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.