Application of Various Normalization Methods for Microscale Analysis of Tissues Using Direct Analyte Probed Nanoextraction.

Direct analyte probed nanoextraction (DAPNe) is a method of extracting material from a microscale region of a sample and provides the opportunity for detailed mass spectrometry analysis of extracted analytes from a small area. The technique has been shown to provide enhanced sensitivity compared with bulk analysis by selectively removing analytes from their matrix and has been applied for selective analysis of single cells and even single organelles. However, the quantitative capabilities of the technique are yet to be fully evaluated. In this study, various normalization techniques were investigated in order to improve the quantitative capabilities of the technique. Two methods of internal standard incorporation were applied to test substrates, which were designed to replicate biological sample matrices. Additionally, normalization to the extraction spot area and matrix compounds were investigated for suitability in situations when an internal standard is not available. The variability observed can be significantly reduced by using a sprayed internal standard and, in some cases, by normalizing to the extracted area.