(E)-Propyl α-Cyano-4-Hydroxyl Cinnamylate: A High Sensitive and Salt Tolerant Matrix for Intact Protein Profiling by MALDI Mass Spectrometry

Article abstract of DOI:10.1007/s13361-015-1325-5

Low-abundance samples and salt interference are always of great challenges for the practical protein profiling by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). Herein, a series of carboxyl-esterified derivatives of α-cyano-4-hy

Full text of DOI:10.1007/s13361-015-1325-5

B American Society for Mass Spectrometry, 2016
J. Am. Soc. Mass Spectrom. (2016)
DOI: 10.1007/s13361-015-1325-5
RESEARCH ARTICLE
(E)-Propyl α-Cyano-4-Hydroxyl Cinnamylate: A High
Sensitive and Salt Tolerant Matrix for Intact Protein Profiling
by MALDI Mass Spectrometry
Sheng Wang,1 Zhaohui Xiao,1 Chunsheng Xiao,2 Huixin Wang,1 Bing Wang,1 Ying Li,1
Xuesi Chen,2 Xinhua Guo1
1College of Chemistry, Jilin University, Changchun, 130012, China
2Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun,
130022, China
Abstract. Low-abundance samples and salt interference are always of great chal-
lenges for the practical protein profiling by matrix-assisted laser desorption/ionization
mass spectrometry (MALDI-MS). Herein, a series of carboxyl-esterified derivatives of
α-cyano-4-hydroxycinnamic acid (CHCA) were synthesized and evaluated as matri-
ces for MALDI-MS analysis of protein. Among them, (E)-propyl α-cyano-4-hydroxyl
cinnamylate (CHCA-C3) was found to exhibit excellent assay performance for intact
proteins by improving the detection sensitivity 10 folds compared with the traditional
matrices [i.e., super2,5-dihydroxybenzoic acid (superDHB), sinapic acid (SA), and
CHCA]. In addition, CHCA-C3 was shown to have high tolerance to salts, the ion
signal of myoglobin was readily detected even in the presence of urea (8 M),
NH4HCO3 (2 M), and KH2PO4 (500 mM), meanwhile sample washability was robust. These achievements were
mainly attributed to improved ablation ability and increased hydrophobicity or affinity of CHCA-C3 to proteins in
comparison with hydrophilic matrixes, leading to more efficient ionization of analyte. Furthermore, direct analysis
of proteins from crude egg white demonstrated that CHCA-C3 was a highly efficient matrix for the analysis of low-
abundance proteins in complex biological samples. These outstanding performances indicate the tremendous
potential use of CHCA-C3 in protein profiling by MALDI-MS.
Keywords: MALDI-TOF MS, Esterified α-cyano-4-hydroxycinnamic acid, Proteins, Sensitivity, Salt-tolerance
Received: 11 July 2015/Revised: 2 December 2015/Accepted: 5 December 2015
MALDI-MS to find biomarkers in blood serum and plasma
[5], urine [6], cerebrospinal fluid [7], extracts from tissues and
cells [8], etc., and to investigate protein distributions in micro-
Introduction
atrix assisted-laser desorption ionization (MALDI) and
M
electrospray ionization (ESI) are two main ionization
organisms [9, 10], foods [11], and tissue [12]. Particularly,
MALDI-MS based imaging technique has been developed
for directly localizing proteins on a tissue and providing post-
translational modification and spatial distribution information
in a single experiment [13, 14]. This exclusive technique has
shown great potentials for direct investigation of biological
function-related macromolecules, such as cytokines, enzymes,
neuropeptide precursors, and receptor in pathological
conditions.
techniques of mass spectrometry (MS) widely used in proteo-
mic analysis [1–4]. Comparison with ESI-MS MALDI-MS has
shown advantages for direct analysis of proteins in complex
biological samples because of its capacity of higher salt-
tolerance and high throughput, as well as production of singly
or lower charged ions. Tremendous interests and efforts have
been stimulated to develop various strategies by using the
In MALDI-MS analysis, the most common sample prepa-
ration procedure is to simply mix samples with a matrix, which
generates matrix-sample co-crystals on the surface of MALDI
plate after ambient drying. Upon laser irradiation, analyte ions
are generated and subjected for detection. However, low-
Electronic supplementary material The online version of this article (doi:10.
1007/s13361-015-1325-5) contains supplementary material, which is available
to authorized users.
