The direct detection and identification of intact proteins in MALDI imaging remains a challenging task due to limited sensitivity and mass range. Here we present new approaches for on-tissue tryptic digestion of proteins. We focus on optimizing the spatial resolution and reliability of peptide identification. We also present strategies for data processing based on the common data format imzML.

Trypsin solution was deposited in several cycles on tissue with a spraying device. An atmospheric pressure matrix assisted laser desorption (AP-MALDI) ion source coupled to a Q Exactive mass spectrometer (Thermo Fisher Scientific GmbH, Bremen) was used for imaging experiments. Tryptic peptides were identified by matching imaged m/z peaks to peptides which were identified in complementary LC-MS/MS measurements of an adjacent tissue sections. All MS measurements were based on accurate mass (< 3 ppm RMS).

A coronal mouse brain section was measured at 50 µm pixel size. Peptide peaks were detected on tissue with a mass resolution of R=80000 (@m/z700). This measurement reveals detailed histological structures such as the ependyma (consisting of a single cell layer) which is clearly defined by several identified peptides. Highly reliable information about protein distribution was also obtained for clinical human tissue originating from brain and gastric cancer biopsies. This data is used to investigate intratumor heterogeneity on a molecular level. A whole body section of an infant mouse was imaged at 50 µm pixel size. Strategies for processing this 40 GB data set by conversion to the common data format imzML will be discussed.

A coronal mouse brain section was imaged at a pixel size of 25 µm. The resulting ion images of tryptic peptides showed excellent correlation with myelin and H&E staining. Peptide peaks were detected on tissue with a mass resolution of R=40000 (@m/z700). The sensitivity could be significantly improved compared to previous experiments and about 150 tryptic peptides which show a clear spatial distribution were identified.

Initial results for formalin-fixed paraffin-embedded tissue will be discussed. Different protocols for deparaffinization and antigen retrieval were evaluated. Tryptic peptides were detected with accurate mass at 35 µm pixel size in mouse brain tissue.

Some of the datasets discussed here were up 40 GB. This requires efficient ways for data processing. An example on how to use the common data format imzML in order to make use of multiple software tools is discussed.