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REVIEW
Recent developments of novel matrices and on-tissue chemical
derivatization reagents for MALDI-MSI
Qiuqin Zhou
1
&Annabelle Fülöp
1
&Carsten Hopf
1
Received: 13 August 2020 /Revised: 17 October 2020 /A ccepted: 22 October 2020
#The Author(s) 2020
Abstract
Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a fast-growing technique for visualization of
the spatial distribution of the small molecular and macromolecular biomolecules in tissue sections. Challenges in MALDI-MSI, such as
poor sensitivity for some classes of molecules or limited specificity, for instance resulting from the presence of isobaric molecules or
limited resolving power of the instrument, have encouraged the MSI scientific community to improve MALDI-MSI sample prepara-
tion workflows with innovations in chemistry. Recent developments of novel small organic MALDI matrices play a part in the
improvement of image quality and the expansion of the application areas of MALDI-MSI. This includes rationally designed/
synthesized as well as commercially available small organic molecules whose superior matrix properties in comparison with common
matrices have only recently been discovered. Furthermore, on-tissue chemical derivatization (OTCD) processes get more focused
attention, because of their advantages for localization of poorly ionizable metabolites and their‚in several cases‚more specific imaging
of metabolites in tissue sections. This review will provide an overview about the latest developments of novel small organic matrices
and on-tissue chemical derivatization reagents for MALDI-MSI.
Keywords MALDI-MSI .MALDI matrix .MALDI imaging .On-tissue chemical derivatization .Tissue preparation
Introduction
Matrix-assisted laser desorption/ionization (MALDI) time-of-
flight (TOF) mass spectrometry imaging (MSI) can provide
unparalleled insight into the spatial distribution of proteins [1],
peptides [2], small molecules [3], lipids [4], glycans [5], and
drugs [6] in tissue sections. The fast-evolving MALDI-MSI
technique has been successfully applied in basic research, in
pharmaceutical research [6], plant biology [7], food analysis
[8], microbiology [9], and in clinical biomarker discovery
[10]. In short, typical MALDI-MSI workflows contain the
following three steps: tissue preparation, data acquisition,
and data analysis. The “4S-criteria”for a desirable MSI ex-
periment [6], namely speed, specificity, spatial resolution, and
sensitivity, often cannot be achieved together, and compro-
mises are required for MALDI-MSI methods. Although de-
velopments of mass spectrometer hardware have a great im-
pact on the performance of MALDI-MSI, proper tissue prep-
aration [11–14] is key for high-quality data acquisition.
During tissue preparation, it is essential to preserve the fidelity
of analytes and prevent their spatial dislocation in tissue.
Additionally, time consumption for tissue preparation should
stay within an acceptable range, in most cases several hours.
Important steps for tissue preparation are illustrated in Fig. 1.
Different workflows from tissue collection to data acquisi-
tion can be followed for tissue preparation. Depending on
tissue type, targeted analyte species and on experimental
goals, washes, on-tissue enzymatic digestion (OTED), on-
tissue chemical derivatization (OTCD), and application of in-
ternal standard are options in addition to the main experimen-
tal workflow (Fig. 1)[12,15,16]. Initially, procedures for
collecting, storing, embedding, and sectioning of tissue sam-
ples have great influence on the resulting quality and need to
be standardized for the same types of tissue. Later, wash pro-
tocols can be applied to remove interfering endogenous com-
pounds, which compete with analytes during ionization and
cause ion suppression effects. For example, wash protocols
Published in the topical collection Mass Spectrometry Imaging 2.0 with
guest editors Shane R. Ellis and Tiffany Porta Siegel.
*Carsten Hopf
c.hopf@hs-mannheim.de
1
Center for Mass Spectrometry and Optical Spectroscopy (CeMOS),
Mannheim University of Applied Sciences, Paul-Wittsack-Str. 10,
68163 Mannheim, Germany
https://doi.org/10.1007/s00216-020-03023-7
/ Published online: 19 November 2020
Analytical and Bioanalytical Chemistry (2021) 413:2599–2617
are indispensable for imaging of peptides and proteins in tis-
sue sections to get rid of physiological salts and abundant
lipids. Furthermore, wash protocols can also be used to en-
hance the subsequent ionization or extraction of the analytes
into the matrix layer [17–19]. OTED belongs to often-used
strategies for identification and visualization of proteins [2]
and glycans [5]. Finally, before starting data acquisition, the
matrix needs to be applied homogenously except that reagents
used in the optional OTCD step can comply the requirement
and replace of the additional matrix application. Choosing a
suitable matrix and a suitable deposition method can avoid
missing analytes or other time-consuming tissue preparation
steps [13,20]. Aiming at improvement of detection specificity
and enhancement of detection sensitivity in some approaches,
OTCD has been specially developed for small molecule
analytes [21]. To compensate ion suppression effects in het-
erogeneous tissue sections, internal standard can be used for
normalization and relative quantification, yielding superiorly
comparable results from pixel to pixel and tissue to tissue [6,
22,23]. This work only selected the latest developments of
novel matrices and on-tissue chemical derivatization reagents
for MALDI-MSI.
Novel matrices for MALDI-MSI
Ideal matrices for MALDI-MSI need several additional fea-
tures in comparison with matrices for MALDI-MS used in
non-imaging fields. There are some fundamental properties
for substances that can be used as MALDI matrix for all ap-
plications: (i) MALDI matrices display strong and efficient
absorption in the region of commonly used UV or IR laser
wavelengths; (ii) minimal matrix background signals and ad-
ducts with target analytes (“chemical noise”) should result
from matrices, especially in the low mass range (typically
m/z < 500), thus enabling visualization of the spatial distribu-
tion of small molecules; (iii) the ability to interact with
analytes is required for matrices to form co-crystals; (iv) ma-
trices must be able to effectively ionize the analytes, resulting
in protonated ions in positive ion mode or deprotonated ions
in negative ion mode. In addition to these general require-
ments for MALDI matrices, a matrix suitable for tissue imag-
ing applications should fulfill the criteria: (A) It should have
high vacuum stability in the mass spectrometer for at least
several hours in a typical imaging situation or for even longer
acquisition times in serial imaging of multiple tissue sections,
for example, entire clinical cohorts, with high spatial resolu-
tion. (B) The application of matrices onto tissue should be
practical in a routine laboratory, i.e., reproducible and not
time-consuming. Sublimation and automatic spray-coating
methods are often used due to easy handling and sufficient
reproducibility. (C) The generation of uniform, small co-
crystals is necessary for images with reproducibility and high
spatial resolution, which is also limited by minimal laser spot
size and the desired sensitivity (since the ablated area, which
decreases with the square of the step size, strongly correlates
with sensitivity). Thus, high spatial resolution requires an op-
timized matrix, which can promote efficient ionization of tar-
get analytes. (D) For the untargeted discovery of biomarkers,
matrices suitable for both positive and negative ion modes
(also called dual-polarity) cannot only save precious tissue
but also provide broader molecular coverage. The ability of
ionizing more types of analytes in single ion mode (positive or
negative) would be beneficial for detecting more molecular
species simultaneously. (E) Owing to physiologic salts natu-
rally present in tissue, salt tolerance allows the direct applica-
tion of matrices without additional wash protocols. Finally,
chemical stability, cost, toxicity, and promotion of analyte
fragmentation need to be considered. In the field of MALDI-
MSI, performance with desired detection sensitivity, spatial
resolution, and molecular coverage is in large part dependent
on matrix choices and matrix application. Because MALDI-
MSI is becoming more and more popular in different disci-
plines and the existing matrices do not always meet all
Fig. 1 Schema of experimental workflow for MALDI-MSI with highlighted important steps for tissue preparation
2600 Zhou Q. et al.
expectations stated above, many laboratories are developing
novel matrices with at least some of the abovementioned
requirements.