Correspondence to: Chunsheng Xiao; e-mail: xiaocs@ciac.ac.cn, Xinhua Guo;
e-mail: guoxh@jlu.edu.cn

S. Wang et al.: High Sensitive and Salt Tolerant Matrix
abundance samples and signal suppression by salt contami-
nants often associate with the process and influence successful
MALDI-MS analysis. Earlier works have developed numbers
of techniques in sample preparation procedures prior to analy-
sis, which included using on-plate enrichment methods [15],
liquid matrixes [16], additives to matrix [17, 18], spotting
sample techniques [19, 20] such as vacuum sublimed method
and nanoliter spots depositing technique to enhance signal
intensities and detection limits, using C18 Ziptip [21],
surface-functionalized nanoparticles [22] to prefractionate
complex biological samples, using hydrophobic polymers such
as Teflon [23] and parafilm wax [24] as sample support for on-
plate peptides/proteins enrichment or desalting. Recently, we
used patterned MALDI supports to obtain simultaneous enrich-
ment, desalting, and selective peptides/proteins analysis [25,
26]. Nevertheless, these techniques require additional labor,
time, and handling procedures in the fabrication or preparation
process, which inevitably bring certain limits to applications
for large-scale protein profiling (e.g., cancer biomarker screen-
ing and identification of microorganisms). Moreover, many
previous methods are not applicable to imaging mass spec-
trometry because of the requirement of direct deposit of matrix
onto surface of the tissue. Therefore, the development of a
simple, rapid, high throughput, low cost, high sensitive, and
salt-tolerant technique is still critically required in protein iden-
tification by MALDI-MS.
were obtained by using CHCA-C3 as the matrix, compared
with super2,5-dihydroxybenzoic acid (superDHB), sinapinic
acid (SA), and CHCA. Furthermore, CHCA-C3 was assessed
to be able to generate legible spectra of proteins of interest even
in the presence of high concentrations of contaminants. Owing
to the high detection sensitivity and enhanced salt-tolerance,
the practical use of CHCA-C3 matrix for protein profiling was
also investigated by direct analysis of crude egg white.
Experimental
Chemicals and Materials
α-Cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid
(SA), 2,5-dihydroxybenzoic acid (DHB), 5-methoxysalicylic
acid, bovine insulin, cytochrome c, and ubiquitin were pur-
chased from Bruker Daltonics (Bremen, Germany). Porcine
insulin, lysozyme, myoglobin, growth hormone, trypsin
TPCK treated from bovine pancreas, pepsin, bovine serum
albumin (BSA), transferrin from human, IgG from rabbit, ace-
tonitrile (ACN), trifluoroacetic acid (TFA), and alkyl alcohols
were purchased from Sigma-Aldrich (Beijing, China).
Thermophilic histone and alkaline phosphatase were obtained
as a gift from Dr. Quanshun Li (Jilin University). The chemicals
were of analytical grade. All aqueous solutions were prepared
using Milli-Q water by Milli-Q System (Millipore, Billerica,
MA).
A fundamental field of research and improvements associ-
ated with MALDI and its application in high efficient MS
analysis is of matrix design. Matrix serves as the requisite
and basis for MALDI analysis. It has been generally accepted
to possess some common characters, including absorbing laser
energy, preventing sample aggregation, good ability for co-
desorption of the analyte, and transferring charge (proton,
electron or cation) to analyte, etc.[27, 28]. These characters of
the selected matrix have a direct impact on the assay perfor-
mance. Two conventional matrixes are α-cyano-4-
hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic ac-
id (DHB). Based on them, several derivatives have been for-
mulated to improve matrix performance. Previously, 4-chloro-
α-cyanocinnamic acid (Cl-CHCA), a CHCA derivative, was
reported to provide lower proton affinity and obtain outstand-
ing detection sensitivity of acidic peptides and higher sequence
coverage for the identification of protein digests [29]. Recently,
O-alkylated dihydroxybenzoic acid (ADHB), an esterified de-
rivative of DHB, has been developed to improve its affinities
for hydrophobic peptides [30]. ADHB as additive matrix was
mixed with the traditional matrix CHCA and improved the
sensitivity of hydrophobic peptides 10- to 100-fold compared
with CHCA.
Synthesis of Substituted CHCA
Typically, 0.57 g of α-cyano-4-hydroxylcinnamic acid
(3.0 mmol) was first suspended in 20 mL of propyl alcohol,
to which 20 μL of concentrated sulfuric acid was added. The
mixture was heated to reflux at 80 °C for 5 h. After cooling to
room temperature, the solution was concentrated by rotary
evaporation. The residue was then dissolved in ethyl acetate,
washed three times with ammonium bicarbonate solution, and
dried over MgSO4. (E)-propyl α-cyano-4-hydroxyl
cinnamylate (CHCA-C3) was obtained as yellow crystal after
purification by column chromatography (0.40 g, 1.7 mmol). 1H
NMR (300 MHz, CDCl3) δ 8.19 (s, 1H), 7.98 (d, J = 8.2 Hz,
2H), 6.97 (d, J = 8.2 Hz, 2H), 5.83 (s, 1H), 4.29 (t, J = 6.5 Hz,
2H), 1.79 (dt, J = 14.1, 7.1 Hz, 2H), 1.04 (t, J = 7.3 Hz, 3H).
