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Citation: Fuentes-Cervantes, A.; Ruiz
Allica, J.; Calderón Celis, F.;
Costa-Fernández, J.M.; Ruiz Encinar,
J. The Potential of ICP-MS as a
Complementary Tool in
Nanoparticle–Protein Corona
Analysis. Nanomaterials 2023,13, 1132.
https://doi.org/10.3390/nano13061132
Academic Editor: Jose M. Palomo
Received: 16 February 2023
Revised: 8 March 2023
Accepted: 21 March 2023
Published: 22 March 2023
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
nanomaterials
Review
The Potential of ICP-MS as a Complementary Tool in
Nanoparticle–Protein Corona Analysis
Ana Fuentes-Cervantes, Julia Ruiz Allica, Francisco Calderón Celis * , JoséM. Costa-Fernández
and Jorge Ruiz Encinar
Department of Physical and Analytical Chemistry, University of Oviedo, Avda. Julián Clavería 8,
33006 Oviedo, Spain
*Correspondence: calderonfrancisco@uniovi.es
Abstract:
The prolific applicability of nanomaterials has made them a common citizen in biological
systems, where they interact with proteins forming a biological corona complex. These complexes
drive the interaction of nanomaterials with and within the cells, bringing forward numerous potential
applications in nanobiomedicine, but also arising toxicological issues and concerns. Proper character-
ization of the protein corona complex is a great challenge typically handled with the combination
of several techniques. Surprisingly, despite inductively coupled plasma mass spectrometry (ICP-
MS) being a powerful quantitative technique whose application in nanomaterials characterization
and quantification has been consolidated in the last decade, its application to nanoparticle–protein
corona studies is scarce. Furthermore, in the last decades, ICP-MS has experienced a turning point
in its capabilities for protein quantification through sulfur detection, hence becoming a generic
quantitative detector. In this regard, we would like to introduce the potential of ICP-MS in the
nanoparticle protein corona complex characterization and quantification complementary to current
methods and protocols.
Keywords: protein corona; ICP-MS; nanomaterials; proteins; nanoparticles
1. The Context of the Protein Corona
1.1. The Protein Corona Formation
Nanotechnology has experienced enormous growth in recent decades, bringing for-
ward the surging use of engineered nanoparticles as advantageous novel materials in
numerous fields such as biomedicine, cosmetics, pharmacology, food, or agriculture [
1
,
2
].
Its unrestrained used has resulted in the ubiquity of these nanomaterials in the environ-
ment [
3
] and biological systems [
4
], and thus nanoparticles may potentially be inhaled,
ingested, or taken up through the skin into the body [
5
]. Then, when nanoparticles come in
contact with biological fluids, they become covered by a complex layer of biomolecules,
such as proteins, lipids, or sugars, forming a sort of “bio-corona” [
6
,
7
]. Within the biolog-
ical corona complexes formed in biological systems, the predominant and most studied
molecules are proteins, which on their own form the so-called nanoparticle protein corona
complex (NPPC) [
5
,
8
]. The protein corona defines the biological identity of the nanoparticle,
the NPPC being the entity that finally interacts with and is “seen” or recognized by the
cell [6].
Upon introduction in a biological fluid, the process of protein adsorption is an almost
instantaneous event given the higher binding energy of the nanoparticle surface compared
to the surrounding biological environment (Gibbs free energy drives the complex stabil-
ity) [
9
,
10
]. The formation of the corona, driven by non-covalent forces, is dynamic and
changes over time because of continuous association and dissociation processes given
the different affinity of the proteins. Initially, higher concentration proteins will interact
with the nanoparticle, but will eventually be displaced by higher-affinity proteins until
Nanomaterials 2023,13, 1132. https://doi.org/10.3390/nano13061132 https://www.mdpi.com/journal/nanomaterials
Nanomaterials 2023,13, 1132 2 of 16
reaching an equilibrium, when protein exchanges would not affect the composition of
the corona [
6
,
9
,
11
,
12
]. In fact, although the formation of the corona takes place over the
period of an hour, occurring minor changes for around 12 h [
13
], plasma proteins have
been found in the corona already within the first minute of exposure [
14
]. Interestingly,
the composition of the corona at such early stages does not significantly change over time
in protein identities, but in quantities [
14
]. The dynamic nature of these phenomena can
be described by using the “hard” and “soft” corona concepts. Those proteins with high
affinity that form the closest layer to the nanoparticle surface are called the hard corona.
This constitutes a tight, strong, nanoparticle–protein binding that is highly stable of very
rapid formation. Low-affinity proteins forming an external layer, which are not bound
to the nanoparticle surface but have a certain degree of interactions, are called the soft
corona [
4
,
6
,
9
] (Figure 1). It takes more time to constitute, and it is more unstable and highly
dynamic, being more complex in its study.
Nanomaterials 2023, 13, x FOR PEER REVIEW 2 of 16
the dierent anity of the proteins. Initially, higher concentration proteins will interact
with the nanoparticle, but will eventually be displaced by higher-anity proteins until
reaching an equilibrium, when protein exchanges would not aect the composition of the
corona [6,9,11,12]. In fact, although the formation of the corona takes place over the period
of an hour, occurring minor changes for around 12 h [13], plasma proteins have been
found in the corona already within the rst minute of exposure [14]. Interestingly, the
composition of the corona at such early stages does not signicantly change over time in
protein identities, but in quantities [14]. The dynamic nature of these phenomena can be
described by using the “hard” and “soft” corona concepts. Those proteins with high an-
ity that form the closest layer to the nanoparticle surface are called the hard corona. This
constitutes a tight, strong, nanoparticle–protein binding that is highly stable of very rapid
formation. Low-anity proteins forming an external layer, which are not bound to the
nanoparticle surface but have a certain degree of interactions, are called the soft corona
[4,6,9] (Figure 1). It takes more time to constitute, and it is more unstable and highly dy-
namic, being more complex in its study.
Figure 1. Scheme representation of the dynamic formation of nanoparticle–protein corona complex.
Proteins in the inner layer have high anity for the nanoparticle surface and form the hard corona.
The proteins in the outer layer form the soft corona, which shows dynamic changes due to ex-
changes with free proteins in the environment.
Nanomaterial interactions with proteins can be controlled by dierent strategies, na-
noparticle surface functionalization being the most common one [15]. This is because pro-
tein corona formation is a process commonly considered problematic, and thus great ef-
fort is being invested by the scientic community on designing and engineering coatings
that minimize protein adsorption on nanomaterial surfaces as much as possible [16].
1.2. The Biological Impact of the Protein Corona
The interactions between nanoparticles and proteins have signicant biological con-
sequences to the original nanoparticle and to the native proteic environment. On the one
hand, the formation of the protein corona on the nanoparticle surface changes the nano-
particle physicochemical properties. It changes the size, shape, and even the aggregation
state of the nanoparticle, which in turn aects how proteins are oriented and presented to
the biological targets [17]. On the other hand, interactions with nanoparticles may trigger
protein aggregation or conformational changes, which may impact the protein functions
and their interaction with other biomolecules, and may even result in an immune response
to eliminate the circulating nanoparticles. In this regard, changes in protein stability and
enzyme activity have been observed when immobilized on the nanoparticle surface [6].
