Review Article

Analysis of Biomolecules through Surface-Assisted Laser

Desorption/Ionization Mass Spectrometry Employing Nanomaterials

Wen-Tsen Chen,a Iva Tomalová,b Jan Preislerb* and Huan-Tsung Changa*aDepartment of Chemistry, National Taiwan University, 1, Section 4, Roosevelt Road,

Taipei 106, Taiwan, R.O.C.bCEITEC MU and Department of Chemistry, Faculty of Science, Masaryk University,

Kamenice 5, Brno, Czech Republic

Received May 10, 2011; Accepted June 14, 2011; Published Online June 22, 2011

Nanomaterials, primarily nanoparticles (NPs), can serve as an alternative matrix for the analysis of

various biomolecules through surface-assisted laser desorption/ionization mass spectrometry (SALDI

MS). SALDI MS has been developed to overcome poor reproducibility and high background in the low-

mass region commonly occurring in matrix-assisted laser desorption/ionization mass spectrometry

(MALDI MS). Various nanomaterials, unmodified or functionalized with recognition ligands, can have a

strong affinity toward certain analytes and thus are applicable for their concentration and enrichment from

complex biological matrices. In mass spectrometry imaging (MSI), the use of NPs instead of the conven-

tional matrices can improve the spatial resolution up to the cellular level. In this review, the nature of NPs,

the methods of sample preparation and approaches for quantitation of biomolecules through SALDI MS

are discussed. Practical applications and limitations of SALDI MS employing NPs for separate samples

and MSI are mentioned. With regard to the nature of MSI analysis, the use of nanostructured surfaces for

MSI is also reflected in this review.

Keywords: Nanomaterials; Biomolecules; Surface-assisted laser desorption/ionization mass spec-

trometry; Mass spectrometry imaging.

INTRODUCTION

Matrix-assisted laser desorption/ionization mass

spectrometry (MALDI MS), nowadays an essential mass

spectrometric technique for the analysis and characteriza-

tion of biomolecules, was developed independently by two

groups in the late 80’s. Karas et al. presented soft ioniza-

tion of amino acids and dipeptides, when embedding these

non-absorbing analytes in a UV-absorbing matrix, first

tryptophan followed by nicotinic acid.1-3 Instead of using

an organic matrix, Tanaka dissolved analytes in a suspen-

sion of cobalt nanoparticles (NPs) in glycerol.4 With many

developments, such as the discovery of new more efficient

matrices, the former technique has become widely and rou-

tinely used whereas Tanaka’s method has not attracted

much attention because of the lower sensitivity and limited

mass range compared to the results obtained with organic

matrices. Among a few others who continued to develop

Tanaka’s original idea, for instance Sunner et al. and Dell et

al., used larger particles (2-150 µm) of graphite or silicon

in glycerol.5-7 Another approach for desorption and ioniza-

tion of small molecules using the organic matrices, desorp-

tion/ionization on porous silica (DIOS), was presented by

Wei et al.8 DIOS employs a porous silica surface obtained

by electrochemical etching and provides “clean” mass

spectra with no or little chemical background.

With the recent development of nanotechnology,

Tanaka’s original idea of using the nano-sized metal parti-

cles has been enjoying a renaissance. Without the low va-

por liquid matrix such as glycerol, Au,9,10 Ag,11 SiO2,12

TiO2,13 Fe3O414,15 and other NPs16 have become widely

used as efficient concentrations and desorption probes for

various analytes. Similar to organic matrices for MALDI

Journal of the Chinese Chemical Society, 2011, 58, 769-778 769

Special issue for the nanotechnology-related analytical chemistry

* Corresponding author. Tel: 011-886-2-33661171; 420-549-49-6629; E-mail: changht@ntu.edu.tw, preisler@chemi.muni.cz

MS, NPs have high molar absorption coefficients allowing

absorption of the laser energy, leading to soft and efficient

desorption of the analytes (Fig. 1).

When performing the MALDI MS analysis, the ma-

trices are usually mixed in to excess over the analyte to

avoid its fragmentation from laser irradiation and the for-

mation of co-crystals of a size up to hundreds of microme-

ters, resulting in the formation of “sweet spots”. This leads

to poor shot-to-shot and sample-to-sample reproducibility.

In addition, because of strong background signals arising

from matrix fragments in the low-mass range, the MALDI

MS has succeeded neither as a quantitative tool nor a tool

for analysis of low-weight molecules.17,18

Compared to traditional matrices, the use of NPs

overcomes these problems and provides further advantages

including large surface areas available for large analyte

loading capacity (e.g. > 1000 small molecules per single

NP),9 low matrix interference,19 concentration of the ana-

lyte20 or the option of direct combination of the analyte

analysis with its extraction,21 simple preparation of homo-

geneous samples or low-cost and large-scale preparation of

the NPs.

The terminology in this field could appear unclear

and inconsistent. Several new terms, such as NALDI (nano-

structured-assisted LDI),22,23 nano-PALDI (nanoparticles-

assisted LDI)24 or GALDI (colloidal graphite-assisted

LDI)12,25,26 have been used in recent years for numerous

methods employing nanomaterials, but the term SALDI

(surface assisted laser desorption/ionization), which was

first introduced by Sunner et al. for his technique to distin-

guish it from MALDI, remains the most common.

In this review, we describe the properties of NPs,

sample preparation methods for SALDI MS as well as the

effect of the selection of NPs on the sensitivity and repro-

ducibility of analysis. Potential applications and limits of

NP-based SALDI MS techniques are discussed. The appli-

cations and practical considerations for mass spectrometry

imaging (MSI) are mentioned in brief.

NANOPARTICLES, THEIR PROPERTIES AND

ROLE IN SALDI MS

NPs are particles with sizes in the range of 1-100 nm

and, as already comprehensively reviewed, they can be pre-

pared in several ways.27,28 Briefly, two main ways could be

used for their preparation – either the bottom-up approach

by disintegration of bulk materials or top-down by synthe-

sis from atomic or molecular species.29 Metal and metal ox-

ide NPs for SALDI MS are usually synthesized by hydro-

thermal synthesis which provides the most controllable

method for the production of high quality NPs with a de-

fined size. Furthermore, NPs can be used as seeds to pre-

pare nanocomposites (multi-elemental NPs) and core-shell

nanostructures.30,31 In SALDI MS, NPs play important

roles in capturing the analytes, absorbing and transferring

the desorption laser energy leading to the analyte desorp-

tion and ionization. With N2 (337 nm) and Nd:YAG (355

nm) lasers being the most commonly used, NPs with high

absorption in near the UV region are most suitable — for

instance, Au, Ag, and TiO2 NPs.

By decreasing the size of NPs, the surface area per

mass unit increases and thus, the number of adsorbed ana-

lytes increases. More analyte molecules can be desorbed

and ionized when using small-diameter NPs.32,33 The laser

threshold for analyte desorption is inversely related to par-

ticle size: the smaller the NP, the higher the calculated sur-

face temperature generated during irradiation and thus, the

smaller the required laser threshold.33 Relative to larger

NPs, smaller NPs provide greater desorption/ionization ef-

ficiency leading to higher sensitivity. An elevated MS

background over the m/z region from 0 to 3500 was ob-

served when using larger-sized Au NPs (Fig. 2). On the

other hand, smaller NPs having large surface energy can

tend to aggregate and adsorb on a vial surface.34 Based on

our experience, we suggest that 14-nm Au NPs are the opti-

mal substrates.20 Relative to the size, the shape of the NPs

plays a lesser role in determining the sensitivity of SALDI

MS when using UV–VIS lasers.

770 J. Chin. Chem. Soc., Vol. 58, No. 6, 2011 Chen et al.

Fig. 1. Scheme of SALDI MS. A rapid laser-induced

heating of nanoparticles leads to the laser

desorption of analytes from their surface and

ionization.

