Protein carbonylation reveals the molecular basis of cryoinjury in buffalo spermatozoa

During cryopreservation, oxidative damage to sperm biomolecules occurs, ultimately compromising its fertilizing ability. Simple proteome analysis may not accurately reflect the cellular functions that are affected, as most carbonylated proteins lose their functions. The carbonylated proteins act as a good marker of oxidative stress. Choosing buffalo spermatozoa as a model, carbonylated proteins from fresh-extended (FESCL) and frozen-thawed (FTSCL) spermatozoa were enriched by affinity chromatography using biotin hydrazide and a monomeric avidin agarose matrix followed by mass spectrometry. Data were analyzed with Proteome Discoverer (v2.2), post-translational modification analysis was performed using SEQUEST, and affected functional activities were predicted using the FunRich tool (v3.0). The mass spectrometric analysis of sperm proteins led to the identification of 415 carbonylated proteins, 151 in FESCL and 405 in FTSCL. Twelve highly abundant carbonylated proteins identified only in FTSCL can be the major contributors to the cryoinjury. The cryopreservation induces the carbonylation of selective sperm proteins. A total of 98 peptides have been precisely annotated that have oxidative modifications. In comparison to FESCL, more proteins in various cellular components, pathways, and processes in FTSCL were carbonylated. The findings indicate the molecular injury to spermatozoa through carbonylated proteins affecting energy metabolism, free-radical scavenging, cytoskeleton, plasma membrane, capacitation, zona-pellucida binding, and sperm-oocyte interaction and are well correlated with the functional parameters of the spermatozoa. The findings provide strong evidence that protein carbonylation is one of the major factors and molecular basis of cryodamage, which compromises the functions of FTSCL. With cryopreservation becoming an inevitable tool, the study becomes more relevant, and this experimental design would give better-quality frozen-thawed spermatozoa with good molecular profiles.