Upon entrance in the biological media, nanoparticles are involved in a myriad of bi-
ological processes. The formation of the protein corona creates a dierent structure that
will drive and inuence the behavior and interaction of the nanoparticles with biological
systems. Thus, the protein corona can alter the interactions of the nanoparticle with the
cell surface moieties (e.g., antibodies), promoting or inhibiting the uptake of the nanopar-
ticle by the cell [18]. For instance, in a case of study with silica nanoparticle, it was ob-
served that the formation of the protein corona reduced the cell uptake by weakening their
NP
Hard corona
NP
Adsorption Adsoption/Desorption until stable
NP
Soft corona
Figure 1.
Scheme representation of the dynamic formation of nanoparticle–protein corona complex.
Proteins in the inner layer have high affinity for the nanoparticle surface and form the hard corona.
The proteins in the outer layer form the soft corona, which shows dynamic changes due to exchanges
with free proteins in the environment.
Nanomaterial interactions with proteins can be controlled by different strategies,
nanoparticle surface functionalization being the most common one [
15
]. This is because
protein corona formation is a process commonly considered problematic, and thus great
effort is being invested by the scientific community on designing and engineering coatings
that minimize protein adsorption on nanomaterial surfaces as much as possible [16].
1.2. The Biological Impact of the Protein Corona
The interactions between nanoparticles and proteins have significant biological con-
sequences to the original nanoparticle and to the native proteic environment. On the one
hand, the formation of the protein corona on the nanoparticle surface changes the nanopar-
ticle physicochemical properties. It changes the size, shape, and even the aggregation state
of the nanoparticle, which in turn affects how proteins are oriented and presented to the
biological targets [
17
]. On the other hand, interactions with nanoparticles may trigger
protein aggregation or conformational changes, which may impact the protein functions
and their interaction with other biomolecules, and may even result in an immune response
to eliminate the circulating nanoparticles. In this regard, changes in protein stability and
enzyme activity have been observed when immobilized on the nanoparticle surface [6].
Upon entrance in the biological media, nanoparticles are involved in a myriad of
biological processes. The formation of the protein corona creates a different structure that
will drive and influence the behavior and interaction of the nanoparticles with biological
systems. Thus, the protein corona can alter the interactions of the nanoparticle with the cell
surface moieties (e.g., antibodies), promoting or inhibiting the uptake of the nanoparticle
by the cell [
18
]. For instance, in a case of study with silica nanoparticle, it was observed
that the formation of the protein corona reduced the cell uptake by weakening their cell
membrane adhesion [
19
]. The protein corona also plays an important role in targeting the
nanoparticles to different organs [13].
Nanomaterials 2023,13, 1132 3 of 16
The biological impact of the nanoparticle interactions with proteins is particularly
relevant in the development and use of the NPPC in biomedicine. The small size of
nanoparticles gives them the ability to efficiently access cell compartments, which has
important biomedical implications, leading to important developments in the field of
nanomedicine [
20
,
21
]. The feature of the protein corona, which can direct the nanoparticles
to specific organs or locations, can be used to design NPPC that can be placed anywhere
desired in the organism or in the cell. This final location will then determine the effect of the
NPPC and the pathways or processes it gets involved in, disrupts, or promotes. Likewise,
it can be used as a vehicle for drug delivery [
18
], or for the promoted accumulation of
nanoparticles in specific areas, e.g., tumors, and serve as a biomarker or therapeutical
target [
9
]. Naturally, the presence of nanoparticles in biological systems and their use in
biomedicine brings a potential safety and toxicological risk. Even for nanoparticles without
the protein corona, cellular uptake is related to cytotoxicity, being affected by properties like
colloidal stability or nanoparticle surface charge. In fact, positively charged nanoparticles
tend to have higher cellular uptake as well as a higher cytotoxicity than negatively charged
nanoparticles [22].
2. Traditional Approaches to Study and Characterize the Nanoparticle–Protein
Corona Complex
Protein–nanoparticle interactions are highly determined, not only by the environment
composition and dynamics, but also by the nature and structure of the nanoparticles. There-
fore, characterization of the starting nanoparticle is a key issue that must be investigated
and understood before affording the study of the protein corona.
2.1. Nanoparticle Characterization
Nanoparticles’ physicochemical properties control the formation and composition
of the protein corona. Whereas the extent of the impact of each property on the protein
corona composition and disposition is not completely understood, there is an extended
consensus that no nanoparticle property alone drives the formation of the corona, but all of
them together [
23
]. Nanoparticle protein adsorptions are mainly driven by non-covalent
forces, affinity constants of proteins and protein structure thermodynamics [
24
]. Thus,
the nanoparticle surface constitutes a major factor influencing the protein corona [
25
]. In
contrast to the previously commented direct influence of nanoparticle surface charge in
the toxicity and biodistribution of nanoparticles, this parameter does not seem to be so
influential on the formation and eventual biological effect of NPPC [22,25].
The hydrophobicity of a nanoparticle’s surface is, on the contrary, a significant factor
limiting the amount and identity of the proteins bound to the nanoparticle. In this regard,
for instance, Lynch et al. found that when plasma is incubated with low hydrophobic
copolymer particles, virtually no protein is retrieved, while significant amounts of protein
are consistently bound to hydrophobic particles [
26
]. Nanoparticle surface chemistry is
also a driving parameter in the formation of the characteristics of the protein corona, as
it controls the number of possible binding sites [
12
]. Nanoparticles commonly need to be
functionalized with ligands and biomolecules that affect their solubility, stability, and define
and control their biocompatibility and biological interactions [
27
]. This functionalization
eventually affects the formation of the protein corona (because it largely confers a stealth
character on the nanoparticle, protecting it and making it inaccessible to proteins) and could
be crucial when developing nanoparticles with biological or therapeutical applications [
28
].
Walkey et al. observed that differently charged ligands have more adsorption capabilities
than neutral ligands [
17
], whereas Galmarini et al. observed a higher number of proteins
forming the corona when nanoparticles were initially functionalized with citrates rather
than with DMSA, stating the influence of the nanoparticle functionalization on the corona
protein composition [
25
]. Despite the clear relevance of the adequate characterization of
nanoparticle functionalization, there are yet no standard methods available to determine
nanoparticle/surface biomolecule ratios [
29
]. The surface charge of nanoparticles also
Nanomaterials 2023,13, 1132 4 of 16
seems to be a key parameter affecting nanoparticle–protein interactions. Indeed, Lundquist
et al. studied the PC composition formed in three different polystyrene particles: with
positive (amine-modified), neutral (the plain unmodified particles), or negative (carboxyl-
modified) charges. Remarkably, they found a certain dependence of the resultant protein
corona depending on surface charge and zeta potential, and they were able to identify
adsorption patterns and similarities [30].
Interestingly, several works have found that nanoparticle size seems to be more
relevant than their functionalization in the formation of the protein corona. Tenzer et al., for
instance, determined nanoparticle size to be dominant in the formation of the protein corona
over surface functionalization, which in turn was dominant over exposure time [
14
]. When
comparing nanoparticles of different sizes with the same functionalization, Walkey et al.
observed that the steric hindrances formed in larger nanoparticles hindered the adsorption
of proteins, which was more favored in smaller ones [
17
]. Nanoparticle shape is a key
parameter that has, for some reasons, been disregarded in protein corona investigations.
However, different studies evidenced that protein corona formation is also dependent on
nanoparticle morphology. As an example, Deng et al. found that protein binding rate is
different for TiO
2
, SiO
2
, and Zn nanoparticles; TiO
2
nanorods; and TiO
2
nanotubes, despite
these nanoparticles having similar surface charges [31].
In this regard, common approaches to study and determine nanoparticle size and size
distribution include dynamic light scattering (DLS) and differential centrifugal sedimenta-
tion (DCS) (Figure 2). DLS is a fast and general-purpose method of nanoparticle dispersions,
and it is of low resolution, though it is limited in terms of sample concentration, small nm
shifts in size, non-spherical particles, or the dynamic range of nanoparticle size [
32
]. In
the case of most precise nanoparticle size studies, more accurate techniques such as DCS
are required.
Nanomaterials 2023, 13, x FOR PEER REVIEW 4 of 16
functionalization on the corona protein composition [25]. Despite the clear relevance of
the adequate characterization of nanoparticle functionalization, there are yet no standard
methods available to determine nanoparticle/surface biomolecule ratios [29]. The surface
charge of nanoparticles also seems to be a key parameter aecting nanoparticle–protein
interactions. Indeed, Lundquist et al. studied the PC composition formed in three dierent
polystyrene particles: with positive (amine-modied), neutral (the plain unmodied par-
ticles), or negative (carboxyl-modied) charges. Remarkably, they found a certain de-
pendence of the resultant protein corona depending on surface charge and zeta potential,
and they were able to identify adsorption paerns and similarities [30].
Interestingly, several works have found that nanoparticle size seems to be more rel-
evant than their functionalization in the formation of the protein corona. Tenzer et al., for
instance, determined nanoparticle size to be dominant in the formation of the protein co-
rona over surface functionalization, which in turn was dominant over exposure time [14].
When comparing nanoparticles of dierent sizes with the same functionalization, Walkey
et al. observed that the steric hindrances formed in larger nanoparticles hindered the ad-
sorption of proteins, which was more favored in smaller ones [17]. Nanoparticle shape is
a key parameter that has, for some reasons, been disregarded in protein corona investiga-
tions. However, dierent studies evidenced that protein corona formation is also depend-
ent on nanoparticle morphology. As an example, Deng et al. found that protein binding
rate is dierent for TiO2, SiO2, and Zn nanoparticles; TiO2 nanorods; and TiO2 nanotubes,
despite these nanoparticles having similar surface charges [31].
In this regard, common approaches to study and determine nanoparticle size and
size distribution include dynamic light scaering (DLS) and dierential centrifugal sedi-
mentation (DCS) (Figure 2). DLS is a fast and general-purpose method of nanoparticle
dispersions, and it is of low resolution, though it is limited in terms of sample concentra-
tion, small nm shifts in size, non-spherical particles, or the dynamic range of nanoparticle
size [32]. In the case of most precise nanoparticle size studies, more accurate techniques
such as DCS are required.
Figure 2. Common methods used to characterize nanoparticle–protein corona complexes.
In contrast to DLS, DCS allows the resolution of multiple peaks in polydisperse sam-
ples as well as the possibility to carry out the analysis even in the presence of large bio-
molecules, as in the case of protein corona studies [23]. Transmission electron microscopy
NP SURFACE CHARACTERIZATION
NP-PC INTERACTIONS
PROTEIN CORONA COMPOSITION
NP POLYDISPERSITY
Isolation
–Centrifugation
–Magnetic separation
–Field Flow fractionation
–Size exclusion chromatography
–SDS-PAGE
–Capillary electrophoresis
–Dynamic light scattering
–Size exclusion chromatography
–Transmission electron microscopy
–Differential centrifugal sedimentation
–UV-Vis
–Dynamic light scattering
–Size exclusion chromatography
–Transmission electron microscopy
–Zeta-potential
–Fourier transform infrared
–Nuclear magnetic resonance
Identification
–Mass spectrometry
–Circular dichroism
–SDS-PAGE
Quantification
–Colorimetric assays
–Bradford
–Mass spectrometry Morphology
–Transmission electron microscopy
Composition
–Nuclear magnetic resonance
–Fourier transform resonance
–Isothermal titration calorimetry
Stoichiometry and affinity
–Isothermal titration calorimetry
–SDS-PAGE
Protein dissociation and association
–Surface plasmon resonance
–Size Exclusion Chromatography
–Nuclear magnetic resonance
–Capillary electrophoresis
PC conformation and dynamics
–Fourier transform infrared
NP CORE CHARACTERIZATION
NP SIZE
Figure 2. Common methods used to characterize nanoparticle–protein corona complexes.
Nanomaterials 2023,13, 1132 5 of 16
In contrast to DLS, DCS allows the resolution of multiple peaks in polydisperse
samples as well as the possibility to carry out the analysis even in the presence of large
biomolecules, as in the case of protein corona studies [
23
]. Transmission electron microscopy
(TEM) is routinely used to determine nanoparticle size, shape, and size distribution. In
fact, it can also provide information on nanoparticle aggregation, as well as morphology.
This is particularly relevant because, as observed by Nandakumar et al. when they studied
the protein corona formation on model AuNPs, the AuNP morphology conditioned the
identity of the proteins forming the protein corona based on their size and structure [
33
].
Interestingly, different morphologies resulted in differences in both the identities and the
quantitates of the proteins forming the corona. In brief, it is therefore fundamental to
control nanoparticle size, shape, and surface properties to control nanoparticle behavior
and produce NPPCs in a systematic and reproducible way (see Figure 2).