Currently, most very oft-used MALDI-MSI matrices like
2,5-DHB and CHCA were developed for specific analytes
during empiric research. Different substance classes, such as
small organic molecules, graphene, graphene oxide, nanopar-
ticles, metal oxides, ionic liquids, and conjugated polymers,
are presently developed, tested for their suitability as MALDI-
MSI matrices, and reviewed by various papers [4,11,20].
Small organic molecules are the most widespread matrices
used in MALDI-MSI. Since recent reviews [11,20]haveal-
ready described some novel organic matrices applied for
MALDI-MSI including 1,5-diaminonapthalene [24], 4-
phenyl-a-cyanocinnamic acid amide [25], alkylated 2,5-
DHB [26], 1,8-di(piperidinyl)-naphthalene [27], and
4,5-(bis(dimethylamino)naphthalen-1-yl)furan-2,5-dione (4-
maleicanhydrido proton sponge, MAPS) [28], this work se-
lected only the latest efforts about novel organic matrices for
MALDI-MSI from 2017. This section contains the rationally
designed and synthesized as well as commercially available
small organic molecules whose properties as bona fide
MALDI matrices for the detection and imaging of analytes
in tissue sections have recently been discovered or character-
ized (Fig. 2and Table 1).
Rationally designed and synthesized novel matrices
(E)-4-(2,5-Dihydroxyphenyl)but-3-en-2-one (2,5-cDHA) was
designed and synthesized by Yang and coworkers [29]among
other derivatives, using Wittig reaction of 2′,5′-
dihydroxybenzaldehyde with (acetylmethylene)-
triphenylphosphorane. 2,5-cDHA was applied to tissue by a
two-step spraying approach, producing small crystals (1–
2μm). Unlike the parent matrix 2,5-DHA, the 2,5-cDHA
proved to be vacuum-stable for more than 24 h in the Bruker
Rapiflex mass spectrometer, whose source has a high vacuum
(4.4 * 10
−7
mbar) and heated (around 35 °C) environment. As
a result, 2,5-cDHA was used for the visualization of proteins
in mouse brain at high spatial resolution (< 10 μm) and for
many hours without concern of matrix loss in the mass spec-
trometer. Additionally, 2,5-cDHA was also used for imaging
of lipids in both positive and negative ion modes.
Huang and coworkers [30] rationally designed and synthe-
sized a series of novel matrix candidates, including
2-(methylamino)benzoic acid (COOH-NHMe), candidate as
a MALDI matrix for imaging. The matrix COOH-NHMe
was synthesized by adding a methyl group to anthranilic acid
using CH
3
I in basic conditions. The new methylated
anthranilic acid derivative features both basic and acidic func-
tional groups and thus showed the ability to be used in both
positive and negative ion modes for the ionization and detec-
tion of lipids and proteins in mouse brain tissue. In compari-
son with commercially available matrices, COOH-NHMe was
able to visualize more lipid and protein species.
Newly discovered novel matrices
N-Phenyl-2-naphthylamine (PNA) is usually used as an anti-
oxidant for natural and synthetic rubber. Liu and coworkers
Fig. 2 Structures of selected novel MALDI matrices for imaging
2601Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI
Table 1 Rationally designed novel matrices and newly discovered novel matrices for MALDI-MSI
Matrix abbr. Matrix Target analyte classes Ion
modes
Properties Deposition Ref.
•Rationally designed small organic substances as novel matrices
2,5-cDHA (E)-4-(2,5-dihydroxyphenyl)but-3-en-2-one Protein, lipid +, −* More vacuum-stable, small crystals for higher
?spatial resolution, improved sensitivity,
?dual-polarity for lipids
Two-step
spraying
[29]
COOH-NHMe 2-(Methylamino)benzoic acid Lipid, protein +, −Dual-polarity, detection of more lipid and
?protein species
Sublimation [30]
•Newly discovered small organic substances as novel matrices
PNA N-Phenyl-2-naphthylamine FA, AA, antioxidant, lipid –Strong UV-absorption, low matrix background
?signals, salt tolerance capacity
Spraying [31]
NEDC N-(1-Naphthyl)ethylenediamine
dihydrochloride
Glucose, Na
+
,K
+
, AA, nucleotide,
?antioxidant, glycerophospholipid
–Salt tolerance capacity, low matrix background
?signals, improved sensitivity for selected
?analyte classes
Spraying [32–36]
3-APH 3-Aminophthalhydrazide Nucleotide, FA, lipid +, −Dual-polarity, improved sensitivity, broad
?molecular coverage, low matrix background
?signals, vacuum stability
Spraying [37]
IR-780 (Poly)phosphoinositide, cardiolipin,
?ganglioside
–Proton affinity, vacuum stability, homogenous
?crystals, salt tolerance capacity
Spraying [28]
DPH 1,6-Diphenyl-1,3,5-hexatriene Lipid, FA with polyene structure –Vacuum stability, high spatial resolution Sublimation [38]
BNDM 1,1′-Binaphthyl-2,2′-diamine AA, organic acid, nucleoside, nucleotide,
?nitrogenous base, cholesterol,
peptide,
?FA, choline, carnitine, polyamine,
?creatine, lipid
+, −Low matrix background signals, improved
sensitivity,
?dual-polarity, broad molecular coverage
Spraying [39,40]
DCH 2,3-Dicyanohydroquinone Lipid + High spatial resolution, vacuum stability,
?chemical stability, sensitivity
Spraying [41]
DCTB (2-[(2E)-3-(4-Tert-butylphenyl)-2-methylprop-
?2-enylidene]malononitrile)
Central nervous system drug + Improved sensitivity, low signal suppression Spraying [42]
DMCA 3,4-Dimethoxycinnamic acid Small molecule + Low matrix background signals, sensitivity, broad
?molecular coverage
Spraying [43]
*Positive ion mode for protein and dual-polarity for lipid. AA amino acid, FA fatty acid
2602 Zhou Q. et al.
[31] developed PNA as a matrix for imaging of small-
molecule metabolites, including free fatty acids, amino acids,
antioxidants, and phospholipids. On account of its strong UV
absorption, low background interference in the m/z range of <
500 and considerable salt tolerance, the unique distributions
and changes of89 small-moleculemetabolites were visualized
in negative ion mode in brain tissue of a middle cerebral artery
occlusion (MCAO) rat model of ischemic stroke.
