Homogenization of tissues via picosecond-infrared laser (PIRL) ablation: Giving a closer view on the in-vivo composition of protein species as compared to mechanical homogenization

Abstract

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.

Protein crystals IR laser ablated from aqueous solution at high speed retain their diffractive properties: applications in high-speed serial crystallography

“…The method has been particularly applicable as a surgical technique with minimized collateral damage to surrounding tissue (Amini-Nik et al, 2010;Petersen et al, 2016). Recently, we have demonstrated PIRL-DIVE ablation for the soft extraction of structurally and even functionally intact biomolecular complexes such as large protein complexes ($520 kDa) and viruses ($40 MDa) as well as unmodified protein species, directly from aqueous solution and tissue (Kwiatkowski et al, 2015(Kwiatkowski et al, , 2016Ren et al, 2015).…”

Proteome analysis of tissues by mass spectrometry

“…The ablation process was shown to be soft enough to extract proteins from tissue without any change in their chemical composition, and samples of human blood plasma were shown to have detectable enzymatic activities after sample treatment. PIRL‐DIVE showed a high quality of tissue homogenate with a higher number of intact proteins and recovery compared to mechanical homogenization (Kwiatkowski et al, ).…”

Digital interference microscopy and density reconstruction of picosecond infrared laser desorption at the water-air interface

“…At the same time, efficient energy redistribution into thermal modes is ensured by keeping the irradiance low enough to avoid nonlinear optical excitation of higher-lying molecular states. Picosecond infrared laser (PIRL) irradiation fulfilling these criteria has been shown to be well-suited for the extraction of large bio-molecules and even intact viruses for subsequent offline analysis, [20][21][22] as an atmospheric online ion source in combination with electrospray ionization (LAESI), 23 and has been suggested as a potential tool for sample-delivery in high-speed X-ray crystallography. 24 Moreover, the absence of ionizing radiation and minimal deposition of thermal and acoustic energy into the non-ablated sample surface make DIVE a valuable tool for precise material removal in surgical applications.…”