On-Tissue Direct Analysis: MALDI Mass Spectrometric Imaging for Tryptic Digest Peptides

Introduction

MALDI imaging, a type of mass spectrometry using the MALDI technique, can display the distribution of biomolecules such as peptides and proteins without having to conduct such operations as the extraction and labeling of the biomolecules. Up to now, biomolecular MS imaging for a variety of tissue specimens has been reported, and recently there have been published reports showing the distribution of disease-specific protein biomarker candidates. However, proteins that have been detected by conducting mass spectrometry directly on a tissue section are extremely difficult to identify using the mass information alone. The typical method for identifying proteins is to conduct PMF (peptide mass fingerprinting) utilizing “tryptic digest peptides” of the target protein, together with in-silico generated database searches. Since multiple proteins are present in a tissue section, PMF cannot be used for identification of these proteins. This makes it necessary to conduct the MS/MS ion search for the tryptic digested peptides. By utilizing a spotter instrument to apply a coat of enzyme solution on the tissue section, it becomes possible to limit the analysis to micro regions of the tissue. This also allows the use of smaller amounts of expensive enzyme solution as compared to the spray method of coating. The use of spray techniques, including air brushing, can also lead to peptide diffusion on the tissue, complicating analyses. Here we present an example of protein identification conducted at a micro region of a rat liver tissue section, and an example of MALDI imaging of a tryptic digest site on a rat brain tissue section. First, trypsin solution (40 μg/mL, 5 mM NH4HCO3) was deposited on a micro region of the liver tissue section using a chemical inkjet printer (CHIP-1000). Repeat deposition of 1.0 nL of trypsin solution (10 nL/spot) was conducted at 500 μm intervals on the tissue section. After allowing the enzymatic reaction to continue for 2 hours at 37 ̊C in an external incubator, matrix solution (5 mg/mL DHB, 50 % methanol, 0.1 % trifluoroacetic acid) was dispensed onto the tissue.

October 28, 2009 GMT