MALDI-TOF/MS analysis of archaebacterial lipids in lyophilized membranes dry-mixed with 9-aminoacridine

Abstract

A method of direct lipid analysis by MALDI mass spectrometry in intact membranes, without prior extraction/separation steps, is described. The purple membrane isolated from the extremely halophilic archaeon Halobacterium salinarum was selected as model membrane. Lyophilized purple membrane were grinded with 9-aminoacridine (9-AA) as dry matrix, and the powder mixture was crushed in a mechanical die press to form a thin pellet. Small pieces of the pellet were then attached to the MALDI target and directly analyzed. In parallel, individual archaebacterial phospholipids and glycolipids, together with the total lipid extract of the purple membrane, were analyzed by MALDI-TOF/MS using 9-AA as the matrix in solution. Results show that 9-AA represents a suitable matrix for the conventional MALDI-TOF/MS analysis of lipid extracts from archaeal microorganisms, as well as for fast and reliable direct dry lipid analysis of lyophilized archaebacterial membranes. This method might be of general application, offering the advantage of quickly gaining information about lipid components without disrupting or altering the membrane matrix.

Fig. 1.

MALDI-TOF MS analysis of phospholipids standards (diphytanylglycerolphosphate ether analogs) isolated and purified from extremely halophilic archaeal microorganisms. Shown are the peaks of the molecular ions [M-H]⁻: BPG at m/z 1520.5 (A); PGP-Me at m/z 900.0 (B); PGS at m/z 886.0 (C); and PG at m/z 805.9 (D). In addition to the peaks of the molecular ions, some fragments that were produced during the desorption/ionization process are shown: the fragment at m/z 731.7 corresponding to PA is present in all spectra, while the fragment at m/z 805.9 in the PGS spectrum (C) corresponds to PG. Under the spectra, chemical structures of the archaebacterial phospholipids are reported.

Fig. 2.

MALDI-TOF/MS analysis of glycolipids standards (diphytanylglycerol ether analogs) isolated and purified from extremely halophilic archaeal microorganisms. The peaks of the molecular ions [M-H]⁻ of S-TGD-1 (B) and S-TGD-1-PA (A) are at m/z 1218.1 and m/z 1932.8, respectively. Present in the spectrum of S-TGD-1-PA (A) is a peak at m/z 1853.0, corresponding to the fragment [M-HSO₃]⁻ produced during the desorption/ionization process. Under the spectra, the previously determined (7, 15) chemical structures of the archaebacterial glycolipids are shown.

Fig. 3.

A: TLC lipid profile of the total lipid extract of purple membranes of Halobacterium salinarum. Lipid abbreviations and molecular ion masses are reported. Lipid assignments are based on previous studies (15, 20). B: MALDI-TOF mass spectrum of the total lipid extract of purple membranes of Hbt. salinarum acquired in the negative ion mode using 9-AA as the matrix.

Fig. 4.

Photograph of fragments of PM/9-AA pellet attached to the MALDI-TOF target. Dimensions of the target are approximately 53 × 41 mm. MALDI-TOF, matrix-assisted laser desorption/ionization time-of-flight; PM, purple membranes; 9-AA, 9-aminoacridine.

Fig. 5.

MALDI-TOF mass spectrum of pellets obtained with lyophilized PM and 9-AA (2:1, w/w) as described in “Experimental Procedures.” The m/z signals corresponding to the different PM lipid components are PG, [M-H]⁻ at 805.9; PGS, [M-H]⁻ at 886.1; PGS sodium adduct, [M-H]⁻ + Na⁺ at 908.2; PGP-Me, [M-H]⁻ at 900.1; PGP-Me sodium adduct, [M-H]⁻ + Na⁺ at 922.1; S-TGD-1, [M-H]⁻ at 1218.3; S-TGD-1-PA, [M-H]⁻ at 1933.5; S-TGD-1-PA, [M-HSO₃]⁻ at 1853.3; and S-TGD-1-PA sodium adduct, [M-H]⁻ + Na⁺ at 1955.5.

Fig. 6.

A: TLC lipid profile of the total lipid extract of red membranes of Halorubrum sp. MdS1 strain. Lipid abbreviations and molecular ion masses are reported. Lipid assignments are based on previous studies (12, 13). B: MALDI-TOF mass spectrum of pellets obtained with lyophilized red membranes of Halorubrum sp. MdS1 strain and 9-AA (2:1, w/w) as described in “Experimental Procedures.” Most lipid components are mainly represented by their sodium adducts. The spectrum was acquired on a Bruker Autoflex mass spectrometer (Bruker Daltonics).