N-(1-Naphthyl)ethylenediamine dihydrochloride (NEDC)
has been known as a commercially available coupling agent
for spectrophotometric analysis. Since Chen and coworkers
[33] discovered NEDC as a high-salt tolerance matrix for the
analysis of glucose as [glucose + Cl
−
]
−
in rat brain
microdialysates in negative ion mode, several contributions
of NEDC could be found in the MS imaging field. Hou and
coworkers [34] applied NEDC to mouse brain sections for
visualization of sodium and potassium distribution. Here, so-
dium and potassium were detected as chloride ion adducts.
Wang and coworkers [36] were able to visualize distributions
of glycerophospholipids and small-molecule metabolites be-
low m/z 400, including glucose in various mouse organs. Li
and coworkers [35] successfully used NEDC for imaging of
the change of lipid metabolism and the levels of amino acids,
nucleotides, and antioxidants in brain tissue of an orthotopic
glioma xenograft model. Barré and coworkers [36]compared
NEDC with 9-AA for imaging of small metabolites in diffuse
large B cell lymphoma xenograft mouse model. With a higher
signal-to-noise ratio for metabolites and less matrix back-
ground, NEDC appears to be a better matrix for adenosine
monophosphate (AMP), adenosine diphosphate (ADP), and
adenosine triphosphate (ATP) than 9-AA.
3-Aminophthalhydrazide (3-APH, also known as luminol)
is a commercially available and very effective chemilumines-
cent substrate. 3-APH was evaluated by Li and coworkers [37]
as a dual-polarity matrix with superior performance than com-
mon matrices such as 2,5-DHB, CHCA, and 9-AA. 3-APH
was able to detect and give a spatial distribution of 159 and
207 metabolites in the mouse brain in the positive and nega-
tive ion modes, respectively. Among the detected metabolites
were nucleotides, fatty acids, glycerolipids,
glycerophospholipids, sphingolipids, and saccharolipids.
Thus, 3-APH was demonstrated to have some improved fea-
tures including high sensitivity, broad molecular coverage,
low background noise, and high vacuum stability.
IR-780 is a commercially available near-infrared fluores-
cent dye. Li and coworkers [28] successfully screened and
optimized the application of IR-780 as a matrix for visualiza-
tion of various high-molecule lipids, including
(poly-)phosphoinositides, cardiolipins, and gangliosides in
acute traumatic brain injury tissues in negative ion mode.
IR-780 was proved to possess various good properties includ-
ing strong UV absorption, high proton affinity, good salt tol-
erance ability, homogeneous co-crystallization, and high
vacuum stability. Thus, it was suggested as an almost ideal
matrix for imaging.
1,6-Diphenyl-1,3,5-hexatriene (DPH) is a commercially
available fluorescent dye for the detection and localization of
lipids in fluorescence imaging. Ibrahim and coworkers [38]
presented DPH as a suitable and effective matrix for imaging
of lipids and fatty acids in rat and mouse brain tissues in
negative ion mode. It was proposed that the interaction with
the acyl chain of lipids and fatty acids is accountable for the
features of DPH as a matrix for lipids and fatty acids with
hydrophobic polyene structure. The possibility of application
via sublimation and the stability for at least 24 h under high
vacuum (10–7 Torr) allowed DPH for the application for high
spatial resolution imaging. Use of relative low laser energy
avoided the fragmentation of lipids.
The enantiomers and derivatives of commercial available
1,1′-Binaphthyl-2,2′-diamine (BNDM) are widely used in
asymmetric syntheses. Sun and coworkers [40] developed
BNDM as a dual-polarity matrix with low background inter-
ference and high sensitivity. BNDM could be applied for im-
aging of 301 negative metabolite ions and 175 positive me-
tabolite ions, including amino acids, organic acids, nucleo-
sides, nucleotides, nitrogenous bases, cholesterols, peptides,
fatty acids, cholines, carnitines, polyamines, creatine, and
phospholipids in rat brain. Additionally, BNDM could give
the spatial information of various metabolites in different
mouse lung cancer tissue sections. In another work, Sun and
coworkers [39] optimized BNDM for the imaging application
of amino acids, phenolic acids, fatty acids, oligosaccharides,
cholines, polyamines, tanshinones, and phospholipids in
Salvia miltiorrhiza Bge.
2,3-Dicyanohydroquinone (DCH) is originally known as a
fluorescent dye and was applied by Liu and coworkers [41]as
a new matrix for imaging of lipids in biological tissues. In
contrast to commonly used matrices, more lipids could be
detected from mouse brain and germinating Chinese yew seed
tissue sections in positive ion mode. Strong UV absorption,
high vacuum stability, profound chemical stability, and for-
mation of homogenous small crystals make for a good matrix
with the superior performance, including high spatial resolu-
tion and reproducibility.
In 2000, Ulmer and coworkers [44]provedDCTB
(2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-
enylidene]malononitrile) as an electron transfer matrix for the
analysis of labile compounds. Recently, Rzagalinski and co-
workers [42] found the theoretical and experimental evidence
that DCTB is a proton transfer matrix. DCTB was applied for
imaging of central nervous system drugs in mouse brain tis-
sue on positive ion mode. Despite the solubility and vacuum
stability drawbacks, DCTB was applied for quantitative im-
aging of the adrenergic receptor agonist xylazine in mouse
brain tissue with significant improvements of signal intensity
over CHCA.
2603Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI
He and coworkers [43] investigated 3,4-dimethoxycinnamic
acid (DMCA) as a promising matrix for imaging of endogenous
low molecular weight metabolites (m/z < 500 Da) on rat liver,
rat brain, and germinating Chinese yew seed tissue, with 303,
200, and 248 metabolites, respectively. Using DMCA, more
low molecular weight metabolites (m/z < 500) could be detect-
ed and imaged than using other commonly used matrices such
as 2,5-DHB, CHCA, 2-mercaptobenzothiazole (2-MBT),
graphene oxide, and silver nanoparticles. Due to the broad mo-
lecular coverage and low background signals, DMCA was
established as a powerful matrix with high ionization efficiency
for small molecules.
On-tissue chemical derivatization reagents
Nowadays, MALDI-MSI draws more and more attention as a
label-free imaging technique that can visualize various metab-
olites in a single measurement. However, the MALDI-MSI
technique faces some challenges such as detection sensitivity
for small molecule analytes, extend of metabolic coverage,
more comprehensive identification of isomers, and so on.
