title: Cysteine shotgun-mass spectrometry (CS-MS) reveals dynamic sequence of protein structure changes within mutant and stressed cells.
abstract: Questions of if and when protein structures change within cells pervade biology and include questions of how the cytoskeleton sustains stresses on cells--particularly in mutant versus normal cells. Cysteine shotgun labeling with fluorophores is analyzed here with mass spectrometry of the spectrin-actin membrane skeleton in sheared red blood cell ghosts from normal and diseased mice. Sheared samples are compared to static samples at 37 °C in terms of cell membrane intensity in fluorescence microscopy, separated protein fluorescence, and tryptic peptide modification in liquid chromatography-tandem mass spectrometry (LC-MS/MS). Spectrin labeling proves to be the most sensitive to shear, whereas binding partners ankyrin and actin exhibit shear thresholds in labeling and both the ankyrin-binding membrane protein band 3 and the spectrin-actin stabilizer 4.1R show minimal differential labeling. Cells from 4.1R-null mice differ significantly from normal in the shear-dependent labeling of spectrin, ankyrin, and band 3: Decreased labeling of spectrin reveals less stress on the mutant network as spectrin dissociates from actin. Mapping the stress-dependent labeling kinetics of α- and β-spectrin by LC-MS/MS identifies Cys in these antiparallel chains that are either force-enhanced or force-independent in labeling, with structural analyses indicating the force-enhanced sites are sequestered either in spectrin's triple-helical domains or in interactions with actin or ankyrin. Shear-sensitive sites identified comprehensively here in both spectrin and ankyrin appear consistent with stress relief through forced unfolding followed by cytoskeletal disruption.
authors.affiliation: Molecular and Cell Biophysics Lab, University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Christine C
authors.initials: CC
authors.lastname: Krieger
authors.forename: Xiuli
authors.initials: X
authors.lastname: An
authors.forename: Hsin-Yao
authors.initials: HY
authors.lastname: Tang
authors.forename: Narla
authors.initials: N
authors.lastname: Mohandas
authors.forename: David W
authors.initials: DW
authors.lastname: Speicher
authors.forename: Dennis E
authors.initials: DE
authors.lastname: Discher
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D003545:Cysteine; D003599:Cytoskeleton; D004910:Erythrocyte Membrane / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D056004:Molecular Dynamics Simulation; D009154:Mutation; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry*; D013049:Spectrin / Q000737:chemistry; D013312:Stress, Physiological
chemical_list: D000199:Actins; D011506:Proteins; D013049:Spectrin; D003545:Cysteine