Advances in the Mechanistic Understanding of Matrix‐Assisted Laser Desorption/Ionization In‐Source Decay Mass Spectrometry for Peptides and Proteins: Electron Transfer Reaction as the Initiating Step of Fragmentation

Because matrix-assisted laser desorption/ionization in-source decay (MALDI-ISD) induces selective cleavage on the peptide backbone, this technique allows reliable identification of peptides and proteins. In the last 15 years, several new matrices have been developed that more efficiently induce MALDI-ISD, opening new research avenues. Fragmentation of peptides by MALDI-ISD can be divided into two categories: reducing and oxidizing matrices induce selective cleavage of N-Cα and Cα-C bonds, respectively. Regarding the dissociation mechanism, MALDI-ISD was believed, until recently, to be initiated by "hydrogen atom" transfer between an analyte peptide and the matrix. Based on this hypothesis, the origin of the hydrogen atoms would be the aniline group of the matrix in MALDI with a reducing matrix and the amide nitrogen of the peptide backbone in MALDI-ISD with an oxidizing matrix. MALDI-ISD involves homolytic cleavage of N-H bonds, though the N-H bond is generally stronger than O-H and C-H bonds. Notably, mass spectrometry experiments cannot distinguish between "hydrogen atom transfer" and "electron transfer and subsequent proton transfer." Recent well-designed experiments and quantum chemistry calculations have strongly suggested that electron transfer between the peptide and matrix is likely to be the initial step of the MALDI-ISD process. Reducing and oxidizing matrices for MALDI-ISD induce fragmentation through peptide radical anions and cations, respectively. The generated fragment ions and radicals subsequently undergo reactions within the MALDI plume, leading to the formation of stable even-electron ions that are detectable in the mass spectrum. As a result, MALDI-ISD fragments are observed as both positively and negatively charged ions, despite MALDI-ISD entailing the fragmentation of peptide radical anions and cations. The proposed mechanism offers a robust framework for understanding the MALDI-ISD process. A more comprehensive understanding of this process is essential to fully harness the potential of the MALDI-ISD technique and would pave the way for further development of methodologies advancing the field of analytical chemistry based on finding new matrices.