Posttranslational modifications and proteolytic processing regulate almost all physiological processes. Dysregulation can potentially result in pathologic protein species causing diseases. Thus, tissue species proteomes of diseased individuals provide diagnostic information. Since the composition of tissue proteomes can rapidly change during tissue homogenization by the action of enzymes released from their compartments, disease specific protein species patterns can vanish. Recently, we described a novel, ultrafast and soft method for cold vaporization of tissue via desorption by impulsive vibrational excitation (DIVE) using a picosecond-infrared-laser (PIRL). Given that DIVE extraction may provide improved access to the original composition of protein species in tissues, we compared the proteome composition of tissue protein homogenates after DIVE homogenization with conventional homogenizations. A higher number of intact protein species was observed in DIVE homogenates. Due to the ultrafast transfer of proteins from tissues via gas phase into frozen condensates of the aerosols, intact protein species were exposed to a lesser extent to enzymatic degradation reactions compared with conventional protein extraction. In addition, total yield of the number of proteins is higher in DIVE homogenates, because they are very homogenous and contain almost no insoluble particles, allowing direct analysis with subsequent analytical methods without the necessity of centrifugation.Biological significanceEnzymatic protein modifications during tissue homogenization are responsible for changes of the in-vivo protein species composition. Cold vaporization of tissues by PIRL-DIVE is comparable with taking a snapshot at the time of the laser irradiation of the dynamic changes that occur continuously under in-vivo conditions. At that time point all biomolecules are transferred into an aerosol, which is immediately frozen.
It was recently shown that sampling of tissues with a picosecond infrared laser (PIRL) for analysis with bottom-up proteomics is advantageous compared to mechanical homogenization. Because the cold ablation of tissues with PIRL irradiation is soft, proteins remain intact and even enzymatic activities are detectable in PIRL homogenates. In contrast, it was observed that irradiation of tissues with a microsecond infrared laser (MIRL) heats the tissue, thereby causing significant damage. In this study, we investigated the question if sampling of tissues with a MIRL for analysis of their proteomes via bottom-up proteomics is possible and how the results are different from sampling of tissues with a PIRL. Comparison of the proteomes of the MIRL and PIRL tissue homogenates showed that the yield of proteins identified by bottom-up proteomics was larger in PIRL homogenates of liver tissue, whereas the yield was higher in MIRL homogenates of muscle tissue, which has a significantly higher content of connective tissue than liver tissue. In the PIRL homogenate of renal tissue, enzymatic activities were detectable, whereas in the corresponding MIRL homogenate, enzymatic activities were absent. In conclusion, MIRL and PIRL pulses are suited for sampling tissues for bottomup proteomics. If it is important for bottom-up proteomic investigations to inactivate enzymatic activities already in the tissue before its ablation, MIRL tissue sampling is an option, because the proteins in the tissues are denatured and inactivated by the heating of the tissue during irradiation with MIRL irradiation prior to the ablation of the tissue. This heating effect is absent during irradiation of tissue with a PIRL; therefore, sampling of tissues with a PIRL is a choice for purifying enzymes, because their activities are maintained.
The analysis of proteomes directly from tissues requires the proteins to be released from the cells and their compartments and solubilized, which usually is achieved by mechanical homogenization. It was recently shown, that sampling of tissues with the novel picosecond infrared laser (PIRL) offers higher yields of proteins with respect to the total amount and total number of individual proteins in comparison to mechanical homogenization. Furthermore, proteins obtained from tissues by homogenization with PIRL are significantly less enzymatically degraded, giving improved access to the original composition of proteoforms. The effective cold vaporization of tissue with PIRL is very soft, which is responsible for the phenomenon, that even enzymatic activities of proteins in the tissue aerosol are maintained. In contrast, the energy following irradiation of tissue with microsecond infrared laser (MIRL) pulses is not thermally and acoustically confined to the ablated volume. In this study, PIRL (1 J·cm-2) and MIRL (40-60 J·cm-2) were compared for sampling different tissue types for bottom-up proteomics. We showed that PIRL at low fluence is optimal for soft tissue and desired in scenarios were enzymatic activities of proteins must be maintained as well as were no residual tissue damage is a requirement. MIRL could be well suited for scenarios were enzymatic activities must be suppressed within the intact tissue and thermal and acoustic damage is not a concern.<br>
The analysis of proteomes directly from tissues requires the proteins to be released from the cells and their compartments and solubilized, which usually is achieved by mechanical homogenization. It was recently shown, that sampling of tissues with the novel picosecond infrared laser (PIRL) offers higher yields of proteins with respect to the total amount and total number of individual proteins in comparison to mechanical homogenization. Furthermore, proteins obtained from tissues by homogenization with PIRL are significantly less enzymatically degraded, giving improved access to the original composition of proteoforms. The effective cold vaporization of tissue with PIRL is very soft, which is responsible for the phenomenon, that even enzymatic activities of proteins in the tissue aerosol are maintained. In contrast, the energy following irradiation of tissue with microsecond infrared laser (MIRL) pulses is not thermally and acoustically confined to the ablated volume. In this study, PIRL (1 J·cm-2) and MIRL (40-60 J·cm-2) were compared for sampling different tissue types for bottom-up proteomics. We showed that PIRL at low fluence is optimal for soft tissue and desired in scenarios were enzymatic activities of proteins must be maintained as well as were no residual tissue damage is a requirement. MIRL could be well suited for scenarios were enzymatic activities must be suppressed within the intact tissue and thermal and acoustic damage is not a concern.<br>
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