Peptides are short chains of amino acids that serve as the building blocks for proteins and create a variety of structural functions in the human body, including hormones, neurotransmitters, and enzymes. When it comes to peptide analysis, LC-MS has proven to be one of the most reliable methods for separating peptide bonds and observing their properties. By implementing the correct testing principles, a major contributor to success is your choice of solvents. In this article, we’ll highlight ways knowledge about LC-MS solvents can significantly improve your sample throughput and sensitivity while minimizing time and effort for quality peptide analyses.
Solvents for Peptides and Preparing Solutions
Although there are thousands of variations, peptides are typically a short string of 2 to 50 amino acids formed by covalent bonds, also known as peptide bonds. They form through a condensation reaction between the amino group of one amino acid and the carboxy group of another amino acid. In order to separate these bonds through LC-MS, the conditions can either unravel their properties for analysis, or potentially destroy your sample. This especially affects peptide samples and the overall impact on sensitivity and resolution. For these reasons, LC-MS solvents can make or break your testing efforts. Selecting one that has compatible properties with your sample and system will ultimately make your results worthwhile.
Water and Organic Mixtures
Aqueous solutions involving LC-MS-grade water and either acetonitrile or methanol are commonly used due to their peptide solubility and compatibility with both liquid chromatography and mass spectrometry. They also set up the ability for solvent gradients, where the mixture’s composition can be adjusted to changing properties during an analysis. This creates a more consistent and effective elution within a chromatography column to accommodate the inherent variables associated with more complex peptide chains, sharpening the resolution between different peptide fragments.
High-purity Acetonitrile
Acetonitrile is one of the most popular choices for LC-MS due to its favorable properties, such as low viscosity and high volatility. These characteristics help produce well-defined peaks for chromatographic and mass spectrographic runs. In peptide analysis, this becomes ideal for solubility of hydrophobic peptides and reduces back pressure, which allows for better flow through the system. The solvent's low ultraviolet absorbance also minimizes interference during detection in the LC to further clear measurable signals for peptide quantification.
Acidic Additives
Additives are often included in the aqueous phase to further boost detection capabilities for a reliable bioanalysis. They balance the pH of the mobile phase while promoting ionization needed for detectability. By increasing the protonation of analytes, acidic additives improve the formation of multiple charged ions necessary for peptide analysis with MS. They also improve the resolution of closely eluting peptides, separating distinct characteristics while identifying complex peptide sequencing. The combination of organic modifiers like dimethyl sulfoxide (DMSO) and formic acid help offer substantial improvements within mass spectrometers by promoting better recovery and repeatability during peptide analyses, which allows you to tackle challenges related to analyte recovery.
Ion-Pairing Reagents
Ion-pairing reagents increase interactions between the peptide chains and the LC column material. These interactions allow for more robust bond separations and distinctions between amino acids. By forming a neutral pair with charged peptides, these reagents improve retention and peak shape in reverse-phase chromatography. Common ion-pairing reagents include trifluoroacetic acid (TFA) and heptafluorobutyric acid (HFBA). These agents work by selectively interacting with positive or negative charges, reducing electrostatic repulsion. While TFA is effective for LC, it can suppress ionization, affecting sensitivity when used in MS applications. HFBA, on the other hand, offers a balance between retention and detection, proving advantageous for specific peptide analyses.
Volatile vs. Non-Volatile Salts
Salts can help both separation and ionization within an MS system, and adjusting their concentrations can avoid adverse precipitation and potential LC instrument damage. Non-volatile salts like phosphate can cause buildup within a system, leading to signal suppression and contamination. They can also destroy components within a chromatography column. Volatile salts, such as ammonium acetate, can stabilize ionization, improve ion exchange, and maintain consistent ionic strength. They also reduce adduct formation for a cleaner spectra and better signal intensity. Incorporating salts within a solution can create better control for more reproducible results but requires meticulous optimization to avoid negative effects on the equipment.
Solvent Considerations for Peptide Analysis
Solvent selection influences both sensitivity and resolution in LC-MS. Sensitivity outlines the detection limit against background noise, while resolution pertains to the ability to distinguish between closely eluting compounds. Solvents facilitate ionization efficiency, so the formulation of your final solution impacts resolution by affecting peak shape and retention times. Using solvents that create stable, narrow peaks can enhance the separation efficiency needed for the quantitation and identification.
