Tag: Ziran Zhai

New Frontiers in Intact Protein Characterization by LC-MS at CAST

Post author By Andrea Gargano
Post date 2025-07-04

CAST scientists Annika van der Zon and Ziran Zhai have just published two manuscripts
showcasing significant advances in the low flow analysis of intact antibodies and protein complexes, offering improved sensitivity and performance. The CAST team will utilize these novel nano SEC-MS and HILIC-MS methods in future bioanalysis projects.

Analyzing Minute Amounts of Protein Complexes with Nanoflow Size Exclusion Chromatography–Native Mass Spectrometry

Characterizing intact proteoforms and protein complexes often faces challenges in maintaining native structures and high sample requirements. CAST scientist Ziran Zhai developed a novel nanoflow size exclusion chromatography–native mass spectrometry (nanoSEC-nMS) method to overcome these limitations.

Key Advancements:

Optimized Capillary SEC Columns & Reduced Peak Broadening: The method includes techniques for preparing high-performance capillary SEC columns and optimizing injection to reduce peak widths^.
Direct Coupling under Challenging Conditions: It enables direct coupling of nanoflow SEC with native MS even in salt-rich environments.
Milder Desolvation for Native Structures: Nanoflow allows for milder ESI desolvation, preserving the native structures of proteins and complexes.
High Sensitivity and Throughput: The method requires limited sample (approx. 100 nL per injection) and significantly enhances native MS throughput, enabling online desalting and oligomer separations within 25 minutes.

Figure 1: Analysis of urine samples and Ovitrelle with the nanoSEC-nMS: (a) EIC of the urine hCG samples; (b) MS spectrum of hCG proteins; (c) deconvoluted results of hCG proteins; (d) EIC of the Ovitrelle sample; (e) MS spectrum of Ovitrelle; (f) deconvoluted results of Ovitrelle.

This nanoSEC-nMS method enables the analysis of proteins and complexes across a broad molecular weight range (10 to 250 kDa) in their native states, preserving noncovalently bound metal ions. This study was published in Analytical Chemistry and can be accessed freely at the link below:

https://pubs.acs.org/doi/10.1021/acs.analchem.5c01019

Precise Glycoform Profiling of Intact Antibodies with HILIC-MS

Traditional methods struggle with comprehensive intact antibody glycoform profiling. To address this,CAST scientist Annika van der Zon et al. at developed a novel hydrophilic interaction chromatography (HILIC) method based on lab made acrylamide-based monolithic columns directly coupled to mass spectrometry.

Key Innovations:

Optimized Monolithic Stationary Phase: The porogen composition was optimized, enhancing separation efficiency
Enhanced Glycoform Resolution: The method achieved baseline separations for single and double Fc glycosylation, and partial separations for glycoforms differing by a single glycan unit.
Sensitive Detection of Minor Glycoforms: It enabled sensitive measurement of low-abundance glycoforms in the nanogram injection range.

Figure 2. Analysis of intact trastuzumAb at the intact level. Base Peak Chromatogram of the analysis and Extracted Ion Currents of selected glycoforms are shown.

This HILIC-MS method significantly enhances glycoform selectivity for intact antibodies, providing a more comprehensive characterization essential for bioanalytical applications. This work was published in the Journal of Analytical Chemistry and can be accessed freely at the link below:

https://doi.org/10.1021/acs.analchem.5c02033

Tags Analytical Separation Science, Andrea Gargano, annika van der zon, glycoform analysis, Intact Protein Analysis, mass spectrometry, protein complexes, proteins, Ziran Zhai

Publications

Nanoflow IEC-HRMS to study complex proteoform mixtures

Post author By Andrea Gargano
Post date 2024-05-22
No Comments on Nanoflow IEC-HRMS to study complex proteoform mixtures

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.