115
ions during MS analysis that can significantly complicate the mass spectra. Tissue
washing procedures have been shown to lessen or remove OCT artifacts, but these
washes may wash away compounds of interest as well [35]. Other materials have
been purposed as embedding materials with limited MS effects, including carboxymethylcellulose (CMC) and gelatin [45, 46]. Use of these materials typically introduces minimal effects, while still assisting in sectioning.
Washing can remove potential chemical interferences and select for specific analytes (Fig. 7.4b) [35, 49, 50, 51]. Ammonium formate (AmF) buffer is used to
remove salts from tissue for the analysis of lipids [49]. The ammonium cation of the
buffer system is volatile and removed from tissue by the vacuum of MS instruments.
In the positive ion mode, protonated ions dominate the spectra after AmF tissue
washing. Decreased salt presence also increases sensitivity in the negative ion
mode. Sensitivity for intact proteins can be increased by removing both lipids and
salts from tissue using Carnoy’s fluid (ethanol, chloroform, and acetic acid), water,
and ethanol [35]. Recently washes have been developed to select for metabolites
[48, 49]. Chloroform and acetone washes remove hydrophobic lipids, leaving
behind many water soluble metabolites. Washes may delocalize some molecules, so
care and validation are recommended. Some washes are being developed to either
select or analytes of interest or eliminate chemical interferences [52–56].
7.6 MALDI Matrix Selection and Application
MALDI matrices are chosen based on their ability to provide sufficient ionization
efficiency for a given analyte class (e.g. low molecular weight metabolites, lipids,
proteins, polymers, or organometallics). A MALDI matrix is typically a small
organic molecule consisting of a UV absorbing chemical moiety. Differences in
observed analyte sensitivities can be attributed to the physical properties of a matrix
such as molecular structure, pH, proton affinity, and peak wavelength absorbance
[3, 57–64]. However, studies have successfully employed various inorganic materials such as nanoparticles or thin layers of metals [65]. 2,5-dihydroxybenzoic acid
(DHB) is widely employed as a MALDI matrix, offering sufficient sensitives for
many analyte classes in positive ion mode MS analysis [63, 66– 69]. 9-Aminoacridine
(9AA) is often used for the analysis of metabolites in negative ion mode and
1,5-diaminonaphthalene (DAN) for lipids with high sensitivity in both polarities
[58, 64]. However, the energy transferred during the ablation process can result in
analyte modification or fragmentation, complicating data interpretation [66, 70].
Volatility is also a consideration when selecting a MALDI matrix because the matrix
layer must remain stable for lengthy acquisition times (hours) to avoid signal loss
during an imaging experiment. 2,5-Dihydroxyacetophenone (2,5-DHA) is an excellent matrix for MS analysis of multiple analyte classes; however, its high volatility
limits acquisition times [3]. (E)-4-(2,5-dihydroxyphenyl)but-3-en-2-one (2,5cDHA) is a vacuum stable matrix providing high sensitivity for lipids, peptides, and
intact proteins [57]. Table 7.1 lists many commonly used matrices.
7 Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry…
ions during MS analysis that can significantly complicate the mass spectra. Tissue
washing procedures have been shown to lessen or remove OCT artifacts, but these
washes may wash away compounds of interest as well [35]. Other materials have
been purposed as embedding materials with limited MS effects, including carboxymethylcellulose (CMC) and gelatin [45, 46]. Use of these materials typically introduces minimal effects, while still assisting in sectioning.
Washing can remove potential chemical interferences and select for specific analytes (Fig. 7.4b) [35, 49, 50, 51]. Ammonium formate (AmF) buffer is used to
remove salts from tissue for the analysis of lipids [49]. The ammonium cation of the
buffer system is volatile and removed from tissue by the vacuum of MS instruments.
In the positive ion mode, protonated ions dominate the spectra after AmF tissue
washing. Decreased salt presence also increases sensitivity in the negative ion
mode. Sensitivity for intact proteins can be increased by removing both lipids and
salts from tissue using Carnoy’s fluid (ethanol, chloroform, and acetic acid), water,
and ethanol [35]. Recently washes have been developed to select for metabolites
[48, 49]. Chloroform and acetone washes remove hydrophobic lipids, leaving
behind many water soluble metabolites. Washes may delocalize some molecules, so
care and validation are recommended. Some washes are being developed to either
select or analytes of interest or eliminate chemical interferences [52–56].
7.6 MALDI Matrix Selection and Application
MALDI matrices are chosen based on their ability to provide sufficient ionization
efficiency for a given analyte class (e.g. low molecular weight metabolites, lipids,
proteins, polymers, or organometallics). A MALDI matrix is typically a small
organic molecule consisting of a UV absorbing chemical moiety. Differences in
observed analyte sensitivities can be attributed to the physical properties of a matrix
such as molecular structure, pH, proton affinity, and peak wavelength absorbance
[3, 57–64]. However, studies have successfully employed various inorganic materials such as nanoparticles or thin layers of metals [65]. 2,5-dihydroxybenzoic acid
(DHB) is widely employed as a MALDI matrix, offering sufficient sensitives for
many analyte classes in positive ion mode MS analysis [63, 66– 69]. 9-Aminoacridine
(9AA) is often used for the analysis of metabolites in negative ion mode and
1,5-diaminonaphthalene (DAN) for lipids with high sensitivity in both polarities
[58, 64]. However, the energy transferred during the ablation process can result in
analyte modification or fragmentation, complicating data interpretation [66, 70].
Volatility is also a consideration when selecting a MALDI matrix because the matrix
layer must remain stable for lengthy acquisition times (hours) to avoid signal loss
during an imaging experiment. 2,5-Dihydroxyacetophenone (2,5-DHA) is an excellent matrix for MS analysis of multiple analyte classes; however, its high volatility
limits acquisition times [3]. (E)-4-(2,5-dihydroxyphenyl)but-3-en-2-one (2,5cDHA) is a vacuum stable matrix providing high sensitivity for lipids, peptides, and
intact proteins [57]. Table 7.1 lists many commonly used matrices.
7 Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry…