The analyses of cellular components, such as protein, peptide, and metabolites in several tissues using MALDI-MS have been developed. However, the lipid identification using MALDI-MS has been limited due to less existence of proper matrix for lipid identification. Therefore, we mentioned here about some efforts to find a proper matrix for phospholipid imaging.
The choice of matrix, one of important issues for MALDI-MS, is not simple for in vitro and in situ lipid analysis due to the special properties of lipids, including lower molecular weights (<1,500 Da), rather wide molecular diversity, very close presented m/z ratio, relative insolubility in aqueous systems and the obvious concentration difference within a crude sample. Various matrices had been screened and suggested to be useful for phospholipid analysis (, ). Besides the low chemical noise and interferences from matrix relevant ions, the important prerequisite of a matrix for lipid analysis are namely, good absorptivity at the given laser wavelength, good solubility in analyte solvents, suitable acidity and basicity and high ionization efficiency of analyte molecules.
Structures and names of organic matrices tested useful for analysis of phospholipids by MALDI-MS.
Advantages and disadvantages of organic matrices in the analysis of phospholipids by MALDI-MS.
Among the matrices shown in , 2,5-dihydroxybenzoic acid (DHB), so far, is regarded as the most suitable for lipid analysis in rat brain and neutrophilic granulocytes because it can produce cleaner spectra and fewer fragments of analyte ions (Schiller et al., 1999
). Furthermore relevant matrix peaks do not interfere with analyte identification (Petkovic et al., 2001
). Additionally, suitable vacuum stability of DHB is a key factor for its popular application in imaging mass spectrometry (IMS) of lipids (Wang et al., 2007
). When used for phospholipids analysis in mouse brain, rat brain, mouse cerebellum, mouse retina, single zooplankter individuals, and lipid mixtures extracted from tissues, matrix solution can be prepared with a number of solvent system, such as ethanol/water (1:1, v/v with or without 0.1% trifluoroacetic acid (TFA)) (Schwartz et al., 2003
; Jackson et al., 2005b
; Woods et al., 2006
; Puolitaival et al., 2008
) ethanol/water (9:1, v/v, with or without 0.1% TFA) (Fujiwaki et al., 2002
) acetonitrile/water (1:1, v/v, with 0.1% TFA) (Chen et al., 2008
) methanol/water (7:3, v/v) (Hayasaka et al., 2008
; Shimma et al., 2008
) and chloroform/methanol (2:1 v/v ) (Ishida et al., 2003
). Addition of small amounts of TFA in the matrix solution was found to enhance considerably signal to noise (S/N) ratio in the case of phosphatidylcholines and phosphoinositides, most likely due to enhanced solubility of lipids (Schiller et al., 1999
; Petkovic et al., 2001
One obvious drawback of DHB as MALDI matrix is to its tendency to form large crystals, where the analyte is not evenly distributed, leading to poor spot-to-spot reproducibility and lowering mass resolution (Bouschen and Spengler, 2007
). DHB has some disadvantages for phospholipid analysis as shown in . Due to its relatively higher acidity, DHB produced weak or no signal intensity and exhibited poor sensitivity for phospholipids, especially for phosphatidic acids (PAs), phosphatiylserines, phosphatidylinositols, phosphatidylglycerols, phosphatidylethanolamines in negative-ion mode (Petkovic et al., 2001
; Estrada and Yappert, 2004a
Figure 4 Positive-ion mass spectra of phospholipids extracted from the cortical region of porcine lenses, (A) matrix: 0.5 M DHB in methanol after addition of CsCl crystals, (B) matrix: 0.17 M p-nitroaniline in chloroform/methanol=2/1 after addition of CsCl crystals. (more ...)
