+ in the solid sample is not easy to determine, but can be roughly estimated using the equilibrium constant of equation ( ), which can be estimated using the pKa of the matrices. For example, the pKa of CHCA in water is 1.17, which corresponds to an equilibrium constant of 6.8 × 10 -2 and a concentration of 0.26 M for [M-H] -and [M+H] + with the assumption that the pKa value in water can be used here even though the proton goes to the matrix molecules (all calculations in the article are given in supporting information). The concentration corresponding to ion to neutral ratio of 3.5 × 10 -2 using 133 mL/mol as the molar volume of CHCA. Evidently, the actual concentration of the ions and thus the ion to neutral ratio should be significantly lower because the values of pKa of carboxylic acids in acids are usually two or more units larger than those in water. In addition, the pKauto of acetic acid has been suggested to be 14.5. Assuming the pKauto of CHCA is close to that number, the concentration of [M-H] - and [M+H] + would be in the order of 10 -7 . However, it has been suggested that small amount of residue solvents especially water can drastically change pKa values of acids. MALDI samples are usually prepared from solutions that contain water, and the samples are usually not dried extensively before MALDI MS experiments. It is reasonable to assume that the crystals contain significant amount of water. Thus, the ion concentration should be significantly higher than 10 -7 .