New Approach for Tandem Mass Spectrometry Precursor Selection

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A research team has developed a new approach for selecting precursor ions in tandem mass spectrometry (MS/MS) experiments called DiffN, which increases the likelihood of biomarker discovery and provides a promising tool for lipidomic analyses.

Researchers at the University of North Carolina at Chapel Hill have developed a novel approach for selecting precursor ions in tandem mass spectrometry (MS/MS) experiments. The study, published in Analytical Chemistry, introduces the DiffN method, which utilizes the relative differential intensity of ions between two samples to target species undergoing the largest fold changes for MS/MS analysis (1).

Traditional data-dependent acquisition (DDA) methods employ a TopN approach, selecting precursor ions based on their absolute intensity. However, this TopN approach may overlook low-abundance species that could serve as important biomarkers. The DiffN approach seeks to address this limitation by focusing on ions that exhibit significant relative changes between two samples.

To implement the DiffN method, the researchers utilized a dual nano-electrospray (nESI) ionization source, enabling parallel analysis of samples contained in separate capillaries. The team successfully developed and validated the DiffN approach using well-defined lipid extracts as a test case.

To demonstrate the efficacy of the DiffN method in a biological context, the researchers applied the dual nESI source and DiffN DDA approach to compare lipid abundance in two colorectal cancer cell lines. The cell lines, SW480 and SW620, originated from a primary tumor and a metastatic lesion, respectively, in the same patient.

A comparison of the TopN and DiffN approaches on the cancer cell samples revealed the advantages of DiffN in biomarker discovery. DiffN exhibited a higher likelihood of identifying lipid species with substantial fold changes, while TopN had a reduced probability of efficiently selecting such species.


The researchers believe that the efficient selection of precursor ions offered by the DiffN approach positions it as a promising candidate for lipidomic analyses. Moreover, they suggest that the DiffN DDA method may also find applications in studying other molecule classes, such as metabolites and proteins, amenable to shotgun analyses.

The development of the DiffN method represents an important advancement in tandem mass spectrometry, enhancing the ability to detect low-abundance species with significant biological implications. By leveraging the relative differential intensity of ions, this novel approach has the potential to uncover valuable biomarkers and expand our understanding of complex biological systems.

Further research and implementation of the DiffN approach in various analytical settings will shed light on its broader applications and impact in the field of mass spectrometry. With the continued advancements in analytical techniques, the quest for comprehensive molecular characterization is steadily progressing, paving the way for transformative discoveries in the life sciences.


(1) Larson, T. S.; Worthington, C. D.; Verber, M. D.; Keating, J. E.; Lockett, M. R.; Glish, G. L. DiffN Selection of Tandem Mass Spectrometry Precursors. Anal. Chem. 2023, 95 (25), 9581–9588. DOI: