Integrated NanoLC and UPLC-MS Platforms for Single Cell and Complex Mixture Analysis

ABSTRACT: Background: Multiple Reaction Monitoring (MRM) using triple quadrupole mass spectrometry (QqQ-MS) has become essential for multiplexed protein quantification. This study presents a comprehensive optimization of Agilent 6495C QqQ-MS equipped with both nano-electrospray ionization (nanoESI) and ultra-performance liquid chromatography ESI (UPLC-ESI) sources for enhanced proteomics analysis. We systematically optimized source parameters and compared spectral intensities using a standard peptide mixture (6×5 LC-MS/MS Peptide Reference Mix). Results: The nanoLC system, operating at sub-microliter flow rates, demonstrated over 100-fold enhancement in spectral intensity compared to UPLC when analyzing equivalent sample amounts, highlighting its superior sensitivity for trace-level detection. Performance validation using 30 stable isotope-labeled peptides at varying concentrations confirmed the significant improvements achieved through systematic parameter optimization. Additionally, we evaluated the Multiple Heart-Cutting (MHC) 2D-LC system's separation capabilities using hyaluronic acid polymers. The MHC approach effectively resolved overlapping peaks by selectively transferring UV-detected peaks from the first dimension to a second dimension with orthogonal selectivity. Successful separation of hyaluronic acid tetramer [M+H]+ peaks within complex polymer mixtures validated the system's performance for challenging separations. Significance: These findings demonstrate that the integration of optimized nanoESI sources with nanoLC systems substantially enhances MRM performance for biomarker discovery and validation. The combination of enhanced sensitivity from nanoLC-QqQ optimization and superior separation capability of MHC 2D-LC presents a powerful analytical platform for precise quantification of target molecules in complex biological matrices, advancing proteomics research and clinical biomarker development.

ORGANISM(S): Homo Sapiens

SUBMITTER: Hyunsoo Kim  

PROVIDER: PXD082365 | panorama | Sun Aug 09 00:00:00 GMT+01:00 2026

REPOSITORIES: PanoramaPublic