For peptides, the simplest case for negative peptide ion detection is that the ion pair is Pep-CO2 -⁞Na + (or K + ). If all the peptide molecules contain such an ion pair, the MALDI MS ion yield would be similar to that of permanent salts. In reality, too much salts are more of a problem for MALDI MS because it reduces the likelihood of single charged ions and increases metal adduct formation. Therefore, maintaining the majority of cations being ammonium ions is important as they can neutralize the carboxylates in the gas phase via proton transfer. For the analysis of peptide ionization using the individual ion pair approach, we can use the case of equation ( ) as an example. Assuming the plume temperature is 1,000 K, the predicted ion yield is at the order of 10 -9 (4.6 × 10 -5 × 10 -4 ), which is significantly lower than the experimentally determined yield of 10 -4 -10 -5 . However, at 2,200 K, the predicted yield is at the order of 10 -4 (1.1 × 10 -2 × 3.2 × 10 -2 ), which is consistent with the observed values. Besides temperature, in the case of large biomolecules, intramolecular stabilization of ion pair by electron donor and acceptor groups may be a reason for the higher than calculated ion yield as the stabilization can increase the ion pair to neutral ratio and making the separation of ions in ion pairs easier. Finally, like in the analysis of matrix ionization and ion extraction, it is also possible that other ionization pathways described in the literature may be at play. For nucleic acids, the negative ions detected by MALDI MS are most likely coming from the Nuc-O3PO -⁞Na + (or K + ) ion pair rather than the Nuc-O3PO -⁞NH4 + ion pair, as the concentration of the latter in gas phase is too low for detection due to the strong tendency for proton transfer. For the ion yield of Nuc-O3PO -⁞Na + , the analysis for permanent salts can be applied. The predicted ion yield would be at the order of 10 -4 (4.6 × 10 -4 × 10) assuming each nucleic acid molecule contains over 10 nucleotides, the temperature is 1,000 K and there are sufficient alkaline metal cations (but not too much to cause multicharged or metal adduct peaks) needed for the formation of the Nuc-O3PO -⁞Na + (or K + ) ion pair. For the case of positive ion detection, the ion yield analysis is similar to that of positive peptide ion detection.