Detection of 25,000 molecules of Substance P by MALDI-TOF mass spectrometry and investigations into the fundamental limits of detection in MALDI

Abstract

Mass spectrometric analysis of peptides with a total sample loading of several tens of thousands of molecules (i.e., low zeptomoles) is demonstrated. At this low level of sample loading, it becomes important to consider several very unique technical and fundamental aspects that are not obvious in compatible experiments with a higher amount of sample loading. We demonstrate that prudent matrix preparation allows analysis of peptides from solutions with picomolar concentrations in matrix-assisted laser desorption ionization (MALDI) mass spectrometry. Using microspot MALDI we demonstrate the introduction and detection of 25,000 molecules of Substance P in a time-of-flight mass spectrometer. A method based on probability theory is presented to estimate the minimum number of ions required for generating a statistically significant isotope peak pattern of peptide ions. It is found that the low boundary of ionization efficiency is 1–2% for Substance P. In addition, comparison of macro- and microspot sample deposition techniques for Substance P shows that under the experimental conditions used, a minimum of ∼5 analyte molecules per μm2 are necessary to generate useful signals. Implications of these results on further mass spectrometric developments towards even more sensitive detection are discussed.