Rationale: For the matrix-assisted laser desorption/ionization (MALDI) imaging of proteins and tryptic peptides it is recommendable to remove salts, lipids, and phospholipids prior to analysis. However, thorough investigations of the influence of commonly used washing protocols on the entire protein content and the spectral quality have not been carried out.
Methods: After the application of various on-tissue washing protocols, proteins and peptides were eluted by use of different solvents. Subsequently, protein quantities of the eluates were determined by a bicinchoninic acid assay. The spectral quality of the tryptic peptide eluates was investigated based upon peak picking. A MALDI time-of-flight (TOF) mass spectrometer was used to generate mass spectra. Skin tissue samples were prepared by embedding them either in carboxymethyl cellulose or in a cutting medium containing polyethylene glycol.
Results: Our work shows the numerical decrease in protein content after applying different on-tissue washing protocols. Protein losses in a range of 17-38% were observed. From evaluating the spectral quality, two washing protocols were shown to be beneficial, enabling the detection of a high number of tryptic peptides. Procedures to thoroughly remove polyethylene glycol (deriving from special embedding media) were determined. Critically, aqueous washing steps conducted as short dips in two different jars were beneficial in achieving complete removal.
Conclusions: Washing steps have a strong impact on improving the spectral quality, but they may lead to a high decrease in the protein content. Our results show that there is a balancing act between avoiding protein loss and obtaining high spectra quality.
Copyright © 2013 John Wiley & Sons, Ltd.