Day: 22 May 2024

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Parallel Gradients 2DLC-HRMS of complex protein digest

2D-LC System
2D-LC System

Investigating the proteins in biological samples can help us understand and identify diseases and improve the effectiveness of medication. To study proteins in these samples, they are typically digested into peptides and subsequently analyzed by liquid chromatography (LC) hyphenated with high-resolution mass spectrometry (HRMS).

Comprehensive two-dimensional LC (LC×LC) offers increased separation power over traditional LC methods. However, most common gradient designs require re-equilibration of every second-dimension run, resulting in high flow rate operations to limit the empty separation space. This also limits MS sensitivity as flow splitting is required to handle such flow rates.

In this work, we developed an LC×LC method using a so-called parallel-gradient design, which omits the need for column re-equilibration and enables the use of the entire separation space. Moreover, this allows for lower flow rates and maintains the sensitivity for low-abundant analytes. The parallel-gradient design achieved higher surface coverages and sensitivity at lower effective peak capacities. Most importantly, both methods were applied to analyze a Human IMR90 lung fibroblast cell line digest to assess its applicability to real complex samples. The parallel-gradient method was able to identify significantly more proteins than the current state-of-the-art methods while using the same analysis time and at a lower solvent consumption. The applicability of the parallel-gradient design could be improved even further by shortening the modulation times, as it was not limited by column re-equilibration.

The study is a collaborative work done thanks for the contribution of many colleagues and students. The link to the publication is reported below.

https://doi.org/10.1021/acs.analchem.4c02172

 

 

 

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Publications

Nanoflow IEC-HRMS to study complex proteoform mixtures

Clinical Materials Molecules
Clinical Materials Molecules

The CAST scientist Ziran Zhai published a manuscript investigating a novel method of using nanoflow strong cation exchange – native mass spectrometry to characterize non-denaturing complex proteoforms mixtures from the intact level. Zhai focuses on three critical aspects: i) extending the MW that can be observed by top-down proteomics, ii) increasing the MS sensitivity to create conditions of detecting low-abundant proteins, and iii) apply mild desolvation conditions to maintain the native structures of proteins and complexes. 

Proteoforms, which are protein products arising from homologous genes due to sequence variations, alternative splicing, and post-translational modifications, play a crucial role in a wide range of critical functions. However, the standard approach to characterize proteins, known as bottom-up proteomics, faces limitations. This approach cannot directly identify proteoforms as the presence of proteins is inferred from peptides. While top-down proteomics and intact protein mass spectrometry offer solutions to these limitations, the most common top-down methods employ denaturing LC-MS approaches, which unfold proteins and lead to the loss of non-covalent protein complexes.

In this work, we directly coupled nanoflow (250 or 500 nL min-1) strong cation exchange chromatography (SCX) to nano-electrospray-ionization (nESI) under native MS (nMS) conditions. Proteins were separated on packed capillary SCX columns and eluted according to their pI values by a salt-mediated pH gradient method. The low flow promoted desolvation/ionization efficiency allowing for sensitive detection of low-abundant proteins and complexes. We successfully applied our method to analyze an E. coli cell lysate and observed hundreds of proteins with masses up to 150 kDa. We believe that the proposed nanoSCX-nMS is a promising approach for characterizing proteoforms and provides a universal strategy to overcome detection limitations in native top-down proteomics.

 

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The study is part of the FFF (From Form to Function) project of Zhai, Astefanei,
Corthals, and Gargano and was funded by the Chinese Scholarship Council (CSC) and was recently published in Analytica Chimica Acta and can be accessed freely at the link below.

https://pubs.acs.org/doi/10.1021/acs.analchem.4c01760.