Differential Mobility Separation for Improving Lipidomic Analysis by Mass Spectrometry

Význam tématu

Lipidomics has emerged as a critical discipline for understanding cellular function, disease mechanisms and biomarker discovery. The structural diversity of over 100,000 lipid molecular species in a narrow mass range poses significant analytical challenges. Conventional shotgun or chromatographic methods often struggle with isobaric and isomeric overlap, limiting confident identification and accurate quantitation of individual lipid species.

Cíle a přehled studie

This study evaluates the integration of differential mobility separation (DMS) via SelexION® technology on QTRAP® mass spectrometers to improve lipid class and subclass resolution prior to MS analysis. Key objectives include:

• Demonstrating baseline separation of major phospholipid subclasses
• Assessing performance on complex biological extracts
• Evaluating the impact on MS/MS spectral clarity and confidence in lipid identification

Použitá metodika a instrumentace

The experimental workflow combines direct infusion and DMS-enabled MS analysis:

• Sample Preparation: Phospholipid standards and bovine heart total lipid extract dissolved in MeOH:CH2Cl2:H2O (45:45:10 v/v) with 3 mM ammonium acetate.
• Instrument: QTRAP® 5500 mass spectrometer equipped with the planar SelexION® DMS cell and Turbo V™ atmospheric-pressure ion source.
• DMS Conditions: Separation voltage up to 3,800 V, high-resolution curtain gas, isopropanol chemical modifier, ramped compensation voltage (CoV) from –15 V to +15 V.
• Data Analysis: Enhanced MS (EMS) and MRM for class separation, followed by Enhanced Product Ion (EPI) scans processed in PeakView™ and LipidView™ software.

Hlavní výsledky a diskuse

1. Phospholipid Subclass Resolution

• Baseline separation of PC, PE, PS, PG, PI and PA standards achieved by DMS, each with a distinct CoV.
• In complex bovine heart extract, DMS generated discrete total ion chromatogram peaks corresponding to each subclass, simplifying MS spectra.

2. Improved MS/MS Specificity

• Without DMS, product ion spectrum of m/z 786.4 contained overlapping fatty acid fragments, yielding multiple candidate structures.
• With DMS, separate CoV windows for PC and PS produced cleaner MS/MS spectra, enabling discrimination of isobaric and isomeric lipids and assignment of precise fatty acyl compositions.

3. Broad Lipid Class Separation

• In positive ion mode, DMS distinguished polar lipids (phospholipids, sphingolipids) from neutral lipids (glycerides, cholesterol esters) with minimal crossover.
• Precursor ion scans for the phosphocholine head group (+184 Da) were resolved into independent SM and PC spectra.

Přínosy a praktické využití metody

• Orthogonal separation prior to mass analysis reduces isobaric interference, improving identification confidence and quantitative accuracy.
• Enables rapid, infusion-based lipid profiling without extensive chromatography or derivatization.
• Compatible with targeted MRM workflows for high-throughput lipidomics in research and QC laboratories.

Budoucí trendy a možnosti využití

Future developments may include:

• Refinement of chemical modifiers to enhance resolution of minor lipid subclasses.
• Integration with LC-MS workflows for multidimensional separation.
• Application of DMS to other metabolite classes and environmental matrices.
• Automated software tools leveraging DMS data for large-scale lipidome mapping.

Závěr

SelexION® differential mobility separation on QTRAP® systems delivers an efficient, orthogonal filter that discriminates lipid classes and reduces isobaric overlap. This approach streamlines shotgun lipidomics, yielding cleaner MS and MS/MS spectra that enhance structural elucidation and quantitation in complex biological samples.

Reference

1. Schneider BB, Covey TR, Coy SL, Krylov EV, Nazarov EG. Planar Differential Mobility Spectrometer as a Pre-Filter for Atmospheric Pressure Ionization Mass Spectrometry. Int J Mass Spectrom. 2010;298(1-3):45-54.
2. AB SCIEX. SelexION Technology: A New Solution to Selectivity Challenges in Quantitative Bioanalysis. Technical Note RUO-MKT-02-3251-A.
3. Schneider BB, Covey TR, Coy SL, Krylov EV, Nazarov EG. Chemical effects in the separation process of a differential mobility/mass spectrometer system. Anal Chem. 2010;82:1867-1880.