Separation and Characterization of Endogenous Nucleosomes by Native Capillary Zone Electrophoresis–Top-Down Mass Spectrometry

Jooß, Kevin; Schachner, Luis F.; Watson, Rachel; Gillespie, Zachary B; Howard, Sarah A.; Cheek, Marcus A.; Meiners, Matthew J.; Sobh, Amin; Licht, Jonathan D.; Keogh, Michael Christopher; Kelleher, Neil L.

doi:10.1021/acs.analchem.0c04975

Cited by 21 publications

(29 citation statements)

References 53 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Native mass spectrometry (nMS) has emerged as a powerful analytical tool for the delineation of protein complexes. − Due to the heterogeneous nature of protein complexes, coupling online and native liquid-phase separations to nMS is an ideal approach for protein complex analysis. Various liquid-phase separation techniques, such as size exclusion chromatography, , ion-exchange chromatography, and capillary zone electrophoresis (CZE), − have been coupled directly to nMS for the extensive characterization of protein complexes in simple to complex samples. However, the separation resolution of the liquid chromatography (LC) and CZE techniques for protein complexes still need to be improved.…”

Section: Introductionmentioning

confidence: 99%

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Han

Sun

2022

Anal. Chem.

View full text Add to dashboard Cite

Protein complexes are the functional machines in the cell and are heterogeneous due to protein sequence variations and post-translational modifications (PTMs). Here, we present an automated nondenaturing capillary isoelectric focusing-mass spectrometry (ncIEF-MS) methodology for uncovering the microheterogeneity of intact protein complexes. The method exhibited superior separation resolution for protein complexes than conventional native capillary zone electrophoresis (nCZE-MS). In our study, ncIEF-MS achieved liquid-phase separations and MS characterization of seven different forms of a streptavidin homotetramer with variations of N-terminal methionine removal, acetylation, and formylation and four forms of the carbonic anhydrase−zinc complex arising from variations of PTMs (succinimide, deamidation, etc.). In addition, ncIEF-MS resolved different states of an interchain cysteine-linked antibody−drug conjugate (ADC1) as a new class of anticancer therapeutic agents that bears a distribution of varied drug-to-antibody ratio (DAR) species. More importantly, ncIEF-MS enabled precise measurements of isoelectric points (pIs) of protein complexes, which reflect the surface electrostatic properties of protein complexes. We studied how protein sequence variations/PTMs modulate the pIs of protein complexes and how drug loading affects the pIs of antibodies. We discovered that keeping the N-terminal methionine residue of one subunit of the streptavidin homotetramer decreased its pI by 0.1, adding one acetyl group onto the streptavidin homotetramer reduced its pI by nearly 0.4, incorporating one formyl group onto the streptavidin homotetramer reduced its pI by around 0.3, and loading two more drug molecules on one ADC1 molecule increased its pI by 0.1. The data render the ncIEF-MS method a valuable tool for delineating protein complexes.

show abstract

Section: Introductionmentioning

confidence: 99%

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Han

Sun

2022

Anal. Chem.

View full text Add to dashboard Cite

show abstract

“…More recently, multistage MS experiments were used to identify endogenous lipids directly bound to membrane complexes . The further development and integration of “online” fractionation − and multistage top-down MS approaches for characterizing native complexes on a global scale would potentially unlock the ability to track dynamic protein interactions, including those made with small molecules, and inform functional relationships between proteins and metabolic pathways, including revealing unknown ligands to assist in receptor deorphanization.…”

