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In this work, 193 nm ultraviolet photodissociation-mass spectrometry (UVPD-MS) was applied to investigate supercharging reagent sulfolane in protein sequence and structure characterization. All datasets were collected by using an Orbitrap MS system equipped with an ArF 193 nm EX50 excimer Laser. The 193 nm UV laser has the advantages of high brightness, good stability, and propagate in air, which can directly excite and dissociate the protein backbone within 5 ns (single pulse). Previous reports demonstrated that the partial protein high-order structures and non-covalent interactions could be retained in the UVPD fragments and the site-specific fragmentation yields (FYs) revealed the dynamic protein structure alterations under different conditions. Therefore, the combination of UVPD with nMS provides a new research paradigm for the characterization of protein structure changes. In this work, we further demonstrated 193 nm UVPD was superior to high-energy collision induced dissociation (HCD) in fragmenting and sequencing the non-denatured carbonic anhydrase II (CA) generated in native electrospray ionization (ESI). UVPD could efficiently activate and fragmentate the non-denatured CA with different charge states in just 5 ns, achieving cleavage sequence coverage 79.8% for CA Z10. Then, 0.25% supercharging reagent sulfolane was added into the protein solution and the CA charge distribution was significantly moved to higher charge states under identical native ESI conditions. However, higher-order structures (HOS) and non-covalent interactions could be obtained as the CA charge distribution was still concentrated and significantly different from the spectra features of denatured CA. The UVPD cleavage sequence coverage of the CA was increased to 87.2%, including the regions that were difficult to be dissociated and characterized without sulfolane addition. In addition, the non-covalent of CA and Zn2+ remained stable, indicating that the addition of sulfoxide had little influence on the Zn2+ binding region. The UVPD sequence coverage of CA Z11 was increased by 7.4% after the addition of sulfoxide, but the site FYs were almost not changed, demonstrating the corresponding local structures were nearly not changed. However, the higher charge states introduced by sulfolane also exhibited the risk of inducing specific structure alteration of part of protein regions. In conclusion, it was infered that supercharging reagent is efficient in modulating the charge state distribution of proteins in native ESI-MS characterization and can expand the MS detection mass range. The introduction of supercharging reagent can further improve the UVPD sequence coverage, including the structure-compact regions hard to be fragmented. Although part of the protein HOS and non-covalent interactions seems retained, the influence of higher charge states on protein structures should be considered in dynamic protein structure investigation.

Nearly all the life activities are regulated by the interactions of the protein with other biomolecules, including protein-protein interactions (PPIs) and protein-ligand interactions (PLIs), therefore understanding interaction and structural characterization of formed complexes represent a key task for modern bioscience. Conventional biophysical techniques like circular dichroism spectroscopy (CD), fluorescence resonance energy transfer (FRET), surface plasmon resonance (SPR), and isothermal titration calorimetry (ITC), are limited in studying protein complexes structures and interactions, considering sample purity is a major issue for obtaining reliable data with conventional methods mentioned above. Additional technologies are required to complement structural biology and interactomics research, while mass spectrometry (MS) can deal with impurities or even mixtures in principle, therefore native mass spectrometry (nMS) is demonstrated as a powerful tool to bridge the gap between structural biology and interactomics research. As the most widespread soft ionization technique, electrospray ionization is utilized in native mass spectrometry, which employs volatile aqueous solution, near physiological pH and gentle desolvation conditions to make the transfer of weakly bound noncovalent complexes into the gas phase while preserving their integrity and native structure possible. In this way, stoichiometry, binding partners, thermodynamics, structural dynamics, and topology information on transferred noncovalent complexes can be acquired. A recent breakthrough in native MS sample preparation demonstrated direct analysis of overexpressed proteins from crude cell lysates, which significantly simplified sample purification procedures. Innovations in mass spectrometry instruments, such as extended m/z range quadrupole stages or modification of Orbitrap instruments allow access to high mass with little compromise in resolving power. This has promoted the native analysis of protein complexes and diverse protein interaction networks to elucidate their architecture, as well as enabled the identification of small differences in proteoforms. While coupling with either ion mobility spectrometry (IMS) or capillary electrophoresis (CE), the analytical ability of native MS can be greatly extended. In this review, the technologies in native mass spectrometry and several cases in recent reports from five aspects were introduced, such as sample preparation, ion source, instrumentation, ion mobility mass spectrometry and coupling with capillary electrophoresis, challenges for the future development of native mass spectrometry.

