MALDI-ToF/ToF mass spectrometry analysis of intact bacteriochlorophylls by using diaminonaphthalene as electron-transfer secondary reaction matrix

Photosynthetic organisms are able to convert photons in chemical energy thanks to unique light-harvesting antenna systems occurring inside the cytoplasmic membrane. These antenna complexes are composed of a wide variety of proteins and different chlorophyll pigments acting as binders [1]. Their structures can be divided into chlorophylls (Chls) class found in cyanobacteria and algae up through plants, and into bacteriochlorophylls (BChls), found in phototrophic bacteria [1]. Both pigments consist of a macrocyclic tetrapyrrolic ring system named porphyrin, coordinating a Mg2+ ion, and several different side chains, usually including phytol [2]. The main difference between these two types of pigments is related to the saturation state of the porphyrin macrocycle, with bacteriochlorophylls having a much more unsaturated structure than chlorophylls. Fast identification of BChls and related compounds may be carried out by matrix assisted laser desorption ionization (MALDI) time-of-flight (ToF) MS because of some characteristic advantages as rapid and easy sample preparation, tolerance to salts, and high sensitivity. However, previous analysis of BChls showed that demetalation of magnesium porphyrins occurs by using conventional acidic matrices. Indeed pheophitinization (i.e., release of the metal ion) was observed by Persson et al., using a-cyano-4-hydroxycinnamic acid (CHCA) as MALDI matrix, for BChls extracted from Chlorobium tepidum green sulfur bacterium that were detected as bacteriopheophytins [3]. Very recently, 1,5-diaminonaphthalene (DAN) was introduced as an electron-transfer secondary reaction matrix for the analysis of chlorophylls [4]. DAN was proved to outperform conventional matrices such as CHCA, dithranol, antracene and even terthiophene, since loss of the metal ion and fragmentation of the phytol-ester linkage are negligible. Here, we report the identification of intact bacteriochlorophylls by MALDI MS in the purple non-sulfur bacterium Rhodobacter sphaeroides, a model system for studying both bacteriochlorophyll biosynthesis and assembly of bacterial photosynthetic complexes [5]. These results show the great capability of MALDI MS to follow bacteriochlorophylls biotransformation occurring in different growth conditions of bacteria.