The detection of some metabolites in tissue sections by
MALDI-MSI is sometimes hindered by their ionization effi-
ciencies, and additionally, also by background interferences
from MALDI matrix or tissue components, sampling size, ion
suppression effects, and in-source fragmentation. Besides, the
broad dynamic range of on-tissue metabolite concentrations
and their diverse functional groups are responsible for distinct
ionization efficiencies. To solve the detection problem of
small metabolites in tissue sections, development of more ef-
fective matrices for MALDI with negligible matrix back-
ground and optimization of matrix deposition methods are just
two strategies to improve their detection sensitivities.
However, more strategies are required. On-tissue chemical
derivatization (OTCD) is an alternative approach to improve
ionization efficiency of targeted analytes in MALDI-MSI for
the in situ detection of analytes. OTCD of small molecule
analytes can increase their molecular mass, resulting in mass
shifting the derivatization product ions out of the “chemical
noise”range. Optimally, the derivatization products can be
ionized more efficiently than analytes without derivatization,
resulting in improved detection sensitivity in MALDI-MSI for
the in situ detection. Finally, broader molecular coverage can
be achieved. Furthermore, the specificity in MALDI-MSI can
be improved by OTCD that allows specific chemical reactions
for the identification of functional groups and their positions
in target analytes. At best, OTCD reagents can serve as a
reactive matrix, which cannot only selectively react with
analytes in tissue sections but also assist in desorption and
ionization in MALDI-MS.
Chemical derivatization in solution is a well-developed
strategy for the detection of analytes in capillary
electrophoresis [45,46], gas chromatography [47], and high-
performance liquid chromatography [45,47] coupled with
optical absorption spectrometry or electrospray ionization
mass spectrometry (ESI) [48–51] for the following purposes:
enabling of detection, improved structure elucidation in terms
of structural isomers, simplified identification of functional
groups, quantification, and improved molecular coverage.
In-solution chemical derivatization methods have found
various applications in MALDI-MS [52]. To date, more and
more chemical derivatization approaches are developed for
on-tissue applications followed by MALDI-MSI measure-
ments. In comparison to in-solution chemical derivatization,
OTCD needs to overcome a couple of challenges including
relatively low derivatization efficiency, interferences due to
excess derivatization reagent, and delocalization due to the
additional spray-coating step and incubation in the humid at-
mosphere. Thus, complex chemical reaction conditions often
used in solution cannot easily be adapted and reconstructed for
on-tissue chemical reactions. Optimally, OTCD can be per-
formed at room temperature without the requirement of addi-
tional specific buffers and with short reaction times.
Generally, OTCD employs an automatic sprayer to apply de-
rivatization reagents directly onto tissue sections before incu-
bation in a humid chamber at room temperature or slightly
increased temperature for several hours. Several review papers
[5,53,54] have already included the work from Holster and
coworkers [55], who developed a linkage-specific two-step
OTCD process for obtaining spatial distribution information
of linkage-specific N-glycan isomers from FFPE tissue sec-
tions by using the MALDI-MSI technique. And although sev-
eral other review papers [20,56–58] have described new de-
velopments of mass spectrometric imaging, which described
among other methods also several MALDI-MSI approaches
using OTCD, we present a comprehensive overview about
recent developments in OTCD of functional groups including
amine, phenolic hydroxyl, carbonyl, carboxylic acid, thiol,
and double bond, for applications in the MALDI-MS imaging
field (Table 2and Figs. 3,4,5,6,7,and8).
Target functional group: Amine
With trans-cinnamaldehyde/4-hydroxy-3-
methoxycinnamaldehyde The functional aldehyde group re-
acts easily with primary amines, forming a stable Schiff’s
base. Using this reaction, Manier and coworkers [59]investi-
gated trans-cinnamaldehyde for the derivatization of isonia-
zid, an anti-tuberculosis drug, in rabbit lung tissue sections.
Later, Manier and coworkers [60] applied 4-hydroxy-3-
methoxycinnamaldehyde (CA) for the visualization of dopa-
mine, norepinephrine, and epinephrine in porcine adrenal
gland tissue sections and γ-aminobutyric acid in rat brain
tissue sections. The pre-coating method of the CA resulted
in enhanced sensitivity with minimal analyte delocalization.
2604 Zhou Q. et al.
Table 2 MALDI-MSI approaches with on-tissue chemical derivatization of analytes in tissue sections
Target analytes in tissue sections OTCD reagent Solution Application and
incubation
Tissue Ref.
Amine as targeted functional groups
Isoniazid CA 50% in MeOH High velocity
spin coating,
30 min at RT
Lung tissue sections of rabbit
infected with M. tuberculosis
and dosed with isoniazid
[59]
Dopamine, norepinephrine,
epinephrine, γ-aminobutyric
acid
CA 23 mg/mL CA and 8.5 mg/mL
trans-ferulic acid in MeOH
Automatic
spraying
Adrenal gland tissue sections of
pig, brain tissue sections of rat
[60]
Glycine, alanine, serine, proline,
valine, threonine,
isoleucine/leucine, aspartate,
glutamine, lysine, glutamate,
tryptophan, dopamine,
γ-aminobutyric acid, taurine,
3-methoxytyramine,
serotonin,
L-dihydroxyphenylalanine
CA 4 mg/mL in 50% MeOH Automatic
spraying,
overnight at
RT
Brain tissue sections from female
C57BL/6J mice
[61]
Glycine, alanine, aminobutenoic
acid, serine, γ-aminobutyric
acid and more other metabo-
lites
CA 20 mg/mL in MeOH Electrospraying Leaf and root tissue sections of
two different maize genotypes
(B73 and Mo17)
[62]
67 small-molecule metabolites
including amino acids,
neurotransmitters, dipeptides
and others
CA 5 mg/mL in 50% MeOH Electrospraying,
overnight at
37 °C
Brain tissue sections of rats [63]
Neuropeptides NBA + hv 5 mg/mL in
ACN/EtOH/FA/H
2
O
Automatic
spraying,
nanosecond
with hv
Brain tissue sections from mouse
brain
[64]
Amino acids excluding lysine,
serine, histidine, threonine,
aspartate and arginine
TAHS 5 mg/mL in ACN Airbrush,
overnight at
55 °C
Liver tissue sections from
xenograft mouse models of
human colon cancer
[65]
Glycine, alanine, serine, proline,
valine, threonine,
isoleucine/leucine, aspartate,
glutamine, lysine, glutamate,
tryptophan, dopamine,
γ-aminobutyric acid, taurine,
3-methoxytyramine,
serotonin,
L-dihydroxyphenylalanine
TAHS 5 mg/mL in 50% ACN Automatic
spraying,
overnight at
RT
Brain tissue sections from female
C57BL/6 J mice
[61]
Phenylalanine, tyrosine TAHS 5 mg/mL in ACN Automatic
spraying, 24 h
at 55 °C
Liver tissue sections from a H460
human NSCLC xenograft
mouse model
[66]
Noradrenaline TAHS 5 mg/mL in ACN Airbrush,
15 min at
55 °C
Adrenal gland tissue sections from
tumor patients
[67]
Glutamine TAHS 5 mg/mL in ACN Automatic
spraying, 24 h
at 55 °C
Tumor and benign tissue sections
from tumor patients
[68]
Dopamine, tyrosine, tryptamine,
tyramine, phenethylamine,
3-methoxytyramine,
serotonin, γ-aminobutyric
acid, glutamate
DPP-TFB**,
TMP-TFB
0.09 mg/mL in 50% MeOH
with 0.06% TEA
Automatic
spraying,
30 min at RT
Brain tissue sections from treated
and control male
Sprague-Dawley rats and
C57BL/6J male mice, brain tis-
sue sections from primate
[69]
Dopamine and amphetamine,
β-N-methylamino-L-alanine
DPP*, TMP,
PBDPP*
1.11 mg/mL in 75% MeOH
with 0.05% TEA, 1 mg/mL
in 83% MeOH with 0.05%
TEA, 0.2 mg/mL in 80%
MeOH with 0.07% TEA
Automatic
spraying,
15 min at RT
Brain tissue sections from the
treated and untreated C57BL/6J
male mice, male Wistar rat pups
or adult Sprague-Dawley rats
[70]
DPP-TFB 5 mg/mL in MeOH [61]
2605Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI
Table 2 (continued)
Target analytes in tissue sections OTCD reagent Solution Application and
incubation
Tissue Ref.