Glycosylation is one of the most common types of post-translational modifications (PTMs) of proteins, which affects protein properties including conformation, stability and solubility, and plays essential roles in functions including molecular recognition, signaling and immune defense. Characterizing the higher order structures and dynamics of glycoprotein complexes at the intact level is essential for studying the biological functions of glycoproteins. Particularly, accessing structural information such as glycosylation pattern, proteoform distribution, protein interaction, and dynamic conformational evolvement requires direct measurement of intact species. Native mass spectrometry (nMS) allows preservation of non-covalent inter-protein interactions and direct analysis of topology, stoichiometry and dynamic assembly/disassembly at the intact complex level, highly complementing the conventional biophysical techniques. The combination of nMS with travelling-wave ion mobility spectrometry (TWIMS) provides not only an additional orthogonal dimension of separation, but also geometric features of the analytes through collisional cross-section (CCS) determination, thereby facilitating the structural characterization. The macro- and micro-heterogeneity caused by glycosylation, as well as binding with small molecules, induce structural changes of glycoproteins and their complexes on a scale smaller than those resulting from unfolding or macromolecular association/dissociation. However, the benefits of nMS-IMS for characterization of small-scale structural changes or differences of glycoprotein complexes remain to be further demonstrated, in aspects including proteoform-specific structural analysis, distinguishing different binding states of glycoprotein complexes in complex with small molecules, and solution-parameter-induced conformational or stability changes. In this work, aiming at characterizing the stability and small-scale structural differences of glycoprotein complexes, two formats of avidin (tetrameric biotin-binding glycoprotein) that were expressed from different species and exhibit differing carbohydrate contents and proteoform patterns were used as model systems, and the benefits of implementing ion mobility analysis in nMS for structural analysis of both intact complexes and subunits released in the gas-phase were evaluated. It was demonstrated that IMS allows separation of subpopulations of proteoforms, which facilitates not only profiling of proteoform distribution for intact glycoproteins by MS, but also investigation of correlation between glycosylation and CCS. We also demonstrated the power of IMS in measuring the geometric features of the protein complexes in complex with small molecules, and characterizing the extent of conformational changes of glycoproteins under conditions including gas-phase dissociation, supercharging, charge-reduction, and charge-manipulated dissociation. These advantages enable the stability evaluation for glycoprotein complexes.

Copyright of Journal of Chinese Mass Spectrometry Society is the property of Journal of Chinese Mass Spectrometry Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Chinese Mass Spectrometry Society is the property of Journal of Chinese Mass Spectrometry Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Journal of Chinese Mass Spectrometry Society is the property of Journal of Chinese Mass Spectrometry Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Dynamic changes in the structures and interactions of proteins are closely correlated with their biological functions. However, the precise detection and analysis of these molecules are challenging. Native mass spectrometry (nMS) introduces proteins or protein complexes into the gas phase by electrospray ionization, and then performs MS analysis under near-physiological conditions that preserve the folded state of proteins and their complexes in solution. nMS can provide information on stoichiometry, assembly, and dissociation constants by directly determining the relative molecular masses of protein complexes through high-resolution MS. It can also integrate various MS dissociation technologies, such as collision-induced dissociation (CID), surface-induced dissociation (SID), and ultraviolet photodissociation (UVPD), to analyze the conformational changes, binding interfaces, and active sites of protein complexes, thereby revealing the relationship between their interactions and biological functions. UVPD, especially 193 nm excimer laser UVPD, is a rapidly evolving MS dissociation method that can directly dissociate the covalent bonds of protein backbones with a single pulse. It can generate different types of fragment ions, while preserving noncovalent interactions such as hydrogen bonds within these ions, thereby enabling the MS analysis of protein structures with single-amino-acid-site resolution. This review outlines the applications and recent progress of nMS and UVPD in protein dynamic structure and interaction analyses. It covers the nMS techniques used to analyze protein-small-molecule ligand interactions, the structures of membrane proteins and their complexes, and protein-protein interactions. The discussion on UVPD includes the analysis of gas-phase protein structures and interactions, as well as alterations in protein dynamic structures, and interactions resulting from mutations and ligand binding. Finally, this review describes the future development prospects for protein analysis by nMS and new-generation advanced extreme UV light sources with higher brightness and shorter pulses.

The direct correlation between proteoforms and biological phenotype necessitates the exploration of mass spectrometry (MS)-based methods more suitable for proteoform detection and characterization. Here, we couple nano-hydrophobic interaction chromatography (nano-HIC) to ultraviolet photodissociation MS (UVPD-MS) for separation and characterization of intact proteins and proteoforms. High linearity, sensitivity, and sequence coverage are obtained with this method for a variety of proteins. Investigation of collisional cross sections of intact proteins during nano-HIC indicates semifolded conformations in low charge states, enabling a different dimension of separation in comparison to traditional, fully denaturing reversed-phase separations. This method is demonstrated for a mixture of intact proteins from Escherichia coli ribosomes; high sequence coverage is obtained for a variety of modified and unmodified proteoforms.