The same method was used to acquire the substituted CHCA
with linear alkyl, i.e., R = C1, C2, C4, C5, C6, C8, C10, C12
(Figure 1). The substituted CHCA with R = isobutyl, isopropyl,
and tert-butyl were synthesized with Knoevenagel reaction
[31].
Herein, a series of new matrices were synthesized by mod-
ifying CHCA with hydrophobic alkyl on the carboxyl group
and evaluated by analysis of various proteins. After screening
the MALDI assay performance, (E)-propylα-cyano-4-hydroxyl
cinnamylate (CHCA-C3) was found to be the most promising
candidate for intact protein analysis. The improved detection
sensitivities by one order of magnitude for protein profiling
Figure 1. Substituted CHCAs with hydrophobic substituent
groups. Cn (n = 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, n denotes the carbon
number in linear alkyl alcohols)

S. Wang et al.: High Sensitive and Salt Tolerant Matrix
Sample Preparation
calibration of the mass weight. All experiments were carried
out at least three times to ensure the reproducibility of the
spectral profiles.
ESI-MS experiments were carried out in ESI positive ion
mode of an electrospray ionization and quadrupole time-of-
flight (ESI-Q-TOF) mass spectrometer (microTOF-Q II,
Bruker Daltonics, Germany). For MS/MS experiment, argon
was used as collision gas. The collision energy was optimized
for the best fragmentation pattern at 7.0 eV.
Proteins were dissolved in 0.1% aqueous TFA (v/v) at appro-
priate concentrations. Two sample deposition methods were
applied in this study: the dried-droplet (DD) and two-layer (TL)
method. For the DD method, matrix solutions of CHCA and
SA were saturated in 50% ACN/0.1% aqueous TFA (v/v), and
superDHB (DHB + 10% 5-methoxysalicylic acid) was dis-
solved in distilled water-ethanol (9:1, v/v) at 20 mg mL–1.
CHCA-C3 solution was prepared in 50% ACN/0.1% aqueous
TFA (v/v) at 1 mg mL–1. All matrix solutions were fleshly
made each time. Equal volumes of analyte solution and matrix
solution (each at 10 μL) were thoroughly mixed by repeatedly
aspirating with pipet for six times, then 1 μL of the mixed
solution was deposited on the steel target plate (MTP 384 target
plate ground steel, Bruker) and analyzed by matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry
(MALDI-TOF MS). For the TL preparation method, 1 μL of
CHCA or SA solution (12 mg mL–1 in 80% acetone/ethanol)
was firstly deposited on the target spot to form a first seed layer,
then 1 μL of the analyte-matrix mixed solution was deposited
on the top of the first layer and left to dry. The TL method was
not applicable to superDHB because of the limit of solubility of
DHB in organic solvent such as acetone. There were no obvi-
ous advantages in the terms of sensitivity improvement by
using TL method for CHCA-C3; hence, CHCA-C3 was per-
formed only with the DD method.
Results and Discussion
Preliminary Evaluation of Substituted CHCA
Matrices
A series of substituted CHCA matrices were synthesized by
modifying CHCA on the carboxyl group with hydrophobic
1
alkyl (Figure 1). The matrices were characterized by H
NMR and tested to be of better solubility than CHCA in
common organic solvent, such as methanol, acetonitrile,
etc. UV-Vis spectra revealed that all the esterified CHCAs
possessed absorption peak at around 338 nm, which is
comparable to that of CHCA (Supplemental Figure S1, see
Supporting Information). A preliminary test of these com-
pounds as MALDI matrices for protein analysis was per-
formed using bovine insulin as the analyte. As shown in
Supplementary Figure S2, the signal-to-noise (S/N) ratio of
different alkyl chain-length of the CHCA derivative matri-
ces and analyte ions were evaluated based on the variation
of laser energy. Broadly speaking, threshold energies re-
quired for highest and stable positive-ion signals of bovine
insulin were about 5% higher than those of corresponding
matrix ions. The threshold energies for matrix ions using
esterified CHCA with carbon numbers of the alkyl groups
n = 2–5 were at energy of 56 1%, which were slightly
lower than those for CHCA-Cn (n = 1 or >5) and CHCA at
60 2%. Simultaneously, spectra of analyte insulin using
the esterified CHCA with carbon numbers of the alkyl
groups n = 2–5 showed higher S/N ratio value. Among
them, CHCA-C3 exhibited the highest S/N ratio at about
1000 compared with CHCA (DD method) with S/N ratio at
about 200. These data suggested that ablation of the ester-
ified CHCA with carbon numbers of the alkyl groups
n = 2–5 required less energy than ablation of esterified
CHCA (n = 1 or >5) and CHCA. For analyte-M+, a similar
trend could be observed. Ion yields of insulin were obvi-
ously improved using esterified CHCA with carbon num-
bers of the alkyl groups n = 2–5. The energy threshold and
optimal S/N ratio using CHCA-C3 were at about 60% and
700, compared with CHCA (DD method) at 66% and 100.
CHCA prepared with two-layer (TL) method was also in-
vestigated. The energy threshold for both matrix and ana-
lyte-M+ declined by 4% and optimal S/N ratio improved 2-
to 3-fold. Thus, CHCA-C3 was selected as the candidate
matrix and used for further evaluation.
Instrumentation
MALDI-TOF MS experiments were performed on an Autoflex
speed TOF/TOF mass spectrometer (Bruker Daltonics,
Germany) with a pulsed Nd:YAG laser at a wavelength of
355 nm. Spectra were obtained by 500 laser shots and a delayed
extraction time of 150 ns in linear and positive ion mode. An
acceleration voltage of 19.38 kV (IS1) was applied for a final
acceleration of 16.88 (IS2). MS/MS experiments were run in
the MS/MS lift mode using collision induced dissociation gas
(air). In general, laser energy was kept about 10% above
threshold to obtain the best signal quality with low noise. For
the evaluation of the limits of detection (LODs), laser energy
was adjusted from the threshold energy of corresponding ma-
trix ions to 100% by variable laser attenuator. The LODs for
proteins was defined as the lowest quantity of analyte detected
as [M + H]+ with S/N ≥ 3 when each series of analyte solution
at different concentration made by 10-fold serial dilutions was
evaluated. After smoothing and baseline subtraction twice, the
S/N ratio of analyte ion in the spectra were automatically
marked by analysis software. Fresh pipet tips were used for
each transfer procedure of solution. Targets for the sample
deposition were processed with careful cleaning (sequential
cleaning with hot water, acetone, hot water, 30% TFA, aceto-
nitrile, and distilled water) and supersonic treatment (methanol,
40 min) before being used. Flex Analysis 3.3080 Software
(Bruker Daltonics) was used to work up data. Commercial
peptide or protein mixture (peptide standard II, protein standard
I, or protein standard II) served as external standards for

S. Wang et al.: High Sensitive and Salt Tolerant Matrix
Sensitivity, Sample Preparation
Morphology/Homogeneity, and Signal-to-Noise
Ratio
SA and CHCA were dense and homogeneous. CHCA-C3 also
exhibited a uniform but slightly thin crystal. As a result, there
was little difference for signal intensities achieved from differ-
ent parts of crystal spots for sample preparation with SA,
CHCA, and CHCA-C3. In the case of superDHB, it formed
large needlelike crystals on the rim of the spot. Thus, it was
difficult to obtain assignable signal information on the center of
spot with the superDHB preparation, and it required search for
Bsweet spots^ in the rim, especially for low concentration of
analyte. Furthermore, the spot-to-spot reproducibility were in-
vestigated by evaluating the relative standard deviation (RSD)
obtained by calculating [tryspin]+ S/N ratio variation. As
shown in Supplementary Figure S4, the RSD were 81.3%,
43.5%, 33.7%, and 38.4% for superDHB, SA, CHCA, and
CHCA-C3, respectively. These results were consistent with
the morphology of matrix-analyte co-crystal as described
above. Hence, our data suggested CHCA-C3 was a good
matrix candidate for highly reproducible analysis of proteins.
The S/N ratio was also compared in detail for these four
matrices. Trypsin (basic protein with isoelectric point ~10.1)
and pepsin (acidic protein with isoelectric point ~1.0) were
typically selected as the reference proteins. As shown in
Figure 2, CHCA-C3 exhibited the highest S/N ratios among
these four matrices for both tested proteins at the correspond-
ing concentrations. Moreover, for trypsin, the detection limit
was 5 fmol with CHCA-C3, whereas that was 50 fmol with
superDHB and CHCA, and 100 fmol with SA. As shown in
Figure 2b, rather weak signal corresponding to [pepsin]+ with
a S/N at about 3.2 was obtained at 100 fmol of sample
loading using superDHB. Although there was no need to find
Bsweet spots^ for CHCA and SA with TL method, the
detection limit of pepsin was at only 1 pmol with S/N ratios
of 7.9 and 23.4 for CHCA and SA, respectively. In contrast,
CHCA-C3 exhibited excellent signal for 1 pmol pepsin with
a S/N ratio at 129.4 and showed the lowest detection limit at
SuperDHB, SA, and CHCA are popular matrices for intact
protein analysis [10, 18, 32, 33]. To further investigate the
applicability of CHCA-C3 matrix in protein analysis, MALDI
MS analysis was performed by screening a series of intact
proteins with molecular weights ranging from 5734.52 to
148,420 Da in comparison with these three classic matrices.
As listed in Table 1, for all the tested proteins, the LODs
obtained by using superDHB are lower than using CHCA
and SA with the DD method. When the TL method was applied
for CHCA and SA, the LOD obtained using CHCA showed
nearly the same order of magnitude as that using superDHB
(100 to 10 fmol), whereas SA resulted in LODs one order of
magnitude higher than that with CHCA and superDHB for the
detection of four small proteins (i.e, bovine insulin, porcine
insulin, lysozyme, and myoglobin). In view of these sensitivity
improvements, CHCA and SA would be performed with TL
method for all of the following comparisons. In contrast,
CHCA-C3 enables obtaining the lowest LODs at 10 to 1 fmol,
which improved the detection sensitivity 10-fold compared
with these three traditional matrices.
