proteome. A very powerful approach, especially when combined with LC-MS analysis, is fractionation based on dipole-moment and mixed-phase interactions, such as the PreOmics® iST-Fractionation Add-on.
Challenges in Peptide Fractionation
Usually, fractionation approaches in proteomics involve eight or more fractions, leading to very time-consuming LC-MS measurements. Balancing proteomic depth and measurement time is crucial for efficient analysis. Peptide loss, reproducibility, bias in fractionation, contamination, and degradation are also common issues encountered with fractionation approaches. Traditional methods require a compromise between efficiency, effort, cost, complexity, performance, and reliability.
The Solution: PreOmics iST-Fractionation Add-on Kit
The PreOmics iST-Fractionation Add-on kit is designed to enhance peptide and protein identifications in complex proteomic samples by simplifying the fractionation process, making it faster and more reproducible, thus improving the depth of proteome analysis. As an all-in-one and ready-to-use solution, the iST-Fractionation Add-on significantly increases peptide and protein identifications from a single sample. The kit can be seamlessly integrated with PreOmics’ established iST technology, known for its efficiency and ease of use in sample preparation for MS analysis. Combined with the iST kit of choice, the iST-Fractionation Add-on elevates proteomic sample preparation to unparalleled efficiency.
Key Features of the iST-Fractionation Add-on Kit
1. Quick and Reliable Methodology: The fractionation process involves three simple steps using specific buffers to separate peptides into three distinct fractions, completing the process in just 10 minutes.
2. Improved Sample Quality: The method results in only three fractions, significantly boosting the number of peptide and protein identifications compared to unfractionated samples.
3. Compatibility: The kit is based on dipole-moment and mixed-phase interactions (patent pending) and is compatible with standard lab equipment, including all necessary reagents and consumables.
4. Increased Identification Efficiency: The kit supports a working range of 1-100 µg and typically achieves 40-50% more protein identifications compared to unfractionated samples, depending on the sample type and MS instrumentation.
Benefits
- Efficiency: The process requires only 10 minutes of hands-on time, providing a significant increase in peptide and protein identifications from a single sample.
- Comprehensive Analysis: This methodology ensures a sensible compromise between processing time and proteomic depth, suitable for a range of processing options.
- Ease of Use: The all-in-one solution includes all buffers and consumables and is shipped at room temperature, facilitating straightforward implementation in various laboratory settings.
Closing Thoughts
Peptide fractionation is a critical step in proteomic analysis, enhancing the detection and identification of proteins, particularly those present in low abundance. By employing advanced fractionation techniques, such as the PreOmics iST-Fractionation Add-on kit, researchers can achieve more comprehensive and reliable proteomic analyses. This streamlined approach improves sample quality, reduces processing time, and significantly increases protein identifications, ultimately advancing our understanding of complex biological systems.
References
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2. Yates JR, Ruse CI, Nakorchevsky A. Proteomics by mass spectrometry: approaches, advances, and applications. Annu Rev Biomed Eng. 2009;11:49-79.
3. Washburn MP, Wolters D, Yates JR. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol. 2001;19(3):242-247.
4. Boersema PJ, Aye TT, van Veen TA, Heck AJ, Mohammed S. Triplex protein quantification based on stable isotope labeling by peptide dimethylation applied to cell and tissue lysates. Proteomics. 2008;8(22):4624-4632.
