Enhancement of Tryptic Peptide Signals from Tissue Sections using MALDI IMS Post-ionization (MALDI-2)

Matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS) allows for highly multiplexed, unlabeled mapping of analytes from tissue sections. However, further work is needed to improve sensitivity and depth of coverage for protein and peptide IMS. Laser-based post-ionization MALDI-2 has been shown to increase sensitivity for several molecular classes but thus far this has not been reported for peptides. Here, we demonstrate signal enhancement of proteolytic peptides from thin tissue sections of human kidney by conventional MALDI (termed MALDI-1), and conventional MALDI augmented using a second ionizing laser (termed MALDI-2). Proteins were digested in situ using trypsin prior to IMS analysis. For identification of peptides and proteins, a tissue homogenate was analyzed by LC-MS/MS for bottom-up proteomics and the corresponding proteins identified. These proteins were next fully ‘digested in silico’ to generate a database of theoretical peptides to then match to MALDI IMS datasets. Peptides were tentatively identified by matching the MALDI peak list to the database peptide list employing a 5 ppm error window. This resulted in 314 ± 45 (n=3) peptides and 1 112 ± 84 (n=3) peptides for MALDI-1 and MALDI-2, respectively. Protein identifications were similarly made by linking IMS data to the LC-MS/MS peptide database. With positive protein identifications requiring two or more peptides per protein, 55 ± 13 proteins were identified with MALDI-1 and 205 ± 10 with MALDI-2. These results demonstrate that MALDI-2 provides enhanced sensitivity for the spatial mapping of tryptic peptides and significantly increases the number of proteins identified in IMS experiments.<br /

Remodeling of the gastric environment in Helicobacter pylori-induced atrophic gastritis

ABSTRACTHelicobacter pylori colonization of the human stomach is a strong risk factor for gastric cancer. To investigate H. pylori-induced gastric molecular alterations, we used a Mongolian gerbil model of gastric carcinogenesis. Histologic evaluation revealed varying levels of atrophic gastritis (a premalignant condition characterized by parietal and chief cell loss) in H. pylori-infected animals, and transcriptional profiling revealed a loss of markers for these cell types. We then assessed the spatial distribution and relative abundance of proteins in the gastric tissues using imaging mass spectrometry and liquid chromatography with tandem mass spectrometry. We detected striking differences in the protein content of corpus and antrum tissues. Four hundred ninety-two proteins were preferentially localized to the corpus in uninfected animals. The abundance of 91 of these proteins was reduced in H. pylori-infected corpus tissues exhibiting atrophic gastritis compared with infected corpus tissues exhibiting non-atrophic gastritis or uninfected corpus tissues; these included numerous proteins with metabolic functions. Fifty proteins localized to the corpus in uninfected animals were diffusely delocalized throughout the stomach in infected tissues with atrophic gastritis; these included numerous proteins with roles in protein processing. The corresponding alterations were not detected in animals infected with a H. pylori ∆cagT mutant (lacking Cag type IV secretion system activity). These results indicate that H. pylori can cause loss of proteins normally localized to the gastric corpus as well as diffuse delocalization of corpus-specific proteins, resulting in marked changes in the normal gastric molecular partitioning into distinct corpus and antrum regions.IMPORTANCEA normal stomach is organized into distinct regions known as the corpus and antrum, which have different functions, cell types, and gland architectures. Previous studies have primarily used histologic methods to differentiate these regions and detect H. pylori-induced alterations leading to stomach cancer. In this study, we investigated H. pylori-induced gastric molecular alterations in a Mongolian gerbil model of carcinogenesis. We report the detection of numerous proteins that are preferentially localized to the gastric corpus but not the antrum in a normal stomach. We show that stomachs with H. pylori-induced atrophic gastritis (a precancerous condition characterized by the loss of specialized cell types) exhibit marked changes in the abundance and localization of proteins normally localized to the gastric corpus. These results provide new insights into H. pylori-induced gastric molecular alterations that are associated with the development of stomach cancer

ICP Mn Reporter

These are LA-ICP IMS data from the kidney of mice infected with S. aureus. This stain of S. aureus is responsive to manganese. Mice were analyzed at 4, 7, and 10 days post infection

ICP Iron Reporter

These data are LA-ICP IMS data corresponding to mouse kidneys infected with S.aureus. These mice were infected with a, iron-reporter. Mice were analyzed at three time points post infection: 4, 7, and 10 day

MALDI 3D Volume

These data are MALDI IMS data from a 3D mouse kidney infected with S. aureus. These were collected using a SimulTOF mass spectrometer

MALDI manganese 7 Day

These data represent MALDI IMS data of biological replicates of mice infected with S. aureus 7 days post infection. This particular strain of S. aureus has been modified with a fluorescent reporter sensitive to manganese deplete conditions. Three biological replicates are tested here

ICP 3D Volume

These data are LA-ICP IMS data from a mouse kidney infected with S. aureus. Each section of the mouse is numbered and data within each folder correspond to each section

MALDI manganese 4 Day

These data represent MALDI IMS data of biological replicates of mice infected with S. aureus 4 days post infection. This particular strain of S. aureus has been modified with a fluorescent reporter sensitive to manganese deplete conditions. Three biological replicates are tested here

FTICR

MALDI FTICR IMS of a mouse section containing an infectious foci. The FTICR was used to obtain accurate mass measurements from the MALDI IMS data in order to have confidence in protein identifications