When CHCA and SA were performed with DD method, it
was noticed that hunting for Bhot spots^ in the matrix-analyte
co-crystals was required to obtain good spectra, especially at
analyte concentration closed to LOD. These might be attributed
to the uneven analyte-matrix co-crystals [34]. When TL meth-
od was applied for CHCA and SA, the spot to spot reproduc-
ibility was well improved [34]. Herein, matrix-analyte co-crys-
tal with superDHB (DD method), SA (TL method), CHCA (TL
method), and CHCA-C3 (DD method) were investigated.
Supplementary Figure S3 shows sample spots of these four
matrices collected from MALDI camera. The crystal layer of
Table 1. Obtained LODs for Proteins Using CHCA-C3, Compared with superDHB, SA, and CHCA
#
Protein
Mass (Da) CHCA (fmola)
SA (fmola)
superDHB (fmola) CHCA-C3 (fmola) Sensitivity improvement
rateb (fold)
DD method TL method DD method TL method DD method
DD method
1
2
3
4
5
6
7
8
9
Bovine insulin
Porcine insulin
Ubiquitin
Thermophilic histone 9716
Cytochrome c
Lysozyme
Myoglobin
Growth hormone
Trypsin
5734.52
5788
8586.76
10
10
10
10
100
100
100
100
100
100
100
100
1000
100
100
1000
100
100
100
100
10
10
1
1
10
10
10
10
10
10
10
10
10
10
10
10
10
10
100
100
100
10
100
1000
1000
1000
100
100
100
1000
100
100
100
10
100
100
100
1000
100
100
100
100
1000
1000
1000
100
1000
1000
1000
1000
100
100
100
100
10
100
100
100
100
100
100
100
100
10
10
10
1
10
10
10
10
10
10
10
10
12361
14260
16952
22124
23982
35000
10 Pepsin
11 Alkaline phosphatasec 56000
12 Bovine serum albumin 66430
1000
1000
1000
13 Transferrinc
14 IgGc
77064
148420
afmol represent the amount of sample loading on the target spot.
bSensitivity improvement rate was obtained by dividing the detection limit using superDHB by that using CHCA-C3.
cAlkaline phosphatase, transferrin, and IgG are glycoproteins.

S. Wang et al.: High Sensitive and Salt Tolerant Matrix
Protein Charge State
It has been reported that CHCA usually has limit detection
ability in analysis of high molecular weight proteins, which
increases the tendency to generate multiple protonation with
relatively low intensity of singly protonated ion [35]. Here,
transferrin (Mw = 77064 Da) as analyte was investigated.
Supplementary Figure S5 shows that the highest charge state
observed using CHCA matrix was 8+ and the relative abun-
dance of [M + H]+ was far below [M + H]2+ and [M + H]3+
(Supplementary Figure S5a). The highest charge state with
superDHB (Supplementary Figure S5c) was 4+ and
[M + H]2+ dominated the spectrum. In contrast, the highest
charge state of protonated transferrin by using CHCA-C3
matrix was 3+ and the [M + H]+ signal dominated the whole
spectrum (Supplementary Figure S5d), which was similar to
the spectrum using SA (Supplementary Figure S5b). The en-
hanced relative abundance of singly protonated protein and
reduced charge number acquired by using CHCA-C3 would
make it more conducive to the identification of objective peaks
and prevent the interference of other multiply charged protein
peaks, particularly for analysis of big and complex protein
sample.
Figure 2. Average S/N ratio for molecular ion signals of (a)
trypsin and (b) pepsin at different protein loading. Each S/N
ratio value was an average of S/N ratio obtained from nine
spectra. The matrices used for analysis are superDHB, SA,
CHCA, and CHCA-C3. The CHCA and SA were performed with
TL method, and the superDHB and CHCA-C3 were prepared
with DD method
Resolution
Peak resolution was a significant parameter for accurate and
reliable analysis of intact protein by mass spectrometry.
SupplementaryTable S1 lists the obtained mass resolution
values (full width at half maximum, FWHM) for the singly
charged molecular ion of trypsin (m/z 23982) and transferrin
(m/z 77064) by MALDI-MS. CHCA-C3 showed comparable
resolution values with superDHB, SA, and CHCA for higher
sample loading at 1 pmol. However, it should be noted that the
resolution values of protein peaks obtained with superDHB,
SA, and CHCA would decrease to varying degrees with the
decrease of sample loading, and even become undetectable at
10 fmol of analyte loaded on the target. In contrast, there was
little difference for resolution values of protein peaks by using
CHCA-C3, even at the sample loading as low as 10 fmol.
