Heat shock protein 27 (Hsp27) and alpha B-crystallin (alphaBC) are small heat shock proteins that stabilize the myofilament during stress. We utilized two-dimensional gel electrophoresis (2-DE), phospho-fluorescence staining, titanium dioxide (TiO(2)) phosphopeptide purification and mass spectrometry (MS) to fully characterize isoelectric point (pI) variants of Hsp27 and alphaBC in rabbit myocardium subjected to brief ischemia/reperfusion (I/R) injury. Four variants of Hsp27 were detected, two of which were phosphorylated: HSP1 (at three sites, Ser15, Ser78 and Ser82) and HSP2 (at Ser15 and Ser82, but not Ser78). Three variants of alphaBC were detected: alphaBC1 was phosphorylated (at Ser59 alone) and alphaBC2 was deamidated (at Asn146). No modifications were found in the remaining variants. Both phospho-Hsp27 variants increased in abundance in tissue subjected to brief I/R injury (15 min I/60 min R) and ischemia without subsequent reflow (15I/0R), and these increases were not affected by addition of the potent antioxidant, N-(2-mercaptopropionyl) glycine (MPG; 15I/60R + MPG and 15I/0R + MPG). Abundance of native and phosphorylated (but not deamidated) alphaBC was elevated following 15I/60R; however, these increases were ameliorated by the presence of MPG, and did not occur in tissue subjected to 15I/0R. Both phospho-Hsp27 variants and phospho-alphaBC translocated to the myofilament following 15I/60R. Increased myofilament association of phospho-Hsp27 was not influenced by MPG, and there was a greater proportion of HSP2 than HSP1 in this fraction. MPG inhibited phospho-alphaBC translocation and increased alphaBC association with the myofilament did not occur during 15I/0R. Increased phosphorylation of Hsp27 is ischemia-specific and not influenced by reactive oxygen species (ROS), while increased expression and phosphorylation of alphaBC are ROS-dependant.