2.2. Protein Corona Composition
When nanoparticles enter biological media, the number of proteins they may get in
contact with is in the thousands. However, they cannot all fit onto the nanoparticle surface:
typically, less than 100 proteins form the hard corona in nanoparticles of >10 nm [
34
,
35
],
resulting in the possibility of subpopulations of a certain nanoparticle with different protein
coronas, potentially showing different
in vivo
behavior [
11
]. The impact of the environment
in the protein corona composition is not limited to the upfront formation of the corona,
but when the NPPC changes its location, the composition of the corona is affected by the
new environment, leading to changes in the identity and quantity of the proteins that
formed the hard corona. Protein composition variation is even more pronounced in the soft
corona, as they have lower and more variable resident times. Therefore, characterization
and understanding the size and composition of the protein corona is critical to predicting
the behavior of the NPPC in biological systems, and to design nanoparticles with specific
properties, such as improved biocompatibility or targeted drug delivery. In this regard, the
identification of both major and minor proteins that form the corona is essential, as is the
study of their abundance and their affinity, e.g., to study their competition to bind when
the system is under kinetic or thermodynamic influence, or understanding the NPPC fate
in biological systems or role in biomedicine [8,18].
It should be stressed that identification of the different proteins is not enough to obtain
a comprehensive characterization of the protein corona. The dynamism in the protein
corona composition (particularly the soft corona), as well as the reasonable assumption
that the most abundant proteins will define the biological roles of the NPPC, demands
quantitative data in order to achieve a comprehensive characterization. Additionally, con-
sidering that protein corona composition is driven by the affinity and interactions between
nanoparticle and proteins, there is not necessarily a correlation in protein abundance in
the media and in the corona [
17
,
18
]. Because of these challenges that entail the study of
NPPCs in biological systems, unbound proteins present in the media must be removed
prior to the proteomic study of the corona, without affecting or altering the NPPC com-
position. The most common methods used to isolate protein corona-coated nanoparticles
include centrifugation, size exclusion chromatography, magnetic separation, and field-flow
fractionation [
36
]. In this regard, purification and isolation methods such as centrifugation
or chromatography are usually preferred to separate proteins from complex mixtures and
plasma. It must be noted though that these purification processes may also result in the
loss of the weakly bound proteins forming the soft corona [37].
Quantification of proteins forming the corona can be performed through standard
approaches such as colorimetric assays, such as BCA, Bradford or Lowry [38]. These tech-
niques are based on the complexation of the protein with metal ions or dyes (respectively),
leading to a color change calibrated with a reference standard protein such as BSA. These
approaches provide information regarding the total protein amount present in the samples
but are limited because of potential reactivity differences between calibrant and the assayed
protein, leading to errors in protein estimation. Gel electrophoresis has also been widely
Nanomaterials 2023,13, 1132 6 of 16
employed for the separation of proteins of the corona from the nanoparticles, and their
identification and classification by molecular weight. Alternatively, Benetti et al. assayed
different detachment approaches, such as using SDS or isopropyl alcohol and NaOH, but
none of them were able to fully detach the complete amount of proteins from the nanopar-
ticle surface [
39
]. Thus, there are some parts of the corona that will not be revealed. This is
why the predominant approach to carrying out the qualitative and quantitative analysis
of the protein corona is mass spectrometry (MS), as proteins are directly digested in the
NPPC, hence it is more efficient than the detachment approaches required for Lowry or
SDS-PAGE assays.
In a typical workflow (Figure 3), before the LC-MS protein corona characterization,
NPPC samples are first washed before their characterization by DLS and TEM to investigate
the homogeneity/heterogeneity of the PC-coated nanoparticles, and SDS-PAGE to study
the protein corona profiles (after separating the proteins from the nanoparticle) [
18
]. Next,
LC-MS/MS is used to characterize the composition of the protein corona (identification of
the different proteins adsorbed onto the nanoparticle) employing proteome approaches.
This study requires the enzymatic digestion of the proteins, either in solution directly to
the NPPC, or in-gel after the SDS-PAGE. Before protein corona characterization by MS
approaches, isolation of the PC-coated nanoparticles must be carried out by size exclusion
chromatography, magnetic separations, field flow fractionation, or centrifugation, the
latter being the most widely used approach. Then, the NPPC is digested and analyzed
with LC-MS. Identification is carried out through sequencing of the proteins following
common proteomics protocols [
7
,
40
]. MS can also provide quantitative protein information,
though regular LC-ESI-MS/MS workflows provide only semiquantitative data by label-free
methods, which estimate protein abundances from the peptide counts and ion intensities
in a single LC-MS/MS analysis. Absolute quantities of the proteins forming the corona
would require specific standardization, which is constricted when addressing the analysis
of a high number of proteins, as is the case in protein corona studies [41].
Nanomaterials 2023, 13, x FOR PEER REVIEW 6 of 16
NaOH, but none of them were able to fully detach the complete amount of proteins from
the nanoparticle surface [39]. Thus, there are some parts of the corona that will not be
revealed. This is why the predominant approach to carrying out the qualitative and quan-
titative analysis of the protein corona is mass spectrometry (MS), as proteins are directly
digested in the NPPC, hence it is more ecient than the detachment approaches required
for Lowry or SDS-PAGE assays.
In a typical workow (Figure 3), before the LC-MS protein corona characterization,
NPPC samples are rst washed before their characterization by DLS and TEM to investi-
gate the homogeneity/heterogeneity of the PC-coated nanoparticles, and SDS-PAGE to
study the protein corona proles (after separating the proteins from the nanoparticle) [18].
Next, LC-MS/MS is used to characterize the composition of the protein corona (identica-
tion of the dierent proteins adsorbed onto the nanoparticle) employing proteome ap-
proaches. This study requires the enzymatic digestion of the proteins, either in solution
directly to the NPPC, or in-gel after the SDS-PAGE. Before protein corona characterization
by MS approaches, isolation of the PC-coated nanoparticles must be carried out by size
exclusion chromatography, magnetic separations, eld ow fractionation, or centrifuga-
tion, the laer being the most widely used approach. Then, the NPPC is digested and
analyzed with LC-MS. Identication is carried out through sequencing of the proteins fol-
lowing common proteomics protocols [7,40]. MS can also provide quantitative protein in-
formation, though regular LC-ESI-MS/MS workows provide only semiquantitative data
by label-free methods, which estimate protein abundances from the peptide counts and
ion intensities in a single LC-MS/MS analysis. Absolute quantities of the proteins forming
the corona would require specic standardization, which is constricted when addressing
the analysis of a high number of proteins, as is the case in protein corona studies [41].
Figure 3. Schematic representation of a typical workow for the synthesis and characterization of
the NPPC.
2.3. Nanoparticle–Protein Corona Interactions
The biological relevance of protein–nanoparticle interaction in the formation of the
protein corona and the relevance and need for a proper characterization of such interac-
tions, which are yet not completely understood, has already been remarked [40]. These
interactions are aected by diverse factors such as protein identities, quantities, anities
and specicities, exchange rates, and redistribution, all these determining the eventual
biological eect of the NPPC. Study and characterization of the protein corona formation
and interaction with the nanoparticle is usually carried out through indirect approaches,
which are based on comparing the nanoparticle before and after the formation of the pro-
tein corona. Then, changes in the nanoparticle properties, such as the size, charge, density,
and mass of spectroscopic features, are analyzed and associated with the protein corona
[37].
SDS-PAGE
NPPC
Corona proteomics characterization
NPPC characterization
Washing
DLSTEM
LC-MS/MS
Tryptic digestion
Figure 3.
Schematic representation of a typical workflow for the synthesis and characterization of the NPPC.
2.3. Nanoparticle–Protein Corona Interactions
The biological relevance of protein–nanoparticle interaction in the formation of the
protein corona and the relevance and need for a proper characterization of such interac-
tions, which are yet not completely understood, has already been remarked [
40
]. These
interactions are affected by diverse factors such as protein identities, quantities, affinities
and specificities, exchange rates, and redistribution, all these determining the eventual
Nanomaterials 2023,13, 1132 7 of 16
biological effect of the NPPC. Study and characterization of the protein corona formation
and interaction with the nanoparticle is usually carried out through indirect approaches,
which are based on comparing the nanoparticle before and after the formation of the protein
corona. Then, changes in the nanoparticle properties, such as the size, charge, density, and
mass of spectroscopic features, are analyzed and associated with the protein corona [37].
The common applicability of DLS or TEM to the characterization of nanomaterials
can also be extended to characterize the NPPC. In the case of DLS, the measurement of
the hydrodynamic diameter of nanoparticles before and after the protein corona formation
provides an estimation of the corona thickness [
23
]. Nevertheless, DLS estimation must
consider the possibility of other potential factors, such as nanoparticle aggregation, affecting
the analysis. TEM can also be used to determine the protein corona formation and size
by assessing nanoparticles size before and after the formation of a protein corona. One of
TEM limitations is the possibility of sample handling and preparation resulting in protein
corona destabilization and degradation. Hence, cryo-electron microscopy (cryo-TEM) is
preferred as it preserves the original state of the protein corona, even making it possible to
discriminate between the soft and hard corona [40].
The study of nanoparticle–protein interactions can also be addressed with separation
techniques such as size exclusion chromatography (SEC) or asymmetric flow field-flow frac-
tionation (AF4). Nanoparticles are too large to be retained in the SEC phase pores, whereas
proteins are not, and their elution time will depend on their molecular weight. Then,
if proteins are associated with the nanoparticles, their retention time will be decreased,
depending on the duration of the interaction [
6
]. This way, SEC can provide information
on the proteins associated with the nanoparticle, which can also be separated from the
non-associated proteins, as well as their association times. Alternatively, zeta-potential
determination before and after the formation of the protein corona can also provide infor-
mation on the thickness and effect of the protein corona on the nanoparticle surface charge.
In this sense, Rampado et al. observed that when nanoparticles are incubated with proteins,
the surface charge value tends toward a value of a few mV below zero, independent from
the nanoparticles’ original charge [42].
3. The Need for Improved Methodologies in Nanoparticle–Protein Corona Analysis
3.1. Lack of Standardization in Nanoparticles Synthesis, Production and Characterization
The need to improve or dispose of more accurate and robust NPPC production and
standard nanoparticle characterization methods is clear, as they would significantly im-
prove reliability, reproducibility, and transparency in biomedicine [
36
]. The use of inade-
quately characterized starting nanoparticles can lead to errors in the subsequent evaluation
of the NPPC biological behavior. Accurate characterization of nanoparticle size distribution
is thus essential to avoid the formation of protein coronas with different composition due
to the initial presence of a wide range of nanoparticle size populations in high polydisperse
samples, or to control potential protein contaminations and the formation of aggregates.
The indirect strategies often used to characterize NPPC imply a great number of steps and
processes that lead to higher variability and potential errors due to nanoparticle alterations
or losses. To minimize this risk, it is important to compare the size distribution of nanopar-
ticles before and after the formation of the protein corona and consider how the size of the
nanomaterial changes on interaction with a biological system.
The current methods to characterize nanoparticles, including DLS, DCS, or TEM, are
highly dependent on instrumental conditions and sample preparation and are seldom
validated, risking the compromise of the results by uncontrolled processes [
43
]. In recent
years, there have been efforts to standardize the characterization of nanomaterials to reduce
this lack of reproducibility and improve the robustness and accuracy of the methodologies.
In the pursuit of this standardization, a group of leading researchers in the analytical
nanotechnology field proposed MIRIBEL, a standardized reporting system to improve
the quality of nanomaterial research and the way it is reported [
44
]. MIRIBEL suggests
including high-quality, reproducible synthesis steps and considering factors such as size
Nanomaterials 2023,13, 1132 8 of 16
and shape, which can affect organ distribution and the adsorption of biomolecules onto
nanomaterials. However, implementing MIRIBEL may be challenging for early-career scien-
tists who lack critical resources and long-term experience in using various characterization
techniques. The lack of standard methods hinders the comparison of different materials
and the evaluation of the applicability of new design alternatives. Furthermore, there is a
growing concern over the increasing nanomedicine products commercially available given
the current lack of such control and standard nanoparticle characterization protocols [32].
3.2. Irreproducibility and Lack of Standardization in LC-MS Protein Analysis
The characterization of protein corona variable composition as a function of nanopar-
ticle physicochemical properties (size, surface charge, morphology, etc.) cannot be assessed
without reproducible and upright LC-MS analyses and datasets. For instance, researchers
examined the interactions between proteins and nanoparticles and the effect of small
changes in surface chemistry on these interactions using SDS-PAGE and LC-MS for protein
separation and quantification, respectively [
25
]. The obtained results demonstrated that,
for most of the proteins studied, their relative quantities were lower when nanoparticles
were present. However, it should be noted that the identification of proteins by mass
spectrometry was only performed once, which may exaggerate the differences between
particle types. Thus, despite the central role of LC-MS in NPPC studies, the biological and
technical variations can significantly compromise the reproducibility of analyses.
Validity of proteomics results depend on the robustness and reproducibility of prepa-
ration and characterization methods and can be compromised by biological and technical
variations. Proteomics results are also dependent on the variability of identified proteins,
which critically depends on the LC-MS instrumentation and data analysis approach [
36
].