LC-MS Compatibility with Solvents
Solvent compatibility involves selecting solvents that align with the instrument's components and ionization techniques. Properties like volatility and the ability to donate protons significantly impact detection, especially with certain techniques like electrospray ionization (ESI). Solvent chemical compatibility also reduces unwanted chemical interactions. Otherwise, an incompatible choice may lead to ion suppression or undesired solvent peaks that interfere with data. Protic solvents like methanol are common choices as they enhance ionization while being generally compatible with several LC-MS techniques.
Certain peptides may be more or less soluble with particular solvents and solutions. Selecting solvents that adequately dissolve peptides without altering their primary amino acid sequence are optimal. Adjusting solution ratios can help dissolve more hydrophobic peptides while maintaining adequate flow and pressure in the system. This balance prevents precipitation and clogging, which could negatively impact results.
High-pH Reverse Phase Chromatography
High-pH reverse phase chromatography is increasingly used to achieve better chromatographic separation of peptides, particularly those with similar properties. At higher pH levels, the acidic amino acid residues are deprotonated, leading to a permanent negative charge causing improved resolution. This method complements traditional low-pH approaches and can separate peptides with subtle differences.
Using high-pH eluents extends column life and reduces column bleed, a common issue at low pH. A key advantage is the ability to analyze basic peptides without excessive tailing, enhancing quantitative accuracy. Be sure your chromatography columns and selected solvents are compatible with high-pH conditions to ensure optimal results and prevent degradation.
Solvent Challenges and Strategies
Due to the complexity of peptide chains, finding a balance with your solvent properties may take the application of several techniques. Selecting the right solvents for analysis can be challenging due to impurities and adduct formation. Addressing these challenges improves accuracy and reliability in your results.
Complex Peptide Chains
Complex peptide chains may require more fine-tuned parameters and solutions with a wider variety of polarity. Without accommodations, this can create problems with isocratic separations like early eluted peaks and poor resolution. Gradient elution is a common technique in LC-MS used to adjust elution variables through a test in order to control separation and retention properties over time. Applying carefully designed solvent gradients with dynamic changes to acetonitrile or methanol ratios provides consistent resolution and better calibration for a wide range of biologically active peptides and.
Temperature Impacts on Performance
Maintaining a temperature between 40-80°C is ideal for peptide analysis without exceeding 100°C and adverse heat that may damage amino acids. Although higher temperatures generally promote mass transfer and reduce viscosity, this leads to greater solvent evaporation and increased sample or column degradation. For these reasons, the boiling point of acetonitrile (81.6°C) is an ideal choice and methanol (64.7°C) can be within a suitable range. The ability to adjust the temperature within a column and a mobile phase allows for better control of retention times, peak shapes, and analyte stability.
Automated Solvent Screening Tools
Automated solvent screening tools save time and streamline the optimization process in an LC-MS system. These tools allow for the evaluation of multiple solvents by adjusting their parameters dynamically. They can either be softwares, robotic systems, or a combination. For example, the AssayMAP Bravo Protein Sample Prep Platform is a piece of machinery that automates liquid flow while precisely filling several protein cartridges simultaneously. UNIFI software facilitates accurate bioseparations, machine calibrations, and peptide mapping. These steps may be tedious and prone to human error and contamination, thus automation is an effective way to save time and resources within high-throughput labs.
Avoiding Adduct Formation
Adduct formation can affect the mass analyzers by causing shifts in mass of peptides and peptide fragments, complicating the interpretation of data. This typically occurs due to strong interaction between solvents and analytes. Choosing solvents like acetonitrile over others can reduce adduct-related issues because of its compatibility with a wide range of analytes. Additionally, adjusting the solvent's pH with buffer systems tailored to your specific peptides can help mitigate adduct formation. Using additives like formic acid can aid in stabilizing the ionization process and reduce adduct presence, leading to cleaner spectra and easier analysis of your peptides.
Dealing with Solvent Impurities
Solvent impurities can easily interfere with the sensitivity and accuracy of your peptide detection. Filtration helps remove particulate contaminants and improves the reliability of your analysis. Consistent lot testing can also aid in identifying and avoiding impurities from unknown sources, preserving the quality of your results. High-purity solvents are ideal choices as they are designed with low trace levels of impurities to reduce the background noise and improve detection limits in a mass spectrum. LC-MS grade solvents, like ours at Birch Biotech, offer a level of purity ideal for peptide analyses.
Want to learn more about our solvents? Read more of our articles or visit us at Birch Biotech.
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