Pigments and proteins in green bacterial chlorosomes studied by matrix‐assisted laser desorption ionization mass spectrometry

Abstract

We have used matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for mass determination of pigments and proteins in chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 mL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI-MS can provide a rapid method for obtaining a semiquantitative determination or finger-print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB.

Chlorobaculum tepidum regulates chlorosome structure and function in response to temperature and electron donor availability

“…Therefore we sought to determine whether the observed BChl c spectral shifts with temperature and light intensity in wild type or C5 cells could also be correlated to BChl c homologue distribution as determined by HPLC analysis. In agreement with previous reports Persson et al 2000 Both wild type and C5 cultures exhibited an overall similar distribution of the four BChl c homologs, with [E,E]-BChl c and [P,E]-BChl c being the two major BChl c species (Fig. 2).…”

“…Chlorophylls were detected by fluorescence (k EX = 368 nm; k EM = 677 nm). Pigments were identified by characteristic retention times based on previously reported values (Persson et al 2000). The alkylation index of BChl c was expressed as the mean number of carbon atoms present on positions 8 and 12 of the chlorin ring (Borrego et al 1999).…”

“…Single chlorosomes contain *200,000 BChl c F molecules that self-aggregate into dimers, parallel dimer layers, and higher order rod elements, the major internal structural unit (Egawa et al 2007;Montaño et al 2003). BChl c molecules exist as a set of homologs which differ in the number and identity of alkyl groups on positions 8 and 12 of the chlorin ring (Nozawa et al 1991) that can be resolved by HPLC (Persson et al 2000). Despite being considered low-light specialists (Overmann and Garcia-Pichel 2006), C. tepidum and other green phototrophic bacteria can acclimate to changes in growth light intensity by altering the distribution of BChl c homologs (Bobe et al 1990;Borrego and Garcia-Gil 1994;Borrego et al 1999).…”

MALDI‐TOF mass spectrometry of bacteria*

“…In another interesting study, pigments and proteins from chlorosomes, the light-harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum were characterized directly from organelles (Persson et al, 2000). By applying a small volume of a concentrated suspension of isolated chlorosome organelles directly onto the target, bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c were FIGURE 10.…”

Electron-Transfer Secondary Reaction Matrices for MALDI MS Analysis ofBacteriochlorophyll ainRhodobacter sphaeroidesand Its Zinc and Copper Analogue Pigments

“…Fast identification and characterization of BChl and related compounds could be carried out by matrix assisted laser desorption/ionization (MALDI) MS because of some significant advantages such as rapid and easy sample preparation, tolerance to salts, and high sensitivity. However, pheophitinization (i.e., release of the metal ion) accompanied by extensive fragmentation was observed by Persson et al [18] using α-cyano-4-hydroxycinnamic acid (CHCA) as MALDI matrix, for BChl extracted from Chlorobium tepidum green sulfur bacterium. Replacing the proton-transfer matrices with electron-transfer (ET) ones, such as terthiophene (TER) [19] and 9,10-diphenylanthracene (9,10-DPA) [20], in MALDI MS of Chl, a highly stabilized radical cation was formed; the demetalation of Chl was greatly reduced, whereas extensive fragmentation (loss of the phytol moiety) remained.…”