Hence, these data suggest CHCA-C3 should be a superior
matrix for detection of low-concentration proteins.
10 fmol with a S/N ratio at 5.9. Thus, these data further confirm
that CHCA-C3 would be a promising matrix for detection of
trace amount of proteins.
The sensitivity improvement efficiency was further evalu-
ated by detecting protein mixtures of five low-concentration
proteins, including bovine insulin, lysozyme, myoglobin, tryp-
sin, and pepsin with superDHB, SA, CHCA, and CHCA-C3.
The used laser energies for different matrices were kept slightly
higher than the signal threshold value to achieve reliable spec-
tra. As shown in Figure 3a–d, when the analyte loadings were
50, 50, 100, 50, and 100 fmol for insulin, lysozyme, myoglo-
bin, trypsin, and pepsin, respectively, the signal peaks of [in-
sulin]+, [lysozyme]2+, [lysozyme]+, [myoglobin]2+, and [tryp-
sin]+ were noted using superDHB and CHCA, whereas the
signal peaks of [myoglobin]+ and [pepsin]+ were not visible.
For the case of SA, only signal peaks corresponding to insulin
and lysozyme were observed. In contrast, the sample prepara-
tion with CHCA-C3 revealed highly resolved spectra of signal
peaks corresponding to all the five proteins. As the concentra-
tions of five proteins further decreased to 10, 10, 50, 10, and
50 fmol (Figure 3e, f), highly resolved signal peaks for the five
low-concentration proteins could still be observed with CHCA-
C3. However, only weak signal peaks of [lysozyme]2+ and
[lysozyme]+ were noted with superDHB and CHCA, and no
assignable signal could be observed with SA.
Salt Tolerance and Sample Washability
High concentrations of contaminants (such as salts, buffers,
and surfactants) are often and inevitably presented in biological
sample. These existing compounds strongly suppress target
ionization, which usually leads to weak ion signal available
or even no detectable signal in MALDI-MS [22, 36, 37]. Salt
tolerance of CHCA-C3 was evaluated by using myoglobin as
the protein analyte in the presence of urea, NH4HCO3, and
KH2PO4, and compared with CHCA, SA, and superDHB. As
shown in Figure 4a–d, high quality spectra were obtained
without any salts with all the tested matrices. In the presence
of 8 M urea (Figure 4e–h), there was no objective signal
observed using superDHB, SA, and CHCA. This is because

S. Wang et al.: High Sensitive and Salt Tolerant Matrix
Figure 3. MALDI mass spectra of protein mixtures of five proteins: bovine insulin [(a), m/z: 5734.5], lysozyme [(b), m/z: 14260],
myoglobin [(c), m/z: 16952], trypsin [(d), m/z: 23982], and pepsin [(e), m/z: 35000]. The sample loading of the corresponding five
proteins were 50, 50, 100, 50, 100 fmol for the left spectra (a), (b), (c), (d), and 10, 10, 50, 10, 50 fmol for the right spectra (e), (f), (g),
(h). Different matrices were marked in the figures and different charge states were marked on corresponding peaks
urea may form a thick layer after solvent evaporation, which
would severely impede UV absorption and energy transfer
among molecules [38]. In contrast, a legible spectrum corre-
sponding to the [M + H]+ was clearly observed with S/N ratio
at 47.8 using CHCA-C3 as the matrix (Figure 4h). Likewise, in
the presence of 2 M NH4HCO3 (Figure 4g–i), no detectable
signal could be observed using SA and superDHB, and a
severely suppressed signal corresponding to [M + H]+ were
detected with S/N ratio at 3.8 using CHCA by searching for
Bhot spots,^ whereas a legible signal was obtained with S/N
ratio at 38.5 using CHCA-C3 (Figure 4h). In addition, phos-
phate is one of the most commonly used buffers, yet MALDI is
relatively insensitive to the phosphate [39]. Thus, KH2PO4
(500 mM) was selected to evaluate the tolerance for phosphate.
Not surprisingly, legible signal was only obtained with CHCA-
C3 matrix (Figure 4m–p). Therefore, the excellent salt toler-
ance makes CHCA-C3 a matrix with great potential for assay
performance or direct protein analysis without tedious
pretreatment.
Moreover, simple washing of the analyte/matrix on the
MALDI target with deionized water is a simple and efficient
procedure to improve ion signals of interest [40]. This is
because the salts are mainly distributed outside the matrix
crystals and readily removed by washing, whereas the biolog-
ical molecules are more or less evenly incorporated in the
matrix crystals (often less soluble in water). Therefore, it is
necessary for the applied matrix to possess good crystal struc-
ture and properties to obtain ideal washing performance. Thus,
sample washability was investigated in the presence of urea,
NH4HCO3, and KH2PO4. After the analyte-matrix-
contaminant was co-deposited and allowed to be completely
dried on the target spot, 2.5 μL of 0.1% TFA aqueous
solution was deposited on the top of the crystal for 5–10 s
before being removed. As shown in Supplementary
Figure S6, significant improved signal intensities were ob-
served after one wash and limited loss of protein sample was
observed even after three washes. This superior sample
washability with CHCA-C3 should be ascribed to the im-
proved hydrophobic nature of CHCA-C3 matrix. The hy-
drophilic salt contaminants can be easily removed by wash-
ing while the protein analytes are preserved to be embedded
in the matrix crystal because of the insolubility of CHCA-C3
in washing solution.