Several neutral, basic or low acidic compounds were also tested their suitability for MALDI-MS analysis of phospholipids (Estrada and Yappert, 2004b
; Jackson et al., 2005a
; Woods et al., 2006
; Wang et al., 2007
) to alleviate difficulties in the detection and identification of phospholipid classes with ionization efficiencies lower than those of sphingomyelins and phosphatidylcholines and to improve the sensitivity of negative-ion mass spectra. Among the matrices shown in , 2,6-dihydroxyacetophenone is one of most employed matrices. 2,6-Dihydroxyacetophenone in 50-70% ethanol or methanol with or without 0.1%TFA is commonly used for lipid analysis (Jackson et al., 2005a
; Shimma et al., 2007
). Cesium iodide (Wang et al., 2007
) and lithium chloride (Jackson et al., 2005a
) can be added. 2,6-Dihydroxyacetophenone can be used for both positive and negative ionization mode analysis of phospholipids (Woods et al., 2006
; Jackson et al., 2007
; Wang et al., 2007
), and works more efficient than DHB in terms of detection sensitivity, resolution and signal-to-noise of mass spectra. 2,6-Dihydroxyacetophenone and another neutral matrix of 6-aza-2-thiothymine (ATT) also can be used to detect noncovalent complexes between chlorisondamine and phosphatidylcholine molecular species phosphatidylcholine32:0 and phosphatidylcholine34: 1 on a rat brain tissue added with 1 nmol of chlorisondamine. Noncovalent complex formation was not observed when highly acidic matrices such as sinapinic acid (SA) and cyano-4-hydroxycinnamic acid (CHCA) were used (Jackson et al., 2005a
-Nitroaniline proved to be another good option for the use of DHB for MALDI-MS analysis of phospholipid in a lens tissue (). Due to its higher absorptivity and its lower acidity than that of DHB, p
-nitroaniline provided superior sensitivity for analysis of phospholipids either positive-ion or negative-ion modes and increased the relative amount of deprotonated species in the negative-ion mode than DHB (Estrada and Yappert, 2004a
). Therefore, it was possible to confirm peak assignments for phospholipid classes (phosphatidylglycerols and phosphatiylseriness) that normally give weak signals when DHB is used. p
-Nitroaniline allowed the identification of phosphatidylethanolamines and phosphatidylethanolamines plasmalogens (PEps) even in mixtures containing sphingomyelins and phosphatidylcholines (Estrada and Yappert, 2004b
). However, p
-nitroaniline had the same drawback as 2,6-dihydroxyacetophenone, a high vapor pressure. Although addition of CsCl in p
-nitroaniline solution can minimize matrix (Rujoi et al., 2004
-nitroaniline is precluded in MALDI-MS of phospholipids due to poor vacuum stability.
Most recently, 2-mercaptobenzothiazole as an advantageous alternative to the use of DHB for MALDI-MS of phospholipids in a brain and liver tissue was developed (Astigarraga et al, 2008
). Several special features of 2-mercaptobenzothiazole allows its superior matrix efficiency to that of the commonly used matrices of DHB, 2,6-dihydroxyacetophenone and p
-nitroaniline for profiling and imaging phospholipids in liver and brain samples both in vitro
and in situ
, directly on tissue slices. Its low vapor pressure allows acquisition times of hours, which makes it possible to create well-defined mass images by scanning the whole tissue slices. Its low acidity allows identification of several additional species in negative-ion mode and enables the detection of the largest number of lipids from liver and brain samples. The formation of small and uniform crystals of 2-mercaptobenzothiazole in the IMS sample preparation is a paramount factor for the achievement of good spatial resolution and uniform spectra. Its crystallization in very small and homogeneous crystals also allows high detection reproducibility.
Some ionic liquid or solid matrices have exhibited some advantages over traditional single solid matrixes such as DHB for in vitro
or in situ
phospholipid analysis by MALDI-MS in a brain tissue and biological fluids. Due to their notable features of strong absorptivity, low vacuum volatility (negligible vapor pressure), promote ionization and homogeneous solution, CHCA-based ionic matrices, such as CHCA/n
-butylamine (nBA-CHCA) (Jones et al., 2005
; Li, et al., 2005
), CHCA/1-methylimidazole, CHCA/pyridine, CHCA/tripropylamine, CHCA/tributylamine and CHCA/aniline (Li et al., 2005
), have been reported to give better quality data in terms of ionization efficiency, signal intensities, sensitivity, resolution, signal reproducibility, number of compounds detected, and contaminant tolerance. The utilization of p
-nitroaniline-based solid ionic matrix, p
-nitroaniline/butyric acid, can reduce mass spectrum complexity by reliably appearing of only protonated molecules [M + H]+
of lipids containing phosphatidylcholine head groups, such as lysophosphatidylcholine, phosphatidylcholine, and platelet-activating factor (PAF) and of monosodium adducts [M + Na]+
as the major molecular ions for anionic phospholipids, such as phosphatidylglycerol, PA, and phosphatiylserine in the mass spectra (Ham et al., 2005
). Another feature of p
-nitroaniline/butyric acid is its ability to simultaneously detect phosphorylated lipids of phosphatidylcholines, sphingomyelins, PAFs and phosphatidylethanolamines in the positive-ion mode.
As summarized above, there are several efforts to analyze phospholipid profiles in several tissues using MALDI-MS. Even though current MALDI-MS has certain technical drawbacks, it will become more reliable methodology for phospholipid imaging sooner or later because a lot of trials to conquer the limitations are currently in progress. Therefore, phospholipid imaging by MALDI-MS could be located in a central position of pharmacological investigation of lipid related diseases, such as diabetes, cancer, and obesity.