Protein corona impurities (dependent on nanoparticle size) can be a significant source
of contamination that may cause errors in proteomics analysis [
40
]. Unfortunately, the
LC-MS technique suffers from variability in the proteomics datasets, which depend not
only on the instrumentation, but also on the core facility [
18
,
36
]. This variability may
lead to misinterpretation of NPPC biological, biomedical, therapeutical, or toxicological
effects. Accordingly, protein quantification remains a challenge due to inherent limitations
in LC-MS, the complex composition of the protein corona, and the lack of standard ap-
proaches. Different labs have access to different instrumentation, equipment, and software,
which can affect data processing workflows and relative protein quantification. In this
regard, the recent study of Ashkarran et al. is particularly relevant [
18
]. They carried out
an interlaboratory study to determine the influence of different LC-MS/MS workflows in
the analysis of identical aliquots of NPPCs by sending them to different proteomics facili-
ties across the USA. Interestingly, the results provided by different laboratories showed
significant differences in terms of identified and quantified species. In this regard, it
must be remarked that mass spectrometry is inherently non-quantitative, hence protein
quantification (necessary in protein corona kinetics studies) is more challenging than iden-
tification [
45
]. In fact, whereas quantification of target proteins is usually carried out using
stable isotope-labeled protein analogous as standards, large-scale quantifications (as is the
case when characterizing protein corona) are based on label-free approaches and are mostly
relative quantitative approaches [
41
]. Ashkarran et al. found that, instead of proteomics
methodology, parameters such as sample preparation, instrumental settings, and raw data
processing are more relevant in the variability of analytical results. Another finding was
the lesser biased in proteomics results when analyzing tissue or cell extracts in comparison
to plasma fluids, given their lower protein dynamism, hence the less challenging nature
of the analysis [
46
]. The high dynamism of protein concentration levels implies an added
difficulty in proteomics analysis given that major proteins such as BSA, which accounts
for more than half of the protein content in plasma [
47
], hinder the detection and accurate
determination of minor proteins. Moreover, strategies for depleting abundant proteins from
plasma are often unsuitable for use when analyzing a large number of samples due to high
cost, difficult handling, and carry-over concerns. This type of study clearly demonstrates
Nanomaterials 2023,13, 1132 9 of 16
the need for standardization protocols and methods in MS protein analysis, especially at the
quantitative level, that enable the comparison of data results across proteomics platforms
and facilities.
4. The Potential of ICP-MS in the Study of the Nanoparticle–Protein Corona Complex
4.1. Characterization of Nanoparticle Composition and Functionalization by ICP-MS
Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful elemental
spectrometry technique that consists of the atomization and ionization of the sample in
a high-energy argon plasma, which enables one to carry out multi-elemental and multi-
isotope analysis. Th einorganic nanoparticles commonly used in bioanalytical applications
are typically metal-based structures synthesized from metal-salt precursors. Thus, their
metal atoms can be detected with ICP-MS to determine the multi-elemental content and
absolute quantitative information on nanoparticle mass and number concentrations, as well
as core/ligand ratios and elemental stoichiometries within the nanoparticle (Figure 4). Most
ICP-MS approaches in protein corona analysis focus on nanoparticle metal quantification
by elemental total analysis after sample digestion to dissolve the nanoparticles and remove
matrix interfering compounds [
48
]. Such sample digestion is also necessary to prevent
transport and nebulization issues in larger nanoparticles that would otherwise lead to
biased quantitative results. The use of micro-flow total consumption nebulizers is a viable
alternative to dispose of these effects [
49
,
50
]. Matrix interferences and contamination prob-
lems from inorganic metal target ions present in the sample entail prior sample purification
approaches such as ultracentrifugation [
51
], or the application of an external magnetic field
when working with magnetic nanoparticles [52].
Nanomaterials 2023, 13, x FOR PEER REVIEW 9 of 16
protein analysis, especially at the quantitative level, that enable the comparison of data
results across proteomics platforms and facilities.
4. The Potential of ICP-MS in the Study of the Nanoparticle–Protein Corona Complex
4.1. Characterization of Nanoparticle Composition and Functionalization by ICP-MS
Inductively coupled plasma mass spectrometry (ICP-MS) is a powerful elemental
spectrometry technique that consists of the atomization and ionization of the sample in a
high-energy argon plasma, which enables one to carry out multi-elemental and multi-iso-
tope analysis. Th einorganic nanoparticles commonly used in bioanalytical applications
are typically metal-based structures synthesized from metal-salt precursors. Thus, their
metal atoms can be detected with ICP-MS to determine the multi-elemental content and
absolute quantitative information on nanoparticle mass and number concentrations, as
well as core/ligand ratios and elemental stoichiometries within the nanoparticle (Figure
4). Most ICP-MS approaches in protein corona analysis focus on nanoparticle metal quan-
tication by elemental total analysis after sample digestion to dissolve the nanoparticles
and remove matrix interfering compounds [48]. Such sample digestion is also necessary
to prevent transport and nebulization issues in larger nanoparticles that would otherwise
lead to biased quantitative results. The use of micro-ow total consumption nebulizers is
a viable alternative to dispose of these eects [49,50]. Matrix interferences and contamina-
tion problems from inorganic metal target ions present in the sample entail prior sample
purication approaches such as ultracentrifugation [51], or the application of an external
magnetic eld when working with magnetic nanoparticles [52].
Figure 4. Use of ICP-MS for the characterization of nanoparticles. Reproduced from ref [27] with
permission from the Royal Society of Chemistry.
The use of separation techniques coupled to ICP-MS opens the door to the analysis
of nanoparticles in polydisperse samples. This is the case with size exclusion liquid chro-
matography, which has been used to determine the size and shape of metallic nanoparti-
cles and quantum dots [53,54]. Asymmetric ow eld-ow fractionation (AF4) coupled
with ICP-MS is another nanoparticle fractionation technique used to avoid nanoparticle
degradation or agglomeration of the nanoparticles that sometimes occurs with SEC dur-
ing separation. With this method, nanoparticles are separated based on their hydrody-
namic size, so that dierent nanoparticles can be separated and identied with AF4-ICP-
MS with high recoveries (>81%) [55]. Furthermore, this approach can be used to charac-
terize and quantify bioconjugates stoichiometries and populations [29]. It must be re-
marked that nanoparticle characterization with ICP-MS requires the nanoparticle to have
an ICP-MS detectable element in its core. In the case of metallic nanoparticles, that element
is the metal forming the core. In the case of non-metallic nanoparticles, ICP-MS detection
Figure 4.
Use of ICP-MS for the characterization of nanoparticles. Reproduced from ref [
27
] with
permission from the Royal Society of Chemistry.
The use of separation techniques coupled to ICP-MS opens the door to the analy-
sis of nanoparticles in polydisperse samples. This is the case with size exclusion liquid
chromatography, which has been used to determine the size and shape of metallic nanopar-
ticles and quantum dots [
53
,
54
]. Asymmetric flow field-flow fractionation (AF4) coupled
with ICP-MS is another nanoparticle fractionation technique used to avoid nanoparticle
degradation or agglomeration of the nanoparticles that sometimes occurs with SEC during
separation. With this method, nanoparticles are separated based on their hydrodynamic
size, so that different nanoparticles can be separated and identified with AF4-ICP-MS with
high recoveries (>81%) [
55
]. Furthermore, this approach can be used to characterize and
Nanomaterials 2023,13, 1132 10 of 16
quantify bioconjugates stoichiometries and populations [
29
]. It must be remarked that
nanoparticle characterization with ICP-MS requires the nanoparticle to have an ICP-MS
detectable element in its core. In the case of metallic nanoparticles, that element is the
metal forming the core. In the case of non-metallic nanoparticles, ICP-MS detection can be
performed if dopped with a detectable element, as is the case, for instance, of dopped car-
bon nanoparticles [
56
]. Protein formation dynamics in these cases can then be approached
through the determination of the elemental ratio between the sulfur from the proteins,
and the detectable element from the nanoparticle core [
57
]. It must be considered that the
nanoparticles may contain sulfur or be functionalized with sulfur-containing ligands to
provide stability and solubility to the nanoparticle. Sulfur/metal determination can also be
used to determine stoichiometries ligand/nanoparticle, providing information of surface
functionalization and ligand density. Nevertheless, in those cases, protein incorporation
would be determined from the variation (increase) in the S/metal ratio respect to the initial
ratio (corresponding to the surface ligands) [58].