Practical Protein Profiling in Crude Egg White
Identifications of target proteins in real organic samples are
often difficult because of sample complexities. Based on the
improved sensitivity and salt tolerance available with CHCA-
C3 for the analysis of integrated protein, MALDI mass spectra
of crude egg white was investigated to illustrate applicability.
Previously, SA with TL preparation was commonly used as
MALDI matrix for the detection of crude egg white [41, 42].
Thus, SA was used as the reference matrix. For direct analysis
of crude egg white without pretreatment, nearly no signals
could be detected with both SA and CHCA-C3, even though

S. Wang et al.: High Sensitive and Salt Tolerant Matrix
Figure 4. MALDI mass spectra for evaluating salt tolerance of matrices. Myoglobin as analyte was detected with CHCA (first row),
SA (second row), superDHB (third row), and CHCA-C3 (fourth row). Sample detected in first rank without any additive salts (a, b, c, d);
the second rank detected in the presence of 8 M urea (e, f, g, h); the third rank detected in 2 M NH4HCO3 (i, j, k, l); and the fourth rank
detected in 500 mM KH2PO4 (m, n, o, p). The salt concentrations indicated in this work are for the protein samples prior to mixing with
the MALDI matrixes. All analyte concentrations were always kept at 1 pmol. Peak intensity can be inferred from the coordinate in the
upper right corner of each mass spectrum
washing steps on plate were carried out. This suggests that
excess salts in crude egg white largely suppress analyte ioni-
zation. Therefore, the crude egg white samples were diluted 10
and 500 times, respectively, with 30% ACN/0.1% aqueous
TFA (v/v) (TA30). The diluent solution and matrix solutions
were mixed in 1:1 volume ratio and then 1 μL of mixture
solution was deposited on the target. One wash with 0.1%
TFA aqueous solution was made on each deposited sample to
remove excess salt. Because matrix crystal might be decreased
by the washing step, an additional 1 μL of pure matrix solution
was used to cover the sample spot to form a uniform and dense
crystal layer. In the egg white, four major proteins occupying
more than 3.0% content are ovalbumin (54.0%, Mr ~45,000,
pI = 4.5), ovoconalbumin (12%–13%, Mr ~77,700, pI = 6.0),
ovomucoid (11.0%, Mr ~28,000, pI = 4.1), and lysozyme
(3.4%–3.5%, Mr ~14,300, pI = 10.7) [43]. Figure 5a shows
that three proteins were detected using SA as matrix for
10 times dilution of crude egg white. The m/z at 77,760,
44,542 and 14,295 are likely from ovoconalbumin, ovalbumin,
and lysozyme, respectively. In contrast, apart from the compa-
rable mass signals for the above-mentioned three proteins de-
tected by SA matrix, an additional protein peak at m/z 47,989
along with a doubly charged protein ion were noted using
CHCA-C3 (Figure 5c), which are likely from G2 ovoglobulin

S. Wang et al.: High Sensitive and Salt Tolerant Matrix
C6) and CHCA were investigated with B3LYP DFT molecular
orbital approach in Gaussian 09 [44, 45]. (Table S2, see
Supporting Information). The theoretical data demonstrated
that the proton affinities of the esterified CHCAs were higher
than that of CHCA, namely a lower transfer ability of proton.
Furthermore, the reaction enthalpies of CHCA and esterified
CHCAs had minimal difference (0.08 eV), indicating the same
ability to get over the primary ionization step for all esterified
CHCAs.
This inconsistency between the thermochemical properties
and the practical MALDI-MS performance thus led us to obtain
further insight into the mechanism of ionization processes of
CHCA-C3. To this end, the MALDI mass spectrum of pure
CHCA-C3 was recorded. As shown in Supplementary
Figure S7a, not only the signals of protonated, sodiated, and
potassiated CHCA-C3 but also protonated CHCA was ob-
served. In order to prove whether the produced CHCA comes
from esterified CHCA, protonated CHCA-C3 (m/z: 231.119)
ion was selected to fragment in MS/MS mode with MALDI
and ESI. The protonated signal peak of CHCA (m/z 190.057)
was dominant and no protonated CHCA-C3 ion was observed
in MALDI MS/MS spectrum (Supplementary Figure S7b). In
the ESI MS/MS spectrum, there was only a very weak ion
signal of protonated CHCA-C3 under a 7.0 eV collision energy
(Figure S7c). These data demonstrated that CHCA-C3 can be
easily fragmented into CHCA under considerably low energy.