Glycine, alanine, serine, proline,
valine, threonine,
isoleucine/leucine, aspartate,
glutamine, lysine, glutamate,
tryptophan, dopamine,
γ-aminobutyric acid, taurine,
3-methoxytyramine,
serotonin,
L-dihydroxyphenylalanine
Automatic
spraying,
overnight at
RT
Brain tissue sections from female
C57BL/6J mice
Glutamate, γ-aminobutyric acid DPP-TFB 1.33 mg/mL in MeOH Manual
spraying,
direct
spraying of
matrix
Brain tissue sections from
SCR-KO and WT mice
[71]
Dopamine, serotonin,
norepinephrine
DPP-TFB 1.3 mg/mL in MeOH Airbrush, direct
spraying of
matrix
Whole brain of the C57BL/6J
mouse
[72]
Dopamine, 3-methoxytyramine DPP-TFB 1.3 mg/mL in MeOH Airbrush, direct
spraying of
matrix
Brain tissue sections from male
C57BL/6J mice and WT mice
[73]
Dopamine, glycine, alanine,
γ-aminobutyric acid, proline,
valine, threonine, taurine,
leucine, aspartate, tyramine,
glutamine, lysine, glutamate,
tryptamine, 3-
-methoxytyramine, tyrosine,
L-dihydroxyphenylalanine
DPP-TFB 5 mg/mL in MeOH Automatic
spraying,
overnight
Brain tissue sections from
glioblastoma multiforme mice
and WT mice
[74]
Dopamine, serotonin,
γ-aminobutyric acid,
histamine, threonine,
phenethylamine,
methylhistamine, agmatine,
adenine, tyramine, lysine,
tryptamine,
L-dihydroxyphenylalanine
DPP-TFB 1.33 mg/mL in 75% MeOH
with 0.05% TEA
Automatic
spraying, 24 h
at RT
Brain tissue sections from rock
crabs Cancer irroratus
[75]
Dopaminergic and serotonergic
neurotransmitters and their
associated metabolites
containing primary and
secondary amine groups
FMP-8*, FMP-9*,
FMP-10*
4.4 mM in 70% ACN Automatic
spraying, no
incubation
Brain tissue sections from treated
and untreated rat and primate
models of Parkinsonism, brain
tissue sections from a patient
with Parkinson’sdisease
[76]
Dopamine, γ-aminobutyric acid FMP-10* 1.8 mg/mL in 70% ACN Automatic
spraying, no
incubation
Brain tissue sections from GPR37
KO mice and WT mice
[77]
Phenolic hydroxyl as targeted functional groups
Cannabinoids and their
metabolites
FMPTS 10 mg/mL in ACN Airbrush Human hair [78]
Catecholamines (dopamine,
epinephrine, norepinephrine)
(N-Me)Py
+
B(OH)
2
* 12 mg/mL in 60% ACN Automatic
spraying
Adrenal gland tissue sections from
pig
[79]
Dopaminergic and serotonergic
neurotransmitters and their
associated metabolites
containing phenolic hydroxyl
groups
FMP-8*, FMP-9*,
FMP-10*
4.4 mM in 70% ACN Automatic
spraying, no
incubation
Brain tissue sections from treated
and untreated rats and primate
models of Parkinsonism, brain
tissue sections from a patient
with Parkinson’sdisease
[76]
Carbonyl as targeted functional groups
Fluticasone propionate DMNTH*, DNPH* 5 mg/mL in 50% ACN with
0.1% TFA, 4 mg/mL in
50% ACN
Spotting, 48 h at
37 °C
Rat lung tissue sections [80]
11-Dehydrocorticosterone and
corticosterone
GirT Adrenal gland tissue sections of
Sprague-Dawley rat and brain
[81]
2606 Zhou Q. et al.
Table 2 (continued)
Target analytes in tissue sections OTCD reagent Solution Application and
incubation
Tissue Ref.
