Infrared laser ablation microsampling was used with data-dependent acquisition (DDA) and ion mobility-enhanced data-independent acquisition (HDMSE) for mass spectrometry based bottom-up proteomics analysis of rat brain tissue. Results from HDMSE and DDA analyses of the 12 laser ablation sampled tissue sections showed that HDMSE consistently identified approximately seven times more peptides and four times more proteins than DDA. To evaluate the impact of ultra-performance liquid chromatography (UPLC) peak congestion on HDMSE and DDA analysis, whole tissue digests from rat brain were analyzed at six different UPLC separation times. Analogous to results from laser ablated samples, HDMSE analyses of whole tissue digests yielded about four times more proteins identified than DDA for all six UPLC separation times.
Infrared (IR) laser ablation at 3 μm wavelength was used to extract enzymes from tissue and quantitatively determine their activity. Experiments were conducted with trypsin, which was ablated, captured and then used to digest bovine serum albumin (BSA). BSA digests were evaluated using matrix-assisted laser desorption ionization (MALDI) mass spectrometry (MS) and sequence coverage of 59% was achieved. Quantification was performed using trypsin and catalase standards and rat brain tissue by fluorescence spectroscopy. Both enzymes were reproducibly transferred with an efficiency of 75 ± 8% at laser fluences between 10 and 30 kJ/m2. Trypsin retained 37 ± 2% of its activity and catalase retained 50 ± 7%. The activity of catalase from tissue was tested using three consecutive 50 μm thick rat brain sections. Two 4 mm2 regions were ablated and captured from the cortex and cerebellum regions. The absolute catalase concentration in the two regions was consistent with previously published data, demonstrating transfer of intact enzymes from tissue.
Another laser and the second from OPOTEK this year: an IR Opolette 2940. It is fixed wavelength at 2940 nm for no other reason than that is the Er:YAG laser wavelength. It is possible to manually adjust the internal optics to generate light from 2700 to 3100 nm.
This laser has its roots in the STTR grant that I had with OPOTEK starting in 2001 (awarded when I was at Emory but moved immediately to LSU in the first year of Phase I). Our goal was to build an OPO with the capabilities of the Mirage 3000B but in a smaller package.
The left port gives access to the Nd:YAG laser fundamental at 1064 nm and the right port is the 2940 nm mid-IR at about 3 mJ per pulse at 20 Hz. Plenty of energy when focused to efficiently ablate thin films (similar to our wavelength tunable IR Opolette which you can see ablating things here.)
J. Dong, Y. H. Rezenom, and K. K. Murray, “Aerosol Desorption Electrospray Ionization,” Presented at the 55th ASMS Conference on Mass Spectrometry, June 4, 2007, Indianapolis, Indiana, Ambient Ionization I, MP 006.