This transition from CHCA-C3 to CHCA can be explained to
undergo a McLafferty-type rearrangement reaction in the gas
phase [46] (i.e., a γ-hydrogen was transferred to the C-terminal
carbonyl group to cause the elimination of an alkene). The
active hydrogen on the carboxyl of CHCA produced through
fragmentation has been proven to be transferred to analyte by
deuterium labeling experiment [47]. Therefore, the ionization
processes using CHCA-C3 as the matrix should involve both
CHCA-C3 and fragmented CHCA, which endows the CHCA-
C3 matrix with comparable ionization ability as CHCA in the
gas phase.
Figure 5. MALDI mass spectra of crude egg white with dilution
10 times using (a) SA, (c) CHCA-C3, and 500 times using (b)
SA, (d) CHCA-C3 matrices. The m/z range is from 10 to
100 KDa. The m/z of corresponding peaks are labeled in (c)
It is generally accepted that the ionization in UV MALDI
adopts a two-step framework (i.e., primary ionization upon
laser ablation and secondary reactions in the desorbed plume
that follows [48]). Although CHCA-C3 has no advantages on
the secondary ionization step, the improved ablation ability
along with relatively lower threshold energy for CHCA-C3
was observed compared with superDHB, SA, and CHCA, as
shown in the above data and discussions. This means that the
laser-induced cluster evaporation could free more matrix and
analyte ions for the secondary ionization process, which may
directly result in improved MALDI-MS performance toward
intact protein. In MALDI-MS, the morphology of matrix-
analyte co-crystal is also of great importance for obtaining a
satisfied assay performance. An even-distributed and dense
crystal would lead not only to good shot-to-shot reproducibility
but also to the elimination of searching for a Bhot-spot.^
Meanwhile, the data in Table 1 shows an increased sensitivity
with SA and CHCA when using TL method instead of DD
method. Thus, it is believed that the homogeneous CHCA-C3-
(1.0%, pI = 4.9–5.3). The S/N ratio of protein signal corre-
sponding to ovoconalbumin and lysozyme using CHCA-C3 are
nearly identical to that obtained with SA. In the case of ovalbu-
min, the S/N ratio obtained with CHCA-C3 is 115.4 compared
with 28.5 with SA. However, with 500 times dilution, no
assignable signal could be detected using SA, while three
proteins corresponding to ovoconalbumin, ovalbumin, and
lysozyme with S/N ratio at 56.8, 22.5, and 15.4, respectively,
were still clearly detected with CHCA-C3. Several repeating
experiments for the egg white over the course of different hours
were performed and the obtained spectra were nearly identical,
indicating that the novel matrix CHCA-C3 was reproducible
and sufficient to meet precise protein analysis.
Mechanism Analysis
The thermochemical properties (the proton affinity and reac-
tion enthalpy) of esterified CHCA (from CHCA-C1 to CHCA-

S. Wang et al.: High Sensitive and Salt Tolerant Matrix
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analyte co-crystal should partly contribute to the sensitivity
improvement. Another benefit of using CHCA-C3 as matrix
is its hydrophobic nature, which has resulted in high salt
tolerance and robust sample washability in analysis of intact
protein. In these cases, CHCA-C3 was proposed to have high
affinity with protein, so that the protein analyte could be tightly
embedded in the matrix crystal and subjected to effective
ionization without the interference of salt or wash process.
Additionally, it has been reported that the increased affinity
between matrix molecules and analyte could dramatically im-
prove the detection sensitivity in MALDI-MS [49–51].
Therefore, though the detailed mechanism is not yet clear, the
improved MALDI-MS performance for analysis of intact pro-
tein using CHCA-C3 matrix should be generally ascribed to
high laser-induced ablation ability, even-distributed matrix-
analyte co-crystal, and increased affinity for protein.
Conclusions
Herein, a novel MALDI matrix CHCA-C3 has been developed
to improve MALDI-MS performance for protein profiling with
a broad mass range. The detection sensitivity of proteins with
CHCA-C3 was improved 10 times compared with traditional
matrices (i.e., superDHB, SA, and CHCA). Moreover, CHCA-
C3 exhibited excellent salt tolerance and sample washability in
the presence of high concentrations of salts, such as urea (8 M),
NH4HCO3 (2 M), and KH2PO4 (500 mM). Based on the highly
enhanced detection sensitivity and robust salt-tolerance,
CHCA-C3 was successfuly used for MADLI profiling of crude
egg white. A study to develop a more effective matrix based on
the findings obtained is underway.
Acknowledgments
The authors acknowledge financial supported for this work by
the National Natural Science Foundation of China (51273080,
21175056, and 51203153) and Open Project of State Key
Laboratory for Supramolecular Structure and Materials
(SKLSSM201433).
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