Selection of NPs

It is well known that various NPs and microspheres,

owing their great surface area, were found to be applicable

as efficient solid phase extraction and concentration probes

for numerous analytes from complicated biological mix-

tures, including trace components. Selective enrichment

and concentration with NPs reduces signal suppression

from multiple analytes, improves sensitivity and can be di-

rectly combined with MALDI MS through simple mixing

the extracted analyte with organic matrices prior to the MS

analysis.35-38

Moreover, when employing suitable NPs, the extract

(target analyte with NPs) can be directly analyzed through

SALDI MS without any further treatment. Nanomaterials

have immense potential for the one-step extraction, con-

centration and detection of diverse analytes, because of the

wide variety of NPs and the possibility of their surface

modification with numerous functional groups. For in-

stance, for thiol-containing molecules Au NPs can be ap-

plied because of the specific Au-S interaction,20 TiO2 NPs

with a high affinity to enediol compounds were used for the

determination of catecholamines13 or for the detection of

alkanes or waxes, the Ag NPs can be useful due to the low

reactivity of silver with alkanes and thus the generation of

silver adducts with no analyte fragmentation.39,40

Two studies comparing a range of nanomaterials for

various analytes have been published in recent years –

Yonezawa et al. investigated the applications of bare metal

NPs (Ag, Au, Cu and Pt) for the SALDI MS of peptides.41

Using a model sample, angiotensin I, the highest perfor-

mance of Pt NPs was demonstrated, whereas the analyte

was not detected with Ag NPs or Cu NPs. Chiang et al.

studied six different nanomaterials (Au NPs, Fe3O4 NPs,

TiO2 NPs, Se NPs, CdTe quantum dots and Pt nano-

sponges) for their applicability in the analyses of small

analytes, peptides and proteins.42 Au NPs were reported as

the most suitable substrate for small analytes; for larger

molecules, the Fe2O3 and Pt nanosponges were demon-

strated as the most efficient substrate allowing the detec-

tion of molecules up to 25 kDa. To increase the specificity

toward an analyte, NP surface modification or conjugation

with recognition biomolecules, such as aptamers, can be

employed.9,43 Modified NPs were reported to provide a

higher ionization efficiency and reduced analyte ion frag-

mentation than the unmodified ones in many cases.19,44-46

Sample preparation

Similar to MALDI MS practice, the samples for

SALDI MS can be prepared in several ways: The analyte

can be mixed with NPs and a suitable buffer prior deposi-

tion (dry droplet method),47 loaded directly onto a plate

coated with single- or multi-layer NPs or having a nano-

structured surface (matrix-first method)48 or deposited first

and subsequently covered by NPs (sample-first method).18

The deposition methods were investigated by Wu et al. for

the analysis of �-cyclodextrins with bare Au NPs.18 The

sample-first method was presented as the most efficient not

only for �-cyclodextrins but also for the determination of

biomolecules as neutral steroids or carbohydrates in high-

salt solutions without need for diluting prior analysis.49

Analysis of Biomolecules through SALDI MS J. Chin. Chem. Soc., Vol. 58, No. 6, 2011 771

Fig. 2. a) Schematic representation of the determination of aminothiols with SALDI MS with MPG-Au NPs as the internal

standard and Au NPs as the matrix. b) Relative standard deviation of [aminothiol + Na]+ ions for the glutathione, 20

�M captured by 14-nm Au NPs (15 nM) in the absence (�) or presence (�) of MPG–Au NPs from 15 different sam-

ple spots. Reprinted with permission from ref. 47.

Usually, a sample diluting or washing step is required be-

fore analysis to avoid interference and signal suppres-

sion.34 In many cases, the nanoparticles (Au NPs, for exam-

ple) only assist in the desorption process and lack the capa-

bility to ionize analytes. Therefore, ammonium citrate is

usually added as a common proton source.14 The presence

of ammonium citrate reduces the formation of multiple

sodiated adducts, but causes interference in the mass spec-

tra. On the other hand, the functional groups of NPs can act

as the ionization agent as demonstrated by Taira et al.50

Manganese oxide magnetic NPs conjugated with hydroxyl

and amino groups were presented as efficient desorption/

ionization agents without the addition of any external

proton donor.

For MSI, reviews were published about the prepara-

tion of samples with traditional organic matrices.51,52 Sev-

eral methods, such as droplet deposition, electrospray de-

position, spraying or sublimation have been developed in

order to achieve high homogeneity and minimize the ana-

lyte migration. When replacing the organic matrices with

NPs, the colloidal dispersion is usually deposited by spray-

ing onto tissue samples. In order to achieve the highest pos-

sible homogeneity of samples with NPs, the concentration

of NPs, the deposition time, as well as the distance between

the nozzle and sample target require further optimiza-

tion.26,53

Sensitivity and reproducibility

Whereas the organic matrices are mixed with the

analyte to a large excess (e.g., 103–105 matrix/analyte),

NPs allow more molecules to be adsorbed on their surfaces

(e.g., ca. 107–109 analytes per single Au NP);32 thus, more

analytes can desorb and ionize from the NP surfaces per la-

ser shot, theoretically leading to greater sensitivity.

Actually, the sensitivity of SALDI MS is usually at

the micromolar (picomole) level — not better than that of

MALDI MS — primarily because of the relatively poor

desorption and ionization efficiency. One easy approach to

improve the sensitivity of SALDI MS is to use NPs as cap-

ture and concentration probes. For example, SALDI MS

performed without and with preconcentration of the ana-

lyte decreased the limits of detection (LOD) from 1.0 �M

to 25 nM, respectively, for glutathione.20 As mentioned

above, another advantage of SALDI over MALDI is the

high sample homogeneity, which eventually leads to im-

proved reproducibility. Unlike organic matrices, NPs do

not form crystals, but simply allow the preparation of

more-homogeneous samples through adsorption of the

analytes onto their surface. In addition, during laser irradia-

tion, only a few peaks associated with clusters from NPs

appear in the low-mass region, whereas many strong peaks

appear from the fragments originated from organic matri-

ces.20,47,54

The quantitative SALDI MS analyses without any

standard are routinely performed, the intensity of the ana-

lyte signal varies usually within less than 15% from spot-

to-spot.13,55 Using an internal standard, Chiang et al. im-

proved the accuracy of SALDI MS analysis to RSD < 10%

(for inter-day measurements).47 For the determination of

glutathione (GSH) with Au NPs he used the N-2-mercapto-

propionylglycine (MPG) as the standard (Fig. 2). Similarly,

4-mercaptobenzoic acid (MBA)–modified Au NPs were

applied as an internal standard for the determination of the

concentration of captopril (CAP) through SALDI MS mea-

surement in the negative ion mode.54 The spot-to-spot vari-

ations in the absence and presence of the internal standard

(n = 15) were 26 and 9%, respectively. The main advantage

of the quantitation through SALDI MS with NPs consists

of its simple sample preparation, low-cost and fast analyses

without the need for any isotopically labeled standards or

eluents; on the other hand the quantitation restricts the rela-

tively low dynamic range, usually not higher than two or

three orders of magnitude.

APPLICATIONS OF NPS IN SALDI-MS

With its advantages of simple sample preparation,

high sensitivity and high reproducibility, SALDI MS has

become an important analytical tool for the analysis of bio-

logical samples. The main shortfall of SALDI MS is its

limit to the low MS region in most cases; of course it de-

pends on the nature of the NPs and their interactions with

the analytes.

