Matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis of cellular glycerophospholipids enabled by multiplexed solvent dependent analyte-matrix interactions

Abstract

A matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) based approach was developed for the rapid analyses of cellular glycerophospholipids. Through multiplexed solvent-enabled optimization of analyte-matrix interactions during the crystallization process, over a 30-fold increase in S/N was achieved using 9-aminoacridine as the matrix. The linearity of response (r(2) = 0.99) and dynamic range of this method (over 2 orders of magnitude) were excellent. Moreover, through multiplexing ionization conditions by generating suites of different analyte-matrix interactions in the absence or presence of different alkali metal cations in the matrix, discrete lipid classes were highly and selectively ionized under different conditions resulting in the de facto resolution of lipid classes without chromatography. The resultant decreases in spectral complexity facilitated tandem mass spectrometric analysis through high energy fragmentation of lithiated molecular ions that typically resulted in informative fragment ions. Anionic phospholipids were also detected as singly negatively charged species that could be fragmented using MALDI tandem mass spectrometry leading to structural assignments. Collectively, these results identify a rapid, sensitive, and highly informative MALDI-TOF MS approach for analysis of cellular glycerophospholipids directly from extracts of mammalian tissues without the need for prior chromatographic separation.

Figure 1

MALDI mass spectra of D18:0-18:0 phosphatidylcholine (PtdCho) acquired on a 4800 MALDI-TOF/TOF Analyzer in the positive ion mode using 9-aminoacridine as matrix. Both 9-aminoacridine and D18:0-18:0 PtdCho were dissolved in: A) methanol; B) isopropanol; C) acetonitrile (9-aminoacridine was saturated in acetonitrile); or D) isopropanol/acetonitrile (60/40, v/v) and spotted onto the MALDI plate and spectra were collected as described in the “Experimental Section”. Each spot contained 500 fmol D18:0-18:0 PtdCho. The prefix ‘‘D’’ stands for diacyl (i.e., phosphatidyl-) species. S/N stands for signal/noise.

Figure 2

Quantitative analyses of standard PC molecular species using MALDI-TOF MS. A) Mass spectrum of an equimolar mixture of D14:0-14:0 PtdCho, D16:0-16:0 PtdCho, D18:0-18:0 PtdCho, D20:0-20:0 PtdCho and D22:0-22:0 PtdCho; B) Mass spectrum of an equimolar mixture of D18:3-18:3 PtdCho, D18:2-18:2 PtdCho, D18:1-18:1 PtdCho and D18:0-18:0 PtdCho. Mass spectra were acquired on a 4800 MALDI-TOF/TOF Analyzer in the positive ion mode using 9-aminoacridine as matrix as described in the “Experimental Section”. Isopropanol/acetonitrile (60/40, v/v) was used as solvent for dissolving both matrix and PCs. The amount of each individual PC molecular species in each spot is 150 fmol. The prefix ‘‘D’’ stands for diacyl (i.e., phosphatidyl-) species.

Figure 3

Mass spectral comparison of PC molecular species present in mouse heart lipid extracts acquired by either ESI or MALDI. Extracts of murine myocardium were examined by: A) ESI MS or B) MALDI-TOF MS utilizing 9-aminoacridine as matrix dissolved in isopropanol/acetonitrile (60/40, v/v). Myocardial lipid extracts of mice were prepared by a modified Bligh and Dyer procedure as described in the “Experimental Section”. Positive-ion ESI mass spectra of PC was acquired using a TSQ Quantum Ultra Plus triple-quadrupole mass spectrometer as described in the “Experimental Section”. The MALDI-TOF mass spectrum of PC was acquired on a 4800 MALDI-TOF/TOF Analyzer in the positive ion mode as described in the “Experimental Section”. The prefix ‘‘D’’ stands for diacyl (i.e., phosphatidyl-) species. ‘‘IS’’ denotes internal standard.

Figure 4

Comparison of MALDI mass spectra of sodiated TAG molecular species present in mouse heart lipid extract acquired on a 4800 MALDI-TOF/TOF Analyzer in the positive ion mode using 9-aminoacridine as matrix. A) A MALDI mass spectrum of TAG extracted from wild-type mouse heart subjected to normal feeding; and B) A MALDI mass spectrum of TAG extracted from wild-type mouse heart subjected to 24 hours of fasting. Myocardial TAG was extracted by a modified Bligh and Dyer procedure followed by hexane extraction of TAG and methanol back extraction to remove choline glycerophospholipid molecular species as described in the “Experimental Section”. ‘‘IS’’ denotes internal standard.

Figure 5

MALDI mass spectra of Cardiolipins (CLs) present in mouse heart lipid extracts acquired on a 4800 MALDI-TOF/TOF Analyzer in the negative ion mode using 9-aminoacridine as matrix. Mouse myocardial CL was extracted by a modified Bligh and Dyer procedure as described in the “Experimental Section”. Extracts were mixed and spotted onto a 9-aminoacridine matrix and examined in the negative ion mode. * indicates the detected CL ion peaks. ‘‘IS’’ denotes internal standard.

Figure 6

MALDI-TOF MS analysis of phospholipids in the negative ion mode using 9- aminoacridine as matrix. A) A MALDI mass spectrum of negatively charged phospholipids from murine myocardium was obtained by examining an aliquot of a Bligh and Dyer extract of myocardium by MALDI MS in the negative ion mode using a 4800 MALDI-TOF/TOF Analyzer as described in the “Experimental Section”. The prefix “D” and “P” stand for diacyl (i.e., phosphatidyl-) and alkenyl-acyl (plasmenyl-) species, respectively. ‘‘IS’’ denotes internal standard. B) A MALDI tandem mass spectrum of fragment ions obtained from 18:0–20:4 PtdIns present in mouse heart lipid extracts acquired on a 4800 MALDI-TOF/TOF Analyzer in the negative ion mode. The tandem mass spectrum was recorded on a 4800 MALDI-TOF/TOF Analyzer in the negative ion mode using 9-aminoacridine as matrix using CID with the metastable suppressor on and the timed ion selector enabled. The voltages of source 1, collision cell and collision cell offset were 8.0 kV, 7.0 kV and −0.035 kV, respectively. The tandem MS spectrum was obtained by averaging 2000 consecutive laser shots (50 shots per subspectra with 40 total subspectra).