Analytical chemists and proteomics researchers have long recognized the vital role of sample preparation in mass spectrometry (MS) analyses. A recent groundbreaking study published in ‘Analytica Chimica Acta’, conducted by a team at the University of Debrecen, Hungary, has shed new light on the debate surrounding desalting in the peptide mapping process, particularly in combination with capillary zone electrophoresis (CZE). The debate has been addressed with evidence suggesting that, contrary to the norm, desalting may be an optional step for certain samples in CZE-MS peptide mapping.

DOI: 10.1016/j.aca.2023.342162

The Hungarian research team, composed of Nagy Cynthia, Andrasi Melinda, Szabo Ruben, and Gaspar Attila, embarked on this study to elucidate the possible benefits and disadvantages of desalting in the workflow of CZE-MS peptide mapping (“CZE-MS peptide mapping: To desalt or not to desalt?”). In their investigation, they analyzed protein digests of varying complexity in different sample matrices, with a keen focus on the implications of removing salts from samples before analysis—a process widely considered as indispensable in high-performance liquid chromatography (HPLC) or CZE shotgun approaches.

The standard practice in the field has long been to desalt samples primarily because salts can interfere with the electrophoretic separation and mass spectrometric detection. However, the CZE-MS technique is known for its high tolerance towards salts. This fact prompted the current investigation into whether desalting is a necessary step in all scenarios.

The study’s findings indicate that while desalting can alter the peptide profile of samples—mostly impacting hydrophilic peptides—it does not necessarily inhibit the analysis. Instead, desalting has been shown to enable remarkable stacking efficiency due to the low conductivity of the sample background, enabling a phenomenon known as field-amplified sample stacking. Interestingly, even non-desalted samples were able to produce a stacking event, facilitated by transient-isotachophoresis arising from high-mobility ions in non-desalted digestion buffers themselves.

Moreover, the research demonstrates that the introduction of additional ammonium ions or acetonitrile into non-desalted digests can enhance the stacking efficiency. The team used complex samples, such as a yeast cell lysate, and the results followed similar tendencies under optimized conditions.

The implications of these findings are significant for the field of proteomics, where CZE-MS is being increasingly applied for its speed, efficiency, and ability to analyze complex biological samples. The study’s results present a bold proposal: that sample clean-up, in particular desalting, can in many cases be omitted without sacrificing and possibly even enhancing detection sensitivity, separation efficiency, and sequence coverage.

This revelation can potentially streamline the workflow for many laboratories, reducing sample preparation time, costs associated with desalting materials, and the loss of peptides during the desalting process. This could prove particularly beneficial for high-throughput environments where speed is of the essence.

The researchers have declared that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. The study adds a critical piece to the puzzle of optimizing mass spectrometry-based proteomic analyses and opens up new pathways for further research into sample preparation methods.

For researchers and lab technicians alike, these findings will incite revisiting established procedures and reevaluating the necessity of desalting in their own workflows. The study hails a potential paradigm shift, empowering scientists to potentially achieve more with less—less sample processing equating to more efficiency and potentially more accurate results.

References

1. Nagy, C. C., Andrasi, M., Szabo, R., & Gaspar, A. (2024). CZE-MS peptide mapping: To desalt or not to desalt? Analytica Chimica Acta, 1288, 342162. https://doi.org/10.1016/j.aca.2023.342162
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3. Smith, L. M., & Kelleher, N. L. (2013). Proteomics and beyond: cell decision-making shaped by reactive electrophiles. Analytica Chimica Acta, 776, 12–21. https://doi.org/10.1016/j.aca.2013.04.031
4. Gilar, M., Olivova, P., Daly, A. E., & Gebler, J. C. (2005). Two-dimensional separation of peptides using RP-RP-HPLC system with different pH in first and second separation dimensions. Journal of Separation Science, 28(14), 1694–1703. https://doi.org/10.1002/jssc.200500123
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Keywords

1. CZE-MS Peptide Mapping
2. Proteomics Sample Preparation
3. Capillary Zone Electrophoresis
4. Mass Spectrometry Analysis
5. Desalting in Proteomics