Peptides, proteins and amino acids

The first report of the use of nanoparticles as matrixes

for SALDI MS without the use of low-vapor liquid was

published by McLean et al.32 Au NPs of different sizes (2, 5

and 10 nm) were used for peptide systems, phosphopep-

tides and small proteins, where the smallest NPs showed

the best performance. Castellana et al. furthermore re-

ported higher selectivity and an improvement in the analyte

ion yields and a decrease in ion fragmentation when modi-

772 J. Chin. Chem. Soc., Vol. 58, No. 6, 2011 Chen et al.

fying the Au NPs with 4-aminophenol.45

Because of the strong Au–S bonding (418 kJ/mol),

Nile Red–adsorbed Au NPs were selected as probes by

Huang et al. for the determination of aminothiols through

SALDI MS.20 In the absence and presence of a concentra-

tion process, the LODs of the SALDI MS method for GSH

at a signal-to-noise ratio of 3 were 1.0 �M and 25 nM, re-

spectively. Chiang et al. further improved the limit of de-

tection of GSH to 2 nM when using mixtures of two differ-

ently sized (3.5 and 14 nm) Au NPs and validated this

method through analyses of GSH in cell lysates and cysteine

in plasma.34 The selective capture and determination of

biothiols and sulfur drugs in urine was also demonstrated

through SALDI MS and atmospheric pressure-MALDI ion

trap MS with silver NPs capped with various functional

groups.56 Hua et al. demonstrated SALDI MS of peptides

with Ag NPs.57 Compared to the MALDI MS analysis with

�-cyano-4-hydroxy-cinnamic acid (CHCA), the peptides

were detected in the presence of sodium dodecyl sulfate

with reduced interference of background ions originating

from the detergent. Chen et al. applied SALDI-MS using

silanized Fe3O4 NPs for the analyses of peptides and pro-

teins.14 Coating of each Fe3O4 NP with a shell of SiO2 mini-

mized the leakage of Fe ions, which can cause interference

during analysis. Moreover, these silanized Fe3O4 NPs pos-

sessing negatively charged functionalities were used to se-

lectively capture oppositely charged peptides from sample

solutions. Taira et al. presented manganese oxide magnetic

NPs functionalized with amino and hydroxyl groups as an

efficient probe for desorption/ionization and post-source

decay structural analysis of small proteins, peptides and

drugs.50

Quantum dots (QDs) have unique and fascinating op-

tical properties, including broad absorption spectra, sharp

and symmetrical emission spectra with long Stokes shifts,

high quantum yields and size-dependent emission wave-

lengths, mainly due to quantum confinement effects. Shrivas

et al. demonstrated their applicability for SALDI-MS using

11-mercaptoundecanoic acid (MUA)–modified CdSe QDs

for the detection of proteins.58 Ke et al. investigated three

different surface-modified TiO2 NPs for SALDI MS and

found TiO2-CdS NPs as an efficient matrix for back-

ground-free detection of peptides and proteins with an up-

per detectable limit of 17 kDa.59

A hybrid approach between SALDI and MALDI MS

was presented by Duan et al.46 Au NPs were modified with

CHCA and used for the analysis of protein digests. Com-

pared to the use of the citrate- or cysteamine-capped Au

NPs, CHCA-modified Au NPs provided higher ionization

efficiency, lower analyte fragmentation and cleaner spec-

tra. Carbon-based particles and surfaces have also been

widely used for SALDI MS. Carbon-based substrates offer

diverse types of substrate materials with different proper-

ties, such as thermal hydrophobicity, conductivity, specific

heat capacity or ionization efficiency.60 Micro-sized graph-

ite carbon dispersed in glycerol was first used by Sunner et

al. for the analyses of peptides and small analytes.5 Carbon

nanotubes (CNTs) were applied to sensitive and reproduc-

ible detection of small peptides and proteins by Xu et al.

and Chen et al.61,62 Silicon wafers coated with layer-by-

layer (LBL) films of Au NPs and poly(allylamine hydro-

chloride) (PAHC) have been employed for the SALDI MS

analyses of peptides and environmental pollutants.63 Com-

pared to dispersed Au NPs, the use of LBL films provided

increased sensitivity (sub-femtomole) for angiotensin I and

cytochrome C and a wider analyte mass range (up to 12

kDa).

Because Pt is inert and has a high-melting tempera-

ture, high crystallinity and low thermal conductivity,

highly stable Pt-based nanomaterials absorbing laser light

were reported as useful matrices for SALDI MS analyses.