0.15 mg/cm
2
, addition
spraying of MeOH with
0.2% TFA
Precoated,
60 min at
40 °C
tissue sections of C57BL/6
mice
Triamcinolone acetonide GirT 5 mg/mL in MeOH with 0.2%
TFA
Automatic
spraying,
150 min at
40 °C
Human incubated cartilage [82]
Testosterone GirT 5 mg/mL in 2.5% acetic acid Airbrush,
90 min at RT
Testis tissue sections of C57BL/6
mice after human chorionic go-
nadotrophin treatment
[83]
Testosterone and
5α-dihydrotestosterone
GirT 5 mg/mL in 80% MeOH with
0.1% TFA
Automatic
spraying,
60 min at
40 °C
Testis tissue sections of C57BL/6
mice and prostate tissue sec-
tions of Sprague-Dawley rats
after human chorionic gonado-
trophin treatment
[84]
Cortisone, aldosterone,
18-oxocortisol and progester-
one
GirT 10 mg/mL in 20% acetic acid Airbrush,
90 min at RT
Adrenal gland tissue sections of
human patients and Sprague
Dawley rats
[85]
Abscisic acid and
12-oxo-phytodienoic acid
GirT 5 mg/mL in 80% MeOH with
2% TFA
Airbrush,
30 min at RT
Immature P. vulgaris L. seed
sections
[86]
Pyruvic acid, glycolaldehyde,
2-pentenal, dithylacetone,
1-hexanal, 1-heptanal,
jasmonic acid, dotriacontanal
and more other metabolites
GirT 10 mg/mL in MeOH with 2%
TFA
Electrospraying Leaf and root tissue sections of
two different maize genotypes
(B73 and Mo17)
[62]
11-Dehydrocorticosterone,
corticosterone
GirT 5 mg/mL in 80% MeOH with
0.2% TFA
Electrospraying,
1 h at 40 °C
Brain tissue sections of
Sprague-Dawley rats
[63]
Aldosterone, cortisol, cortisone,
18-OH-corticosterone
GirT 10 mg/mL in 20% acetic acid Airbrush,
60 min at RT
Tissue sections of human adrenal
glands
[67]
Carboxylic acid as targeted functional groups
Docosahexaenoic acid,
arachidonic acid, oleic acid,
palmitoleic acid,
eicosapentaenoic acid, linoleic
acid
PA 2mMwith10 mMof
activation reagents in ACN
Electrospraying
or airbrush
Brain tissue sections from rats [87]
3-Maleylpyruvate,
N-acetyl-L-glutamate,
palmitic acid, oleic acid,
stearic acid and more other
metabolites
PA 6 mM PA and 30 mM
activation reagents in ACN
Electrospraying Leaf and root tissue sections of
two different maize genotypes
(B73 and Mo17)
[62]
Fatty acids (C16:1, C16:0,
C18:2, C18:1, C18:0, C18:3,
C20:4, C20:0, C22:6, C22:4)
DMPI 3 mM with 1 mM HATU in
80% ACN
Electrospraying Tumor and normal tissue sections
of patients with thyroid
carcinoma, brain tissue sections
of rat
[88]
Thiol as targeted functional groups
α-Chain and ß-chain of reduced
insulin, glutathione, cysteine,
cysteinylglycine
CHC-MAL*** 10 mg/mL in 50% ACN Automatic
spraying
Liver and pancreas tissue sections
of pig, tumor tissue sections of
mouse xenograft
[89]
Double bond groups
PC 36:1, PS 36:2 isomers Benzaldehyde Vapor of benzaldehyde Custom-made
reaction
chamber,
triggered by
254 nm
Brain tissue sections of
C57BL6/N mice
[90]
PC 34:1, PC 36:1 isomers BPh* 20 mg/mL in solution (ACN:
isopropanol: H
2
O, 6:3:1
with 0.5% TFA)
3 min with
UV-light
Brain tissue sections of
C57BL6/N mice and tegument
of S. mansoni
[91]
PC 34:1, PC 36:1 isomers Ozone Gas High-pressure
linear ion trap
with ozone
Brain tissue sections of
ND2:SmoA1 transgenic mice
containing tumors
[92]
2607Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI
CA was also used by Esteve and coworkers [61] for the com-
parison with THAS and DPP-TFB. Dueñas and coworkers
[62] also applied CA for a proof of concept experiment, in
which various classes of compounds containing a primary
amine group were visualized in tissue sections ofmaize leaves
and roots. Guo and coworkers [63] combined a laser-assisted
tissue transfer (LATT) technique with the CA derivatization
for imaging of up to 67 small molecule metabolites including
amino acids, neurotransmitters and dipeptides, and others in
brain tissue sections of rats.
With NBA and the nanosecond laser irradiation Li and co-
workers [64] introduced the photoactive compound, 2-
nitrobenzaldehyde (NBA) in combination with the nanosec-
ond laser irradiation at a wavelength of 355 nm for the deriv-
atization of primary amine groups in peptides and proteins.
The nanosecond laser irradiation enables the generation of the
reactive 2-nitrosobenzoic anion (NS
−
) which can rapidly react
with primary amines in peptides and proteins, resulting in a
mass shift of 133 Da. It was called as a nanosecond photo-
chemical reaction (nsPCR). Base on the established micro-
electrophoresis and thermophoresis theory, they also proposed
the on-demand matrix-removal effect for the NBA-based
nsPCR strategy. The NBA-based nsPCR strategy could be
applied to the brain tissue sections of the mouse brain to vi-
sualize and identify neuropeptides in the mouse brain with
massive enhanced results.
With TAHS Originally, p-N,N,N-trimethylammonioanilyl N-
hydroxysuccinimidyl carbamate iodide (TAHS) was synthesized
by Shimbo and coworkers [94] using N,N-dimethylamino-p-
phenylenediamine and N,N′-dihydroxysuccinimidyl carbonate
and iodomethane for the subsequent methylation and applied it
for the analysis of amino acids in ESI-MS. According to this
synthesis method, Toue and coworker [65] synthesized THAS
for derivatization of amino acids in liver tissue sections from
xenograft mouse models of human colon cancer in positive ion
mode. After the derivatization with TAHS and subsequent ap-
plication of 2,5-DHB matrix, the detection sensitivity of amino
acids could be improved from failure in detection to easily de-
tection. Most amino acids could be easily detected in tissue
sections, especially because of the introduction of the positively
charged quaternary amine group. However, some amino acids,
such as lysine, histidine, threonine, aspartate, and arginine [61,
94], could hardly be detected. Further application of TAHS
could be found for the analysis of phenylalanine and tyrosine
[66] in liver tissue sections from a H460 human NSCLC xeno-
graft mouse model, noradrenaline [67] in adrenal gland tissue
sections from tumor patients, and glutamine [68]intumorand
benign tissue sections from patients with cholangiocarcinoma.
With DPP-TFB Conversion of the primary amino groups into
charged quaternary amino groups is a good option to increase
ionization efficacy. Shariatgorji and coworkers [69,70]ap-
plied the commercially available pyrylium salts for the deriv-
atization of primary amino groups in neurotransmitters and
amino acids. They selected pyrylium salt such as 2,4-
diphenyl-pyranylium tetrafluoroborate (DPP-TFB), which re-
acts selectively and rapidly with primary amine groups under
mild conditions. The resulting charged derivatization product
possessed the ability of self-assisted laser desorption ioniza-
tion in positive ion mode, but only with a higher concentration
of DPP-TFB solution and in the presence of high amounts of
metabolites. Therefore, an additional matrix such as CHCA
[69]or2,5-DHB[71–75] was applied onto brain tissue sec-
tions after derivatization, leading to the enhanced sensitivity in
MALDI-MSI analysis. DPP-TFB [69–75] was applied in var-
ious animal models to localize and (semi-)quantitatively esti-
mate changes of the metabolites such as dopamine, tyrosine,
tryptamine, tyramine, phenethylamine, 3-methoxytyramine,
Table 2 (continued)
Target analytes in tissue sections OTCD reagent Solution Application and
incubation
Tissue Ref.
