Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes

Ventouri, Iro K.; Veelders, Sharene; Passamonti, Marta; Endres, Patrick; Roemling, Regina; Schoenmakers, Peter J.; Somsen, Govert W.; Haselberg, Rob; Gargano, Andrea

doi:10.1016/j.aca.2023.341324

Cited by 11 publications

(7 citation statements)

References 37 publications

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“…For SEC separation, the setup was similar to that described by Ventouri et al [41]. A TOSOH (Griesheim, Germany) TSKgel SuperSW3000 column with dimensions 300 mm x 1.0 mm I.D., packed with 4-µm particles with 250 Å pore size, was employed for separations.…”

Section: Chromatographic Systemmentioning

confidence: 99%

“…The two methods are known to target different sample dimensions of the glycated proteins, namely the number of sugars for HILIC as demonstrated in several works on glycoprotein analysis [31][32][33][34][35] and the size and presence of aggregates of the protein in native SEC-MS [36][37][38][39]. In this work, HILIC and SEC-MS analysis are performed at low flow rates (0.5 and 15 µL min -1 respectively) following methods recently published by our group ( [40] and [41] respectively) as under these conditions the desolvation of proteins is favored and lower adducts are observed. The samples were analyzed by LC-MS after glycation without pre-processing, minimizing the risk of losing low-abundant AGEs.…”

Section: Introductionmentioning

confidence: 99%

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Identification of Heavily Glycated Proteoforms by Hydrophilic-Interaction Liquid Chromatography and Native Size-Exclusion Chromatography – High-Resolution Mass Spectrometry

Zhai,

Schoenmakers,

Gargano

2023

Preprint

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The non-enzymatic glycation of proteins and their advanced glycation end products (AGEs) are associated with protein transformations such as in the development of diseases and biopharmaceutical storage. The high heterogeneity that glycated proteins carry makes their detection and identification challenging. In this study, we investigated two novel LC-HRMS methods to study glycated reference proteins at the intact protein level: low-flow hydrophilic-interaction liquid chromatography (HILIC) and native size-exclusion chromatography (SEC). Three model proteins (RNase-A, hemoglobin, and NISTmab) were exposed to conditions that favored extensive glycation and the formation of AGEs. After glycation, complicated MS spectra were observed, along with a sharply reduced signal response, possibly due to protein denaturation and the formation of aggregates. When using HILIC-MS, the glycated forms of the proteins could be resolved based on the number of reducing monosaccharides. Moreover, some positional glycated isomers were separated. The SEC-MS method under non-denaturing conditions provided insights into glycated aggregates but offered only a limited separation of glycated species based on molar mass. Overall, more than 25 different types of species were observed in both methods, differing in molar mass by 14 to 162 Da. 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.

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Section: Chromatographic Systemmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Identification of Heavily Glycated Proteoforms by Hydrophilic-Interaction Liquid Chromatography and Native Size-Exclusion Chromatography – High-Resolution Mass Spectrometry

Zhai,

Schoenmakers,

Gargano

2023

Preprint

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“…They combined offline sSEC with reverse phase chromatography (RPC) for top-down analysis of a complex protein mixture extracted from the cardiac sarcomeric subproteome, yielding identification of 4044 proteoforms ranging from 10 to 223 kDa, and a significantly higher resolution than that of one-dimension RPC was achieved. (Ventouri et al, 2022). Targeted nTDP research have emerged in the past few years (Gault et al, 2020;Roberts et al, 2021;.…”

Section: Native Top-down Analysis Of Proteomesmentioning

confidence: 99%

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

Liu

Xia

2022

Mass Spectrometry Reviews

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Progress in structural biology research has led to a high demand for powerful and yet complementary analytical tools for structural characterization of proteins and protein complexes. This demand has significantly increased interest in native mass spectrometry (nMS), particularly native top‐down mass spectrometry (nTDMS) in the past decade. This review highlights recent advances in nTDMS for structural research of biological assemblies, with a particular focus on the extra multi‐layers of information enabled by TDMS. We include a short introduction of sample preparation and ionization to nMS, tandem fragmentation techniques as well as mass analyzers and software/analysis pipelines used for nTDMS. We highlight unique structural information offered by nTDMS and examples of its broad range of applications in proteins, protein‐ligand interactions (metal, cofactor/drug, DNA/RNA, and protein), therapeutic antibodies and antigen‐antibody complexes, membrane proteins, macromolecular machineries (ribosome, nucleosome, proteosome, and viruses), to endogenous protein complexes. The challenges, potential, along with perspectives of nTDMS methods for the analysis of proteins and protein assemblies in recombinant and biological samples are discussed.

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“…The knowledge about proteins obtained under their native conditions is also important for drug designs and medical demands. There are relatively few analytical/biophysical methods (e.g., native gel electrophoresis, size exclusion chromatography, surface plasmon resonance, nuclear magnetic resonance, or cryo-electron microscopy) that enable the investigation of proteins without disrupting their native form. Beyond these methods, native mass spectrometry can provide very versatile information: molecular weight, stoichiometry, formation of protein complexes, and interactions between proteins and ligands. , …”

Section: Introductionmentioning

confidence: 99%

Taylor–Aris Dispersion-Assisted Mass Spectrometry for the Analysis of Native Proteins

Szabo,

Gyemant,

Nagy

et al. 2024

Anal. Chem.

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As has recently been shown, Taylor−Aris dispersion-assisted mass spectrometry (TADA-MS) can offer direct injection MS determinations in fields where the targets of the analyses are large molecules present in a matrix that would otherwise cause serious interferences. In the present study, we demonstrated the exceptional utility of TADA-MS in native protein analysis: (i) a dramatic improvement in detection sensitivity was found due to its ability to strongly reduce matrix interferences, (ii) more "native-like" conditions can be used during analyses, (iii) the direct injection of non-MS-compatible matrices is allowed into MS, and (iv) a considerable simplification and economization of the workflow is ensured. We investigated the behavior of different types of proteins and protein complexes present under native conditions, demonstrating the unambiguous benefits and simplicity of the method.

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Micro-flow size-exclusion chromatography for enhanced native mass spectrometry of proteins and protein complexes

Cited by 11 publications

References 37 publications

Identification of Heavily Glycated Proteoforms by Hydrophilic-Interaction Liquid Chromatography and Native Size-Exclusion Chromatography – High-Resolution Mass Spectrometry

Identification of Heavily Glycated Proteoforms by Hydrophilic-Interaction Liquid Chromatography and Native Size-Exclusion Chromatography – High-Resolution Mass Spectrometry

Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes

Taylor–Aris Dispersion-Assisted Mass Spectrometry for the Analysis of Native Proteins

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