Kawasaki et al. applied SALDI MS using Pt nanoflowers

for the analyses of various biomolecules, mainly proteins.64

Further, sulfonate group-modified PtFeCu NPs were em-

ployed as selective capture probes for positively charged

proteins as well as efficient laser desorption/ionization

(LDI) agents.65

Chiang et al. employed HgTe nanostructures as a new

matrix for SALDI MS–based analyses of proteins and their

complexes with small analytes.16 The mass limit when us-

ing SALDI MS with HgTe nanostructures as matrices

reached as high as 150 kDa. The applicability of this

method provided a LOD of 200 pM for angiotensin I and

was validated through the detections of recombinant pro-

teins transformed in E. coli, a specific complex between

bovine serum albumin (BSA) and L-tryptophan and a car-

bonic anhydrase–acetazolamide complex (Fig. 3).

Other analytes

Besides the proteins and peptides, SALDI MS with

NPs has become popular for the fast analyses of other mol-

ecules such as carbohydrates, lipids, metabolites, drugs or

Analysis of Biomolecules through SALDI MS J. Chin. Chem. Soc., Vol. 58, No. 6, 2011 773

polymers.

Selective ionization of olefinic compounds, for ex-

ample cholesterol or carotenoids, directly from complex

mixtures by Ag NPs was described by Sherrod et al.66 Su et

al. analyzed and quantified small neutral carbohydrates in

urine through SALDI MS using Au NPs capped with vari-

ous agents.55 Lee et al. found that TiO2 NPs having diame-

ters of less than 20 nm interacted with enediol compounds,

resulting in increases of absorption in the UV–Vis region.13

The SALDI MS approach provided LODs on a micromolar

level for several catechins and was applied to determine the

concentration of EGC and EGCG in tea samples. Chiu et al.

demonstrated and tested on real urine samples for the deter-

mination of three estrogens – estrone (E1), estradiol (E2),

and estriol (E3) – with SALDI MS using Ag NPs (34 ± 3

nm).11 NPs featuring anisotropic shapes are also attractive

matrices for the analysis of small analytes with SALDI MS.

For example, Watanabe et al. applied SALDI MS using

ZnO NPs with cubic or rectangular parallelepiped shapes

for the detection of verapamil hydrochloride, testosterone,

phospholipids, oligosaccharides and synthetic polymers.67

(ZnS)-semiconductor NPs capped with different functional

groups were investigated for the analysis of cyclodextrins

and small proteins.68 The best efficiency was exhibited

when using NPs 3-mercaptopropanoic acid as a matrix.

Cysteine capped ZnS NPs doped with Mn2+ were further

developed by this group for the detection of coccidiostats

and peptide mixtures.69 Amini et al. employed oxidized

graphitized carbon black (GCB) particles for the analysis

of pharmaceutical compounds.70 The advantages of using

GCB materials rather than CNTs are their low cost and

greater content of carboxylic acid groups on their surface,

thereby enhancing the efficiency of the ionization process

and the desorption of hydrophobic compounds.

Coatings of nanostructured diamond-like carbon on

digital versatile disks have been employed as targets for the

SALDI MS analyses of various analytes, including amino

acids, carbohydrates, lipids, peptides, and other metabo-

lites.71 Tseng et al. presented a hybrid of immobilized silica

and 2,5-dihydrobenzoic acid (DHB) on iron oxide mag-

netic NPs as an efficient material providing superior soft

ionization of a variety of new types of synthetic materials

used for solar cells, light emitting devices, dendrimers and

glycolipids, including analytes with either thermally labile

structures or with poor protonation tendencies.19 Wen et al.

employed SALDI MS using silicon powder (5–50 nm) for

the analysis of several small molecules, including drugs,

pesticides, peptides and acids.12 Single-crystalline EuF3

hollow hexagonal microdisks were reported as an efficient

material for SALDI MS, allowing the analysis of polyeth-

ylene glycols with an upper detectable range of 35 kDa.72

“Advanced” applications

Nanoparticles for SALDI MS have found applica-

tions not only as an effective matrix for the detection and

quantitation of analytes but also for the investigation of re-

actions of nanoparticles with analytes or for structural

characterization of analytes.

