Sampling of Tissues with Laser Ablation for Proteomics: Comparison of Picosecond Infrared Laser and Microsecond Infrared Laser

Abstract

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.

Tissue Sampling and Homogenization in the Sub-Microliter Scale with a Nanosecond Infrared Laser (NIRL) for Mass Spectrometric Proteomics

“…Compared to our previous studies [ 4 , 15 , 16 ] the total number of identified proteins are in the same order of magnitude, but with a significantly smaller ablation volume in this study. With regards to the study of Kwiatkowski et al (2016) [ 4 ] the ablation volume was reduced 100-fold down to 0.5 µL.…”

“…Compared to mechanical homogenization, this is a very gentle method of sample extraction and homogenization, avoiding time-consuming preparation steps. Over the past decade, this tissue sampling and homogenization has been successfully demonstrated with a PIRL, nanosecond infrared laser (NIRL) and even a high-energy microsecond infrared laser (MIRL) with subsequent mass spectrometric proteomics [ 4 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 ] or directly coupled to real-time MS instruments, such as the “SpiderMass” technology [ 22 , 23 , 24 , 25 , 26 ].…”

“…In our previous studies we utilized a picosecond infrared laser (PIRL) [ 4 , 13 , 16 , 19 ] as well as a microsecond infrared laser (MIRL) [ 15 , 16 ] for tissue sampling and homogenization for subsequent proteomics and lipidomics. In a first study by Kwiatkowski et al (2015), it was shown that tissue sampling with the PIRL makes proteins accessible in a wide range from a few kilodaltons to several million daltons.…”

“…Regarding the enzymatic activity of the proteins based on ablated kidney tissue, it was shown that proteins are denatured during tissue sampling with the MIRL, whereas the PIRL ablation had no denaturing effect. Due to the six orders of magnitude longer pulse duration and a much higher pulse energy, MIRL ablation heats the tissue causing denaturation of proteins in the cells neighboring the zone of ablated cells [ 16 ].…”

Food authentication in real life: How to link nontargeted approaches with routine analytics?

“…However, it can be assumed that their importance will increase in the coming years. Laser systems working with different wavelengths can be used to ablate large biomolecules, such as DNA or proteins, as well as smaller metabolites and elements [32,33]. Some analytical platforms also enable the direct nontargeted measurement of food samples without any extraction, for example by means of infrared (IR) spectroscopy [34] (near IR [NIR] [35], mid IR [36]), Raman spectroscopy [37]) as well as different ionization sources for MS: direct analysis in real time (DART) [38], desorption electrospray ionization (DESI), rapid evaporative ionization mass spectrometry (REIMS) [39], liquid extraction surface analysis (LESA) [40], or laser ablation electrospray ionization (LAESI).…”