Section: Future Directions and Challengesmentioning

confidence: 99%

Protein–Small Molecule Interactions in Native Mass Spectrometry

2021

View full text Add to dashboard Cite

Small molecule drug discovery has been propelled by the continual development of novel scientific methodologies to occasion therapeutic advances. Although established biophysical methods can be used to obtain information regarding the molecular mechanisms underlying drug action, these approaches are often inefficient, low throughput, and ineffective in the analysis of heterogeneous systems including dynamic oligomeric assemblies and proteins that have undergone extensive post-translational modification. Native mass spectrometry can be used to probe protein–small molecule interactions with unprecedented speed and sensitivity, providing unique insights into polydisperse biomolecular systems that are commonly encountered during the drug discovery process. In this review, we describe potential and proven applications of native MS in the study of interactions between small, drug-like molecules and proteins, including large multiprotein complexes and membrane proteins. Approaches to quantify the thermodynamic and kinetic properties of ligand binding are discussed, alongside a summary of gas-phase ion activation techniques that have been used to interrogate the structure of protein–small molecule complexes. We additionally highlight some of the key areas in modern drug design for which native mass spectrometry has elicited significant advances. Future developments and applications of native mass spectrometry in drug discovery workflows are identified, including potential pathways toward studying protein–small molecule interactions on a whole-proteome scale.

show abstract

“…This foundational work toward classification of small molecules using IM-MS data holds great promise with future interpretation of increasingly complex, larger, and heterogeneous samples, especially as IM-MS instrumentation continues to improve and native IM-MS is applied to investigate endogenous and/or unknown bound small molecules and ligands (see section 2.3.2). 142,167,440,541 2.4.2.2. Classification of Large Biomolecular Ion Conformation Using Native IM-MS Data.…”

Section: Ion Reactions For Improved Separationmentioning

confidence: 99%

“…In these sections we draw upon the numerous comprehensive reviews of these specific topics. Although many online solution-phase separation approaches (such as online chromatography methods − and capillary zone electrophoresis − ) have been introduced to help solve this problem, our discussion is confined to instrumentation and techniques commonly available within mass spectrometers themselves or with small modifications. As this review is written from an academic viewpoint, we refer readers not only to recent native MS work on biotherapeutics ,− but also to many recent efforts and helpful perspectives on this topic from industry scientists representing a variety of biopharmaceutical companies, drawing upon these insights where possible. ,,,− Importantly, though implementation of the methods we describe below faces unique challenges in industry (namely, rigorous standardization and commercialization), we hold that educating potential users on the theoretical aspects of these approaches is important and beneficial to all who utilize native MS, regardless of background.…”

Section: Introductionmentioning

confidence: 99%

Approaches to Heterogeneity in Native Mass Spectrometry

Rolland

Prell

2021

Chem. Rev.

View full text Add to dashboard Cite

Native mass spectrometry (MS) is aimed at preserving and determining the native structure, composition, and stoichiometry of biomolecules and their complexes from solution after they are transferred into the gas phase. Major improvements in native MS instrumentation and experimental methods over the past few decades have led to a concomitant increase in the complexity and heterogeneity of samples that can be analyzed, including protein–ligand complexes, protein complexes with multiple coexisting stoichiometries, and membrane protein–lipid assemblies. Heterogeneous features of these biomolecular samples can be important for understanding structure and function. However, sample heterogeneity can make assignment of ion mass, charge, composition, and structure very challenging due to the overlap of tens or even hundreds of peaks in the mass spectrum. In this review, we cover data analysis, experimental, and instrumental advances and strategies aimed at solving this problem, with an in-depth discussion of theoretical and practical aspects of the use of available deconvolution algorithms and tools. We also reflect upon current challenges and provide a view of the future of this exciting field.

show abstract

Separation and Characterization of Endogenous Nucleosomes by Native Capillary Zone Electrophoresis–Top-Down Mass Spectrometry

Cited by 21 publications

References 53 publications

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Automated Capillary Isoelectric Focusing-Mass Spectrometry with Ultrahigh Resolution for Characterizing Microheterogeneity and Isoelectric Points of Intact Protein Complexes

Protein–Small Molecule Interactions in Native Mass Spectrometry

Approaches to Heterogeneity in Native Mass Spectrometry

Contact Info

Product

Resources

About