Native LC/MS: Taking the Next Step in Protein-Based Applications

Proteins are dynamic entities that act as key mediators of cellular phenotypes. Efforts to characterize these proteins began by using mass spectrometry (MS) to determine their molecular masses and peptide sequences. Proteins, however, are not characterized by their peptide sequence alone. Non-covalent protein complexes, those involving molecular interactions other than the direct sharing of electrons, impact protein stability, function, and ultimately the biological impact of the molecules involved. Scott Berger, Senior Biopharmaceutical Market Development Manager at Waters, explains that when desiring to holistically analyze these complexes “maintaining these interactions during sample preparation, analyte separation, and mass detection is a fundamental characterization challenge. Traditional reversed-phase LC/MS-based techniques typically disrupt these non-covalent interactions, removing the ability to properly analyze these complexes.”

Native liquid chromatography/mass spectrometry (LC/MS) has changed this dynamic. LC/MS tweaks existing MS methods by allowing proteins to retain their structure as they enter the mass spectrometer. In doing so, scientists can now begin to investigate protein complexes and interactions with other biomolecules. This, in turn, has facilitated efforts to develop novel biotherapeutics and assure quality controls in these therapeutics.

What is native LC/MS?

Native LC/MS requires a modification to existing MS approaches. Native LC/MS uses softer ionization conditions that preserve non-covalent interactions within and between proteins as they make the transition to becoming gas-phase ions within the mass spectrometer source region.¹ “In native LC/MS, a biological analyte is ionized through applying an electrical potential assisted spraying process, requiring the LC mobile-phase to contain volatile ‘MS-friendly’ and ‘complex-friendly’ components,” Berger explains. “Known as electrospray ionization mass spectrometry (ESI-MS), a narrow capillary emitter disperses a fine spray of highly charged aqueous droplets containing volatile salts and buffers such as ammonium acetate and ammonium formate. At moderate levels, these volatile salts do not significantly disrupt the electrospray plume, nor disrupt the non-covalent interactions of the analytes. Once the charged droplets are dispensed, vacuum-promoted evaporation in the MS source region facilitates removal of the bulk solvent and the volatile salts, enabling individual ionized analyte molecules to be delivered to the mass spectrometer.”

Chromatography in native LC/MS—Enhancing protein separation

Modifications to ESI-MS have also enabled further differentiation of proteins by other characteristics. These traits include hydrophobicity, ionic charge, and size—all of which can impact protein structure and activity. These alterations were produced because ESI-MS cannot resolve protein isoforms without an initial separation step.²

The aforementioned separations can be accomplished through a chromatography step before mass spectrometry. One such approach, size-exclusion chromatography-mass spectrometry (SEC-MS), separates protein aggregates by molecular weight first before performing mass spectrometry.³ Zoe Zhang, Senior Manager of Biopharma Applications and Tech Marketing at SCIEX, draws attention to ion exchange chromatography-mass spectrometry (IEX-MS) as well, “Proteins can also be distinguished by their proteoform, one of many molecular forms in which a protein product from a single gene can be found. A protein’s charge represents one of the variables that can delineate one proteoform from another. IEX-MS separates these proteoforms based on their net charge.”

Waters has also developed a novel implementation of ion mobility-based separations that adds additional value to native LC/MS analysis. The ion mobility elements within the mass spectrometer enable these ESI-generated molecular ions to be separated in the gas phase not only by their mass and charge, as in traditional MS analysis, but also by their shape as measured by their average collisional cross-section (CCS) surface area value.⁴ Berger explains how this characteristic can be used to further separate proteins and complexes within the MS this way, “Ion mobility resolves protein ions that would generate the same measured molecular mass by determining their effective resistance to traveling in a low-pressure region of the instrument. Think of the equivalent of aerodynamic drag. Here, two proteins with the same molecular weight will have differential ion mobility based on a protein’s conformational arrangement or binding state. In other words, a more compact protein or complex will travel faster than those with a more extended structure. This enables us to monitor the higher order structure, interactions, and structural dynamics of proteins.”

