Effects of Matrices and Additives on Multiple Charge Formation of Proteins in MALDI–MS Analysis

The sinapinic acid (SA) matrix has frequently been used for protein analysis in matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS). However, the SA matrix does not result in the formation of distinctive multiple protein charge states, whereas the 2-nitrophloroglucinol (2-NPG) matrix is capable of this. The formation of multiple charge states in the MALDI–MS analysis of proteins is advantageous in that it results in higher accuracy. In this study, the mass spectra of several common standard proteins, namely cytochrome c, myoglobin, bovine serum albumin (BSA), and immunoglobulin G (IgG), were compared using various matrices (2,5-dihydroxybenzoic acid, α-cyano-hydroxycinnamic acid, SA, and 2-NPG). Furthermore, the mass spectra of two large standard proteins (BSA and IgG) using various acid additives (H₃PO₄, HNO₃, H₂SO₄, HCl, and trifluoroacetic acid) with the 2-NPG matrix were also compared. Among the different matrices, 2-NPG provided the broadest range of multiple protein charge states, while, among the different additives, the 2-NPG matrix in combination with HCl generated the broadest multiple charge states as well as the most intense protein peaks.