Introduction:

Multiplexed MALDI-immunohistochemistry (IHC), is a technique where Photo-cleavable peptide tags attached to antibodies are bound to proteins within tissue and released via UV cleavage prior to MALDI imaging and provide localisation information for the original protein. Typically, these proteins will have a molecular weight outside of the instrument mass range and would traditionally have required on-tissue digestion prior to analysis which can reduce localisation and specificity. 

Traditional immunofluorescence histochemistry is typically limited to 2-5 markers per tissue section, this is due to spectral overlap in excitation and emission wavelengths, in contrast MALDI-IHC is able to image >40 markers per tissue section and is only limited by the number of available masses and availability of tags for all proteins of interest.

Objectives:

Here we demonstrate the compatibility and advantages of MALDI-IHC analysis on a SELECT SERIES™ MRT, equipped with a continuous rasta MALDI source.

Methods:

Sections of Human Tonsil (Ambergen) and Human Kidney (ccRCC ISUP grade 3) FFPE tissues were prepared in accordance with the AmberGen MALDI HiPLEX-IHC Miralys™ Imaging laboratory Workflow user guide (Control number v173(J)) protocol. The tonsil control sections were purchased pre stained, the Kidney sections were stained in-house. The samples were analysed using a SELECT SERIES MRT MALDI, in positive ionization mode, with a mass range of 50-2400. Due to the tags small mass distribution, a fixed quad setting of 1000da was set. The laser repetition rate was 2khz with a scan speed of 10 s/s. Images were acquired at 50 µm pixel size and 20µm pixel size with a laser focus setting of 4.0mm and 5.8mm respectively.

Results:

Here we demonstrate full compatibility of MALDI-IHC analysis on a SELECT SERIES MRT and highlight potential advantages over a standard OA-MALDI-Tof. The data generated has a high mass accuracy consistently >500ppb. Coupled with a high mass resolution averaging >200,000 FWHM.

From the tissue sections we analyzed the bio-localization of the tags was consistent with the expected biology of the tissue. Excellent signal strength was achieved with both the 50µm and 20µm pixel sizes suggesting that good data could be achieved with smaller pixel sizes or faster analysis times if desired.

Conclusions:

The high mass accuracy allows for unambiguous identification of released tags from endogenous signals and the high mass resolution significantly reduces potential signal overlap with endogenous signals of similar mass. This also provides higher confidence in protein localization information compared to traditional immunofluorescence, where tissue auto-fluorescence (background fluorescence of molecules in the tissue) can cause high interfering signals and result in non-specific detection. The SELECT SERIES MRT produced excellent signal strength suggesting that good data could be achieved with smaller pixel sizes or faster analysis times if desired.