Insights generated through native LC/MS

Native LC/MS provides valuable insights into the non-covalent interactions between proteins. These interactions are elucidated thanks in part to MS’s ability to identify biomolecules at higher mass-charge ratios. According to Zhang, “The time-of-flight (TOF)-based approach employed in MS produces a wide mass range for detecting proteins, rendering native LC/MS useful for studying intact proteins and conducting large species analyses.” Native LC/MS can also provide information about the stoichiometry of the subunits within complexes, and application of elevated voltages in MS can inform us how the protein complexes are assembled by the pattern of subcomplexes that form when they break apart. Berger adds that with “native LC/MS, we can perform structural studies where we look at the subunit binding patterns of a complex, or look at the changes in the cross-sectional area of an individual protein, a proxy measurement of protein unfolding pathways and overall protein stability.”

Applications—quality controls

The benefits that native LC/MS provides for protein characterization can be applied to biotherapeutic R&D. For instance, lot-to-lot variation represents a major problem when developing protein-based biotherapeutics.⁵ This is especially prevalent when producing antibodies for various applications, reducing specificity.⁶ Native LC/MS provides several opportunities to improve the consistency of therapeutic protein production. For instance, ESI-MS has been used to characterize the types and abundances of different glycoforms present during the production of Trastuzumab, a therapeutic monoclonal antibody.⁷ Zhang explains that “many non-covalent interactions occur between proteins or between protein and small drugs that may significantly impact drug efficacy and may lead to safety concerns. Identifying these interactions using native LC/MS is therefore important to refine the development of therapeutics under native conditions to treat disease.”

Applications—antibody-drug conjugates

Antibody drug conjugates (ADCs) can rely on non-covalent interactions to maintain the antibody structure when bound drugs are covalently linked to Cys residues generated by reduced inter-chain disulfide bridges.⁸ Berger touts the ability of native LC/MS to maintain the capability to look holistically at the ADC molecule, “The more robust ADCs can maintain the canonical double heavy chain/double light chain paired structure even when lacking the bridging disulfides, when they are separated, and mass detected using conditions that won’t break the molecule into pieces. Performing native LC/MS with online size exclusion and appropriate volatile buffers helps us determine how many cysteine residues per molecule contain Cys coupled drugs, whereas reverse-phase LC/MS analysis would have released the disulfide liberated subunits for isolated analysis.”

Conclusion

Native LC/MS represents an upgrade over reverse-phase MS for characterizing intact proteins. Whereas conventional MS techniques require protein fragmentation for characterization, native LC/MS allows proteins to retain their structural properties. Native LC/MS has facilitated efforts to improve quality controls for protein-based biotherapeutics and develop ADCs as novel medications for anticancer therapies. Nevertheless, native LC/MS faces limitations as a tool for quantifying protein abundances and when teasing apart heterogeneous protein complexes.

References

1. Boeri Erba E, Petosa C. The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes. Protein Science. 2015;24(8):1176-1192. doi:10.1002/pro.2661

2. Füssl F, Strasser L, Carillo S, Bones J. Native LC-MS for capturing quality attributes of biopharmaceuticals on the intact protein level. Curr Opin Biotechnol. 2021;71:32-40. doi:10.1016/j.copbio.2021.05.008

3. Hong P, Koza S, Bouvier ESP. A Review Size-Exclusion Chromatography for the Analysis of Protein Biotherapeutics and Their Aggregates. Journal of Liquid Chromatography & Related Technologies. 2012;35(20):2923-2950. doi:10.1080/10826076.2012.743724

4. Ieritano C, Lee A, Crouse J, et al. Determining Collision Cross Sections from Differential Ion Mobility Spectrometry. Anal Chem. 2021;93(25):8937-8944. doi:10.1021/acs.analchem.1c01420

5. Thompson S, Chesher D. Lot-to-Lot Variation. Clin Biochem Rev. 2018;39(2):51-60.

6. Böttcher S, van der Velden VHJ, Villamor N, et al. Lot-to-lot stability of antibody reagents for flow cytometry. J Immunol Methods. 2019;475:112294. doi:10.1016/j.jim.2017.03.018

7. Damen CWN, Chen W, Chakraborty AB, et al. Electrospray Ionization Quadrupole Ion-Mobility Time-of-Flight Mass Spectrometry as a Tool to Distinguish the Lot-to-Lot Heterogeneity in N-Glycosylation Profile of the Therapeutic Monoclonal Antibody Trastuzumab. Journal of the American Society for Mass Spectrometry. 2009;20(11):2021-2033. doi:10.1016/j.jasms.2009.07.017

8. Marei HE, Cenciarelli C, Hasan A. Potential of antibody–drug conjugates (ADCs) for cancer therapy. Cancer Cell International. 2022;22(1):255. doi:10.1186/s12935-022-02679-8