PC 34:1 isomers Ozone Ozone from an ozone generator Glass flask
flushed with
the flow of
ozone, up to
30 min
Brain tissue sections of BALB/c
mice, human colon tissue sec-
tions
[93]
CA, 4-hydroxy-3-methoxycinnamaldehyde; NBA, 2-nitrobenzaldehyde; TAHS,p-N,N,N-trimethylammonioanilyl N-hydroxysuccinimidyl carbamate
iodide; DPP-TFB, 2,4-diphenyl-pyranylium tetrafluoroborate; DPP, 2,4-diphenyl-pyranylium; PBDPP, 1,4-phenylene-4,4′-bis (2,6-diphenyl-4-
pyrylium); TMP, 2,4,6-trimethylpyrylium; FMP-8: 4-(10-bromoanthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide; FMP-9, 4-(anthracen-9-yl)-2-
fluoro-1-ethylpyridin-1-ium iodide; FMP-10, 4-(anthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide; FMPTS, 2-fluoro-1-methylpyridinium p-
toluenesulfonate; (N-Me)Py+B(OH)
2
, 4-(N-methyl)pyridinium boronic acid; DNPH, 2,4-dinitrophenylhydrazine; DMNTH, 4-dimethylamino-6-(4-
methoxy-1-naphthyl)-1,3,5-triazine-2-hydrazine; GirT, Girard’sreagentT;PA, 2-picolylamine (PA); DMPI, N,N-dimethylpiperazine iodide; CHC-
Mal,(E)-2-cyano-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)ethyl)-3-(4-hydroxyphenyl)-acrylamide (CHC-Mal); BPh, benzophenone; ACN, aceto-
nitrile; TFA, trifluoroacetic acid; EtOH, ethanol; FA, formic acid; TEA, triethylamine; RT, room temperature; HATU, 2-(7-azabenzotriazol-1-yl)-
N,N,N´,N´-tetramethyluronium hexafluorophosphate; MeOH, methanol
*Reactive matrix; **Reactive matrix in high concentration; ***Reactive matrix for small thiol-containing molecules
2608 Zhou Q. et al.
serotonin, γ-aminobutyric acid, glutamate, glycine, alanine,
proline, valine, threonine, taurine, leucine, aspirate, gluta-
mine, lysine, L-dihydroxyphenylalanine, histamine,
methylhistamine, agmatine, adenine, and norepinephrine.
With FMP reagents Shariatgorji and coworkers [76]synthe-
sized a series of pyridinium salts including 4-(10-
bromoanthracen-9-yl)-2-fluoro-1-methylpyridin-1-ium iodide
(FMP-8), 4-(anthracen-9-yl)-2-fluoro-1-ethylpyridin-1-ium
iodide (FMP-9) and 4-(anthracen-9-yl)-2-fluoro-1-
methylpyridin-1-ium iodide (FMP-10), which readily reacted
with primary or secondary amine groups and were proven as
the best candidates among them. The derivatization reaction
follows the nucleophilic aromatic substitution reaction of the
2-fluoro-1-methyl pyridinium (FMP) cation with primary and
secondary amine groups, which are often the functional
groups of neurotransmitters and their associated metabolites
from brain tissue sections. Simultaneously, the polyphenyl
group in FMP reagents complies with the requirement of a
MALDI matrix, which has a strong light absorption in the
range of ultraviolet-visible in the solid phase. Hence, the
chemically derivatized brain tissue sections can be directly
measured with MALDI mass spectrometer without spraying
of an additional matrix. FMP reagents were dubbed as reactive
Fig. 3 OTCD reactions targeting
amines
2609Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI
matrices. The improved sensitivity after derivatization with
FMP reagents enabled a comprehensive map of neurotrans-
mitters and their associated metabolites from brain tissue sec-
tions. The ability to form small FMP derivative crystals en-
abled the application at a high spatial resolution of 10 μm.
Zhang and coworkers [77] from the same laboratory applied
FMP-10 to visualize the dopamine and γ-aminobutyric acid
changes in parkinsonian mice lacking GPR37 in comparison
with wild type mice.
Target functional group: phenolic hydroxyl
With FMP Beasley and coworkers [78] applied 2-fluoro-1-
methylpyridinium p-toluene sulfonate (FMPTS) for derivati-
zation of the phenolic hydroxyl group of cannabinoids in hu-
man hair. After rapid derivatization reaction at room temper-
ature and spraying of CHCA as the MALDI matrix, positively
charged N-methylpyridinium derivate contributed to im-
proved ionization efficiency of several cannabinoids and their
metabolites including Δ
9
-tetrahydrocannabinol (THC) in hair,
which were soaked in solutions with the respective analytes.
Furthermore, THC could be visualized with FMPTS derivati-
zation in hairs from a known cannabis consumer.
Derivatization of diol in catechol with (N-Me)Py
+
B(OH)
2
Kaya
and coworkers [79] synthesized 4-(N-methyl)pyridinium
boronic acid ((N-Me)Py
+
B(OH)
2
), which was applied for de-
rivatization of catecholamines including dopamine, epineph-
rine, and norepinephrine in porcine adrenal gland tissue sec-
tions. Boronic acid derivative can readily react with the diol
moiety of catechol (ortho isomer of dihydroxybenzene),
forming five-membered boronate esters. Due to the positive
charge in (N-Me)Py
+
B(OH)
2
and the UV-absorption ability of
the derivatization products in the range of the laser wavelength
of the mass spectrometer, (N-Me)Py
+
B(OH)
2
was used as a
reactive matrix without an additional MALDI matrix.
Additionally, the unique isotopic pattern of boron-containing
derivatized catecholamines assisted in data analysis.
Target functional group: carbonyl
With Girard’s reagent T Cobice and coworkers [81]applied
Girard’s reagent T (GirT) for derivatization of 11-
dehydrocorticosterone and corticosterone in rat adrenal gland
and mouse brain tissue sections. The reactive hydrazine group
in GirT reacts with the ketone group easily, forming hydrazine
derivatives. Due to the charged quaternary amine in GirT, the
resulting derivatization products (GirT-dehydrocorticosterone
and GirT-corticosterone) showed enhanced ionization effi-
ciency. Derivatization with GirT to visualize ketone-
containing analytes was demonstrated in various tissues, such
as mouse testis [83,84], cartilage [82], rat prostate [84], rat
Fig. 4 OTCD reactions targeting phenolic hydroxyl groups
2610 Zhou Q. et al.
adrenal gland [85], immature P. vulgaris L. seed [86], maize
root and leaf [62], human adrenal gland [67,85], and rat brain
[63]. In the case of structural isomers [67], tandem MS imag-
ing was used to distinguish aldosterone from cortisol and cor-
tisone from 18-OH-corticosterone.
DMNTH and DNPH Flinder and coworkers [80] applied differ-
ent hydrazines including 2,4-dinitrophenylhydrazine (DNPH)
and 4-dimethylamino-6-(4-methoxy-1-naphthyl)-1,3,5-tri-
azine-2-hydrazine (DMNTH) on mouse lung tissue sections,
which were spotted with fluticasone propionate. DNPH and
DMNTH were demonstrated as reactive matrix and can be
obtained by purchase and chemical synthesis in the lab, re-
spectively. In comparison with DNPH, DMNTH showed su-
perior results and resulted in a detection limit of 50 ng/μLina
humid environment at 37 °C for 48 h. The detection sensitivity
Fig. 5 OTCD reactions targeting carbonyls
Fig. 6 OTCD reactions targeting carboxylic acids
2611Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI
was improved after additional application of conventional
MALDI matrix CHCA.
Target functional group: carboxylic acid
With 2-picolylamine Wu and coworkers [87] evaluated 2-
picolylamine (PA), well known as derivatization reagent in
solution for application as OTCD before MALDI-MSI.