Watanabe et al. demonstrated that the use of ZnO NPs

enhances the fragmentation of oligosaccharides in a post-

source decay analysis, producing far more fragments than

MALDI MS analysis did when using the DHB as the ma-

trix.67 Lin et al. provided strong evidence for the chemical

reactions of functionalized Au NPs with analyte.73 Via

SALDI MS, Hg2+ ions were demonstrated to induce 3-mer-

captopropionic acid (MPA)�Au NPs aggregation in the

presence of 2,6-pyridinedicarboxylic acid (PDCA) and

H2O2-induced fluorescence quenching of Au nanodots

774 J. Chin. Chem. Soc., Vol. 58, No. 6, 2011 Chen et al.

Fig. 3. SALDI mass spectra of (a, b) solutions contain-

ing HgTe nanostructures, BSA, and (a) L-tryp-

tophan (L-Y) and (b) D-tryptophan and (c, d)

Triton X-100–protected HgTe nanostructures

and human carbonic anhydrase I (hCAI) in the

(c) presence and (d) absence of acetazolamide

(ACZ). When using HgTe nanostructures as

SALDI matrices, weak protein–ligand interac-

tions can be observed, such as the specific com-

plex formed between BSA and L-Y or the hCAI-

ACZ complex. Reprinted with permission from

ref. 16

modified with 11-mercaptoundecanoic acid (MUA). PDCA-

Hg2+-MPA coordination was proved as responsible for Au

NPs aggregation, while the formation of 11-MUA disulfide

compounds that are released into the bulk solution as re-

sponsible for H2O2-induced fluorescence quenching. In ad-

dition to providing information about the chemical struc-

tures, SALDI MS was also reported as being selective and

sensitive for the quantitation of Hg2+ ions and H2O2.

Mass Spectrometry Imaging

MSI has emerged in the last decade as an effective

tool for simultaneous tracing and visualizing the spatial

distribution of proteins, peptides, drugs and their metabo-

lites in tissues.74,75 Rapid development of MSI methodol-

ogy and its countless applications have become possible

mainly due to the recent introduction of commercial high-

throughput mass spectrometers capable of fast data acqui-

sition and equipped with reliable solid-state UV lasers with

high repetition rates, power density and pulse-to-pulse sta-

bility adequate for MALDI. This approach eliminates the

need for labeling because molecular mass is used as an en-

dogenous label, allowing also detection of post-transla-

tional modifications. Besides the above mentioned disad-

vantages, such as the high background noise in the low

mass range, in MALDI MSI other critical limitations of the

spatial resolution are the size of the organic matrix crystals,

analyte migration and laser focus size. With their confined

size and the absence of the crystallization process, MSI em-

ploying nanoparticles were reported to overcome some of

these obstacles and to be able to achieve cellular resolu-

tion.24 So far, the use of several micro- and nano-materials,

including Ag, Au colloidal graphite or magnetic NPs have

been demonstrated for the analysis of small molecules in

MSI.

Cha et al. used an aerosol spray containing cca. 1 �m

colloidal graphite particles for the direct lipid profiling and

imaging of cerebrosides and sulphatides in rat brain tissue

and flavonoids on different plant surfaces and tissue sec-

tions.25,53 Zhang et al. utilized a similar approach for the

imaging of organic acids, flavonoids and oligosaccharides

in various fruit species.26 Cha et al. also presented the use

of colloidal Ag for MSI of epicuticular wax metabolites on

the surfaces of leaves and flowers.39 Taira et al. achieved

cellular (15 µm) resolution when employing 3.7 nm diame-

ter functionalized iron-based magnetic NPs as SALDI ma-

terial in the analysis of lipids and peptides in rat cerebellum

tissues through MSI.24 The same NPs were used by Ageta

et al. to detect different sulfatide species in rat hippocam-

pus;76 gold NPs functionalized with alkylamine were re-

ported as suitable for the detection of minor glycosphingo-

lipids in mouse brain sections77 and silver NPs function-

alized with alkylcarboxylate and alkylamine for fatty acids

in mouse retinal and liver sections.78

Rowel et al. demonstrated direct detection of drugs

and their metabolites in latent fingerprints (LFP) applying

MSI with a hydrophobic silica dusting agent containing

carbon black.79 LFPs were studied also by Tang et al. who

presented Au NPs as suitable for both their visualization

and molecular MSI.80 This approach enables the conserva-

tion of individual identity and also provides information

about drugs or hazardous substances. In 2011, Tang et al.