Lastly, it is worth noting the increase in the use of the single particle-ICP-MS (spICP-
MS) technique [
59
,
60
]. spICP-MS is based on the introduction of a very diluted sample into
ICP-MS with a fast mass scanning speed configuration. Nanoparticle concentration and size
can be obtained through signal intensity and pulse frequency, respectively. This technique
allows elemental analysis of all types of matrices, because it provides the data needed to
determine the size, size distribution, and particle number concentrations of nanoparticles
with little time spent measuring the samples in suspension [
61
,
62
]. To translate ion intensity
to ion mass, and assure the reproducibility and validity and establish metrological trace-
ability of the methodology, adequate calibration approaches are required [
60
,
63
]. The most
common calibration approaches are the use of well-characterized reference materials of
nanoparticles with the same nature as the analyte, or the use of standard element solutions
of the ICP-MS detectable element present in the analyte. Ideally the use of a nanoparticle
reference material would provide the most straightforward calibration, given it is of known
geometry and size and has the same composition as the nanoparticle analyte [
63
]. How-
ever, due to the scarcity of commercially available well-characterized reference materials,
elemental standards solutions are often used for calibration in spICP-MS [
60
,
64
,
65
]. Their
use for calibration, however, requires the determination of the nanoparticles transport
efficiency, using strategies such as the determination of particle frequency or particle size
methods using nanoparticles standards [
66
], methods based on the determination of the
solvent transport efficiency such as dynamic mass flow (DMF) [
64
], or the use of low-
volume, high-efficiency sample introduction systems such as total consumption nebulizers
(TCN) [67].
It has already been discussed that determination of the protein corona thickness
through the comparison of the nanoparticles and the NPPC size provides valuable infor-
mation for studying the adsorption and interactions of the proteins with the nanoparticle.
However, nanoparticle size determination with spICP-MS is approached through the mea-
surement of the metal (or another detectable element) present in the nanoparticle core. In
fact, size determination is based on the intensity of the detected events, and it is indepen-
dent, as commented, of the nanoparticle environment. That is, for a certain nanoparticle,
spICP-MS analysis would provide the core size exclusively and not the hydrodynamic
size (ligands, protein corona, etc.). spICP-MS is moreover limited in terms of nanoparticle
size, as it cannot determine sizes below 10 nm. [
61
]. Alternatively, strategies based on the
combination of Taylor dispersion analysis (TAD) and ICP-MS have been proposed for the
size determination of <10 nm nanoparticles [
68
]. In TDA, nanoparticle size is calculated
through the determination of the diffusion coefficient, and the use of ICP-MS provides a
significant enhancement in sensitivity compared to traditional approaches such as DLS.
Because this approach determines the nanostructure size in the base to the gaussian fit of
the peak obtained in TDA analysis, which is related to the hydrodynamic radius, it can
differentiate between nanoparticles with and without the protein corona. In fact, the tech-
nique has been applied to the study of NPPC, proving useful to determine the nanoparticle
Nanomaterials 2023,13, 1132 11 of 16
size and the protein corona thickness, though it is still limited in terms of discriminating
whether the increase in size is due to aggregation or the formation of a larger corona [
69
].
This approach is also restricted to studies with high protein concentration, and to small
size complexes (<70 nm) for good accuracy.
4.2. Characterization of NPPC Protein Components by ICP-MS
ICP-MS sensitivity and specificity to detect trace levels of metals and other elements
in biological samples (e.g., P or S) makes it useful for measuring the concentration of
the proteins present in NPPC and so study its dynamic changes over time, making it a
valuable tool for understanding the interactions between nanoparticles and proteins. In
contrast to common molecular MS approaches for protein quantification (i.e., electrospray),
ICP-MS ionization can be made species-independent. Once nebulization issues are under
control [
50
], ICP-MS response becomes directly proportional to the mass concentration of
the detected element, and independent of the protein structure, molecular weight, or charge
state, requiring no specific protein standards for absolute quantification. Protein quantifi-
cation analysis can be carried out through any ICP-detectable atom related to the protein
(except C, H, O, and N). Once the element/protein molar ratio is known, the elemental
signal of the target compound can be expressed as protein concentration. In this regard,
sulfur detection can be considered as a generic ICP-MS approach for protein quantification,
because S is present in cysteine (Cys) and methionine (Met) amino acid residues, and
hence in most proteins. Therefore, when the number of these components (Met and Cys) is
established, from the amino acid sequence, absolute protein quantification can be carried
out by correlating with the sulfur quantified with ICP-MS [
70
]. This information can be
obtained by parallel analysis with molecular sources such as ESI-MS [
71
–
73
]. It is worth
remarking that the ICP-MS detectable element, however, can also be added externally, via,
e.g., chemical reagents such as DOTA containing rare earth elements. In this regard, Liu
et al. used ICP-MS to study the internalization of NPPC of Ru-containing NPs, used as drug
nanocarriers, and the effect of their interaction with a cellular membrane glycopeptide,
using an Eu-containing DOTA tagged to the peptide [
74
]. Through the detection of Ru and
Eu with ICP-MS, they were able to study the effect of the corona on the internalization
process and macrophage’s uptake.
It is important to consider that the separation dimension provided by chromatography
is a prerequisite to detect and quantify each protein individually to be able to unequivocally
correlate the elemental signal to a specific biomolecule. An example of this is the work
carried out by Matczuk et al., who used capillary electrophoresis coupled to ICP-MS to
determine the number of protein molecules bound to AuNPs of different sizes (5–50 nm).
The use of CE permitted separation of the conjugate between AuNPs and albumin from
the free AuNPs (in excess) and thus estimate the number of albumin molecules bound
per AuNP, relating the peak areas obtained from the complex AuNP:albumin and the free
unbound AuNPs [
75
]. Likewise, Fernández-Iglesias and Bettmer were able to estimate
the total amount of proteins forming the hard corona of citrate-stabilized AuNPs, ranging
from 10 to 60 nm, when incubated with human serum [
76
]. They calculated the total
protein quantity from the sulfur concentration quantified with ICP-MS. To do so, they first
confirmed with SEC-ICP-MS that the sulfur signal corresponded solely to protein species.
