Tag: HRMS

Parallel Gradients 2DLC-HRMS of complex protein digest

Post author By Andrea Gargano
Post date 2024-05-22
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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

Tags 2D-LC, Andrea Gargano, Bob Pirok, HRMS, Rick van den Hurk, Two-dimensional liquid chromatography

Publications

Nanoflow IEC-HRMS to study complex proteoform mixtures

Post author By Andrea Gargano
Post date 2024-05-22
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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.

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.

Tags Andrea Gargano, HRMS, Intact Protein Analysis, ion exchange chromatography, native mass spectrometry, proteoforms, Ziran Zhai

Publications

Analysis of Heavily Glycated Proteins by HILIC and SEC-HRMS

Post author By Andrea Gargano
Post date 2024-04-11
No Comments on Analysis of Heavily Glycated Proteins by HILIC and SEC-HRMS

Trypsin digestive enzyme molecule (human). Enzyme that contributes to the digestion of proteins in the digestive system. 3D rendering based on protein data bank entry 1trn. Ball-and-stick model, black background.

The CAST scientist Ziran Zhai recently published a manuscript in which he investigated the usefulness of two novel CAST methods, namely low-flow HILIC [1] and SEC-HRMS [2], to characterize extensively glycated proteins from the intact level. Zhai focuses on four critical aspects: i) using denaturing HILIC-MS to separate glycoconjugates (including, in some cases, the separation of isomers), ii) using native SEC-MS to study the aggregates formed during glycation, iii) identifying the advanced glycation end-products (AGEs), and iv) monitor the dynamic changes of AGEs.

Advanced glycation end products (AGEs) are a family of compounds of diverse chemical nature that are the products of nonenzymatic reactions between reducing sugars (here glucose) and, in the case of our study, proteins. Sugars can attach at different positions in a protein following a Maillard reaction, distributing over several amino acids and in many different chemical species. Previous studies focused on digesting glycated proteins to identify the AGEs and glycoconjugates from the peptide level. However, these strategies make it difficult to monitor the co-occurrence of multiple glycation events and, therefore, cannot monitor the evolution of the glycation process.

In this study, three model proteins (RNase-A, hemoglobin, and NISTmab) were exposed to conditions that favored extensive glycation and the formation of AGEs. As shown, with HILIC-MS, the glycated forms of the proteins could be resolved based on the number of reducing monosaccharides, and the SEC-MS method under non-denaturing conditions provided insights into glycated aggregates (Figure 1). More than 25 different types of species were observed in both methods, among which 19 of these species have not been previously reported. By tracing the progress of glycation, the dynamic changes of the specific AGEs could be monitored over time.