also demonstrated solvent-free gold-NP-assisted LDI MSI

as a sensitive and minimally destructive method for the di-

rect detection and imaging of ink and visible and/or fluo-

rescent dyes printed on banknotes or written on questioned

documents.81

An innovative method was introduced by Northen et

al.82 Nanostructure-initiator mass spectrometry (NIMS)

was presented as a tool for spatially confined mass analy-

sis. It uses nanostructured surfaces with trapped ‘initiator’

molecules or ‘clathrates’ to release and ionize intact mole-

cules adsorbed on the surface. This surface responds to

both ion and laser irradiation. The lateral resolution (ion-

NIMS about 150 nm), sensitivity, matrix-free and reduced

fragmentation of NIMS allows direct characterization of

peptide microarrays, direct mass analysis of single cells,

tissue imaging, and direct characterization of blood and

urine. The method and its applications have been re-

viewed.83 Vidova et al. also avoided the time consuming

step of MSI - matrix deposition and used the commercially

available nanostructured surfaces as the substrates for the

imprinting of tissue section for lipid analysis (Fig. 4).22

CONCLUSIONS AND PERSPECTIVES

In this review, we have examined several SALDI MS

approaches using NPs for the detection of biomolecules

and MSI. It is obvious that the nature, the size of the NPs as

well as the sample preparation play a crucial role in deter-

mining the sensitivity of the process.

In contrast to MALDI MS, SALDI MS with NPs can

provide great reproducibility, allowing quantitative deter-

mination of the concentrations of small analytes. For the

Analysis of Biomolecules through SALDI MS J. Chin. Chem. Soc., Vol. 58, No. 6, 2011 775

detection of small analytes (< 500 Da), SALDI MS using

Au NPs is superior to MALDI MS and is applicable to

metabolomics and MSI, mainly because of the absence of

background noise in MS spectra originated from organic

matrices. Functionalized NPs with an affinity towards a tar-

get analyte can be applied for both enrichment and MS

analysis of biomolecules making nanomaterials attractive

in terms of sample treatment and analysis of trace analytes.

In addition, the SALDI approach has been reported as al-

lowing ionization of molecules hardly ionizable by MALDI.

On the other hand, single-crystalline EuF3 hollow

hexagonal microdisks and HgTe nanostructures are all ef-

fective substrates for the detection of molecules having

values of m/z of up to 35 and 150 kDa, respectively. The

SALDI MS approach using HgTe nanostructures also al-

lows the detection of weak complexes of proteins with

small analytes revealing its potential for investigating the

complexes of proteins with other proteins and DNA. Their

toxicity is a concern though. The further development of

new nanomaterials allowing the selective capture of larger

proteins and their more efficient desorption/ionization is

essential. A combination of SALDI MS with further analyt-

ical methods is expected over the coming years as well as

more studies of the desorption-ionization process which

has not yet been fully explained and is essential for further

developments.

MSI performed in conjunction with NPs provides

better spatial resolution mainly due to the absence of the

matrix-analyte co-crystallization process and no analyte

migration. New approaches employing nanostructured sur-

faces avoid the matrix deposition steps and therefore make

the MSI analyses significantly easier and faster. We note

that the number of studies dealing with MSI and nanomate-

rials remains very low and more application and method-

ological studies are needed for overall conclusions.

ACKNOWLEDGEMENTS

This study was supported by the National Science

Council of Taiwan under contracts NSC 98-2113-M-002-

011-MY3 and NSC 99-2923-M-002-004-MY3, the Czech

Science Foundation P206/10/J012, the Ministry of Educa-

tion, Youth and Sports of the Czech Republic

MSM0021622415 and CZ.1.05/1.1.00/02.0068. Iva

Tomalová is supported by Brno City Municipality Scholar-

ships for Talented Ph.D. Students.

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