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Collision-Induced Unfolding of High-m/z Native-like Protein Ions within a Trapped Ion Mobility Spectrometer

ABSTRACT: Native mass spectrometry (nMS) is a powerful tool for the rapid characterization of protein ions and protein-ligand complexes. By coupling nMS with ion mobility spectrometry (IMS), and collisional activation, insights into protein con-formation and stability can be rapidly obtained. Originally incapable of this workflow, recent work enabled this collision-induced unfolding (CIU) process workflow on commercially available Bruker timsTOF instruments. This early work, however, faced challenges in transmitting larger proteins and only sought to unfold small proteins up to 29 kDa. In this study, we continue the development of this technique and optimized instrument settings, enabling the ionization and transmission of proteins up to 8,000 Th, and successfully unfolded proteins such as superoxide dis-mutase, beta-lactoglobulin, and ovalbumin on a timsTOF. When this TIMS activation technique is applied to large pro-tein ions, however, limited unfolding was observed for bovine serum albumin and no unfolding was observed for immunoglobulin G

INSTRUMENT(S): timsTOF

ORGANISM(S): Assorted

SUBMITTER: Nicholas Borotto

PROVIDER: MSV000098552 | MassIVE | Thu Jul 17 15:40:00 BST 2025

REPOSITORIES: MassIVE

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Project description:Large-scale studies of protein phosphorylation have generally focused on determining the sites and stoichiometry of phosphorylated proteins derived from different biological specimens such as cell lines and tissue samples. While such information is important for understanding the role of phosphorylation in biological systems, it fails to expose the relationship between protein phosphorylation and protein function. Because of the close link between protein function and protein folding stability, knowledge about phosphorylation-induced protein folding stability changes can lead to a better understanding of the functional effects of protein phosphorylation. As part of this work, the Stability of Proteins from Rate of OXidation (SPROX) and Limited Proteolysis (LiP) techniques were utilized to identify phosphorylation-induced conformational and stability changes in proteins derived from a human breast cancer cell line MCF-7. This was accomplished by comparing the conformation properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. The SPROX technique, which probes the global unfolding/refolding properties of proteins, identified 168 proteins with dephosphorylation-induced stability changes, and the LiPs technique, which probes the more local unfolding/refolding properties of proteins, identified 251 proteins with dephosphorylation-induced stability changes. A total of 49 proteins identified here with dephosphorylation-induced conformational changes, were previously identified in phosphoproteomic studies to be differentially phosphorlyated in different human breast cancer subtypes. A total of 98 proteins identified here overlapped with protein hits previously found to be differentially stabilized in four human breast cancer phenotypes, suggesting that the phosphorylated changes observed here may be disease related. The experimental approach and results reported here create a novel approach for identifying functionally significant PTMs in proteins.

Project description:Large-scale studies of protein phosphorylation have generally focused on determining the sites and stoichiometry of phosphorylated proteins derived from different biological specimens such as cell lines and tissue samples. While such information is important for understanding the role of phosphorylation in biological systems, it fails to expose the relationship between protein phosphorylation and protein function. Because of the close link between protein function and protein folding stability, knowledge about phosphorylation-induced protein folding stability changes can lead to a better understanding of the functional effects of protein phosphorylation. As part of this work, the Stability of Proteins from Rate of OXidation (SPROX) and Limited Proteolysis (LiP) techniques were utilized to identify phosphorylation-induced conformational and stability changes in proteins derived from a human breast cancer cell line MCF-7. This was accomplished by comparing the conformation properties of proteins in two MCF-7 cell lysates including one that was and one that was not dephosphorylated with alkaline phosphatase. The SPROX technique, which probes the global unfolding/refolding properties of proteins, identified 168 proteins with phosphorylation-induced stability changes, and the LiPs technique, which probes the more local unfolding/refolding properties of proteins, identified 251 proteins with phosphorylation-induced stability changes. A total of 49 proteins identified here with phosphorylation-induced conformational changes, were previously identified in phosphoproteomic studies to be differentially phosphorlyated in different human breast cancer subtypes. A total of 98 proteins identified here overlapped with protein hits previously found to be differentially stabilized in four human breast cancer phenotypes, suggesting that the phosphorylated changes observed here may be disease related. The experimental approach and results reported here create a novel perspective in understanding large-scale molecular influence of phosphorylation and a shortcut in finding possible functionally significant PTMs in cellular proteins.

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Collision-Induced Unfolding of High-m/z Native-like Protein Ions within a Trapped Ion Mobility Spectrometer

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