After activating of a carboxylic acid with 2,2-dipyridyl disul-
fide (DPDS) and triphenylphosphine (TPP), 2-picolylamine
transferred the activated fatty acid to the stable derivatization
product containing an amide bond. By using the electrospray
deposition method, six fatty acids, such as docosahexaenoic
acid, arachidonic acid, oleic acid, palmitoleic acid,
eicosapentaenoic acid, and linoleic acid, were visualized in
rat brain tissue sections with improved detection limits and
decreased delocalization in positive ion mode. Thus, a spatial
resolution of 20 μm was reached. The approach was also
applied by Dueñas and coworkers [62] for visualization of
fatty acids, such as 3-maleylpyruvate, N-acetyl-L-glutamate,
palmitic acid, oleic acid, stearic acid, and more other metabo-
lites with carboxylic acid in tissue sections of maize leaves
and roots.
With DMPI Wang and coworkers [88] synthesized N,N-
dimethylpiperazine iodide (DMPI) using CH
3
IandN-
methylpiperazine. 2-(7-Azabenzotriazol-1-yl)-N,N,N´,N´-
tetramethyluronium hexafluorophosphate (HATU) was used
in this work to generate active esters from carboxylic acids
derived from fatty acids such as C16:0 and C18:0. The active
esters reacted then with DMPI, resulting in derivatized fatty
acids with stable amide bonds and charged quaternary amine
groups, which assisted in improved detection of fatty acid
derivatives in positive ion mode. In this way, fatty acids and
phospholipids could be simultaneously imaged in positive ion
mode. By using optimized spraying parameters using an
electrospray device and optimized derivatization conditions,
this approach was proved to have advantage over the approach
with 2-picolyamine [87] and applied in tissue sections from
patients with thyroid cancer.
Target functional group: thiol
Fülöp and coworkers [89] designed, synthesized, and evaluated
(E)-2-cyano-N-(2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)ethyl)-3-(4-hydroxyphenyl)-acrylamide (CHC-Mal) as a thi-
ol derivatization reagent. Proteins with free thiol groups, such
as α-chain and ß-chain of reduced insulin, were derivatized and
imaged in porcine pancreas tissue sections in positive ion mode.
Besides, free thiol groups from small molecule metabolites,
present in tissue or generated by spray-coated reducing agent,
were derivatized and visualized in porcine liver tissue sections
and tumor tissue sections of a mouse xenograft model in posi-
tive ion mode with clearly improved detection sensitivity. In
this work, cysteine, cysteinylglycine, and glutathione were de-
tectable even without application of additional 2,5-DHB ma-
trix. In other words, CHC-Mal can be used as a reactive matrix
for small thiol-containing metabolites.
Fig. 7 OTCD reactions targeting thiols
Fig. 8 OTCD reactions targeting double bonds
2612 Zhou Q. et al.
Target functional group: double bond
With benzaldehyde/BPh Bednařík and coworkers [90]intro-
duced benzaldehyde for distinguishing and imaging of lipid
double bonds. A laser-induced post-ionization strategy
(MALDI-2) was used for improved protonation of analytes.
Benzaldehyde undergoes the Paternò-Büchi photoreaction
with lipid double bonds in a reaction chamber equipped with
a UV lamp. After derivatization and subsequent collision-
induced dissociation of the Paternò-Büchi photoreaction reac-
tion products, the double bond position were verified and
imaged in mouse brain tissue. Wäldchen and coworkers [91]
introduced benzophenone (BPh) as a reactive matrix for the
identification of the double bond position of unsaturated phos-
pholipids. BPh could not only act as a MALDI matrix but also
react with double bond groups of unsaturated phospholipids
directly after laser irradiation (330–370 nm) during measure-
ment. Thus, no additional equipment is required for the laser-
induced Paternò-Büchi photoreaction, in which C=C double
bond form a four-membered oxetane ring. Using tandem MS
imaging (MS
2
I), spatial distributions of phospholipids with
defined double bond position could be visualized in brain
tissue sections of mice and tegument of S. mansoni.
With ozone Paine and coworkers [92] directly introduced
ozone in the mass spectrometer, leading to ozone-induced
dissociation. With this unique approach, specific lipid isomers
were visualized in the rat brain. Later, Bednařík and co-
workers [93] developed the approach for ozonization of un-
saturated lipids under concentrated ozone atmosphere in a
reaction chamber. The Criegee ozonide ions are formed after
the addition of ozone to C=C double bond in PC 34:1 and the
subsequent rearrangement. Higher-collisional energy dissoci-
ation (HCD) of Criegee ozonide ions results in an aldehyde
and carboxylic acid fragments which can indicate the C=C
double bond position. Δ9 and Δ11 isomers of PC 34:1 in
brain tissue sections of BALB/c mice and human colon tissue
sections could be visualized using this technique by MS
2
I.
Conclusion
In this review, we have summarized recent advances in the de-
sign, discovery, and characterization of novel small organic ma-
trices and OTCD reagents to improve MALDI-MSI perfor-
mance. Although many novel matrices are still not designed
for broader application, MALDI-MSI with novel matrices dem-
onstrated to have superior results than conventional matrices in
many aspects, for example, vacuum stability, detection sensitiv-
ity, molecular coverage, salt tolerance, matrix background sig-
nals, ion suppression, and crystal formation. Novel matrices have
the potential to improve image quality and expand the applica-
tion area of MALDI-MSI. Nevertheless, the possibility of
theoretical simulations for matrix design is still missing for ion-
ization efficiency, interaction of matrices with other competent
metabolites in tissue sections, crystal formation, and other matrix
performances. At the same time, OTCD processes for MALDI-
MSI were proven to be promising methods for visualization of
poorly ionizable analytes, distinction of isomers, and character-
ization of isomers. Accompanying problems found in OTCD
processes, such as low reaction efficiency, poor reproducibility,
and additional time consumption, constrain attempts to transfer
and optimize versatile in-solution chemical derivatization for
OTCD for MALDI-MSI. However, OTCD processes in
MALDI-MSI workflow have clear advantages in the specific
detection of analytes in tissue sections. Thus, more and more
attention will be paid for transferring and optimizing chemical
derivatization in tissue sections, opening new possibilities for
MALDI-MSI to become an indispensable tool in different re-
search fields.
Funding Open Access funding enabled and organized by Projekt DEAL.
This work was funded by the German Federal Ministry of Research
(BMBF) as part of the Innovation Partnership M
2
Aind, project SM
2
all
(03FH8I01IA) within the framework FH-Impuls (to CH).
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict of
interest.
Open Access This article is licensed under a Creative Commons
Attribution 4.0 International License, which permits use, sharing, adap-
tation, distribution and reproduction in any medium or format, as long as
you give appropriate credit to the original author(s) and the source, pro-
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2617Recent developments of novel matrices and on-tissue chemical derivatization reagents for MALDI-MSI