Then, they carried out the washing and purification of the NPPC using centrifugation and
sedimentation through sucrose, the latter being seemingly more efficient and requiring
fewer washing steps. Finally, ICP-MS determination of the S/Au signal ratio was translated
into number of proteins per nanoparticle, assuming an average number of 40 sulfur atoms
per protein molecule.
Regardless of the large amount of potential information provided by ICP-MS, such as
molar ratio NP/protein, absolute protein concentration, protein corona, or nanoparticle
aggregation and/or size distribution, it is still necessary to combine this approach with
previously described analytical methodologies for a better and complete characterization
of the NPPC. For a better understanding of the different NP populations in protein corona
Nanomaterials 2023,13, 1132 12 of 16
studies, separation techniques should precede ICP analysis. Electrophoresis is commonly
applied to determine binding kinetics and size exclusion, or hydrodynamic chromatogra-
phy is suitable for species characterization [
77
,
78
]. Asymmetric flow-field flow fractionation
can provide in a single run complementary information about the NP:protein ratio from
free (i.e., non-surrounded by proteins) particles and different populations formed during
bioconjugation [
29
]. In this regard, López-Sanz et al. used AF4-ICP-MS in combination with
techniques such as UV-Vis spectroscopy, TEM, and ultracentrifugation (UC) to study and
characterize AuNPs in cell culture media [
79
]. The combination of AF4 with ICP-MS pro-
vided valuable information on AuNPs transformations, such as oxidation processes in the
cell media resulting in the formation of ionic Au, confirmed by ICP-MS after ultracentrifu-
gation. The complementarity of ICP-MS with other techniques was further demonstrated
in the characterization of protein corona formation. They observed longer AuNP retention
time in AF4-ICP-MS, which was associated with an increase in their hydrodynamic size.
The use of UV-Vis and TEM provided the necessary information to determine whether this
increase in size was due to the formation of the protein corona or by aggregation of the
nanoparticles, respectively.
5. Concluding Remarks and Prospects
Nowadays, it is unquestionable that the use of NPs in biological applications requires
a precise knowledge of the NP–protein corona system (NPPC) that is formed when NPs en-
ters biosystems. Consequently, NPs acquire a new biological identity through the formation
of this protein corona, which affects their colloidal stability, biodistribution, interactions,
toxicity, and clearance. The understanding of the fate of the NPPC in biological systems, the
development of NPPCs with biomedical applications, or the understanding of the NPPC
toxicological effect require accurate and reliable characterization of nanoparticle physico-
chemical properties, the protein corona composition, and the dynamics of the interaction
between nanoparticles and the different proteins that constitute the corona. To this end,
the combination of multiple analytical methodologies offering complementary information
certainly seems to be necessary. Knowledge of nanoparticle protein corona composition
commonly relies on approaches that lack standardization methods that guarantee or control
the reproducibility and reliability of the results. In particular, important difficulties persist
in the effective quantification of the different proteins from the corona. ICP-MS turns up as
a complementary tool that could provide accuracy and robustness of quantitative results
and workflows given its species-independent nature. In fact, thanks to the presence of
ICP-detectable elements in widespread inorganic NPs (Au, Ag, Ti, Ce, Cd, Se, Zn, among
other) and proteins (S), ICP-MS can provide valuable information, including NP elemental
composition, size, concentration, and populations on the one hand, and absolute protein
amounts on the other.
The combination of several state-of-the-art imaging techniques enable visualization of
the individual biomolecules associated with the surfaces of NPs [
40
] and clearly demon-
strate the great variability of the protein corona within the same sample. Notably, such
highly valuable qualitative information provided by imaging techniques could be com-
pleted with the quantitative nature of the ICP-MS signal. However, ICP-MS itself cannot
provide a comprehensive picture of the diverse NPPC populations present. It requires
separation techniques that are able to first isolate free NPs from NPPS and then to assess
the different protein: NP populations present in the sample. We anticipate that analytical
platforms comprising adequate separation techniques (e.g., CE, SEC, AF4, HPLC) coupled
on-line with ICP tandem MS detection (well suited for the sensitive and free-of-interference
analysis of S) could be well suited to face the remarkable analytical challenge. We have
also to keep on mind that the accurate and precise absolute protein quantification provided
by ICP tandem MS falls short without information regarding the identity of the proteins
involved in the NPPC. For that purpose, liquid chromatography coupled to mass spec-
troscopy (LC-MS/MS) remains the dominant methodology and is thus essential to achieve
a detailed characterization of the protein corona.
Nanomaterials 2023,13, 1132 13 of 16
In summary, we believe that ICP-MS could fill some gaps in NPPC characterization
that are clearly identified and currently demanded and could be established as a useful
and resourceful complementary tool in the current challenge of protein corona assessment.
Author Contributions:
Writing—original draft preparation, A.F.-C., J.R.A. and F.C.C.;
writing—review
and editing, F.C.C., J.R.E. and J.M.C.-F. All authors have read and agreed to the published version of
the manuscript.
Funding:
This work was funded by the Spanish Ministry of Science and Innovation (PID2019-
109698GB-I00) and the Principality of Asturias GRUPIN IDI 2021/000081. A.F.C. acknowledges the
FPU19/00006 doctoral scholarship from the Spanish Ministry of Universities. F.C.C. acknowledges
the Principality of Asturias for the Margarita Salas Joven postdoctoral grant (AYUD/2021/58459).
Data Availability Statement:
The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Monopoli, M.P.; Walczyk, D.; Campbell, A.; Elia, G.; Lynch, I.; Bombelli, F.B.; Dawson, K.A. Physical
−
Chemical Aspects of Protein
Corona: Relevance to in Vitro and in Vivo Biological Impacts of Nanoparticles. J. Am. Chem. Soc.
2011
,133, 2525–2534. [CrossRef]
2.
Malakar, A.; Kanel, S.R.; Ray, C.; Snow, D.D.; Nadagouda, M.N. Nanomaterials in the Environment, Human Exposure Pathway,
and Health Effects: A Review. Sci. Total Environ. 2021,759, 143470. [CrossRef]
3.
Lead, J.R.; Batley, G.E.; Alvarez, P.J.J.; Croteau, M.-N.; Handy, R.D.; McLaughlin, M.J.; Judy, J.D.; Schirmer, K. Nanomaterials in
the Environment: Behavior, Fate, Bioavailability, and Effects—An Updated Review. Environ. Toxicol. Chem.
2018
,37, 2029–2063.
[CrossRef]
4.
Auría-Soro, C.; Nesma, T.; Juanes-Velasco, P.; Landeira-Viñuela, A.; Fidalgo-Gomez, H.; Acebes-Fernandez, V.; Gongora, R.;
Almendral Parra, M.J.; Manzano-Roman, R.; Fuentes, M. Interactions of Nanoparticles and Biosystems: Microenvironment of
Nanoparticles and Biomolecules in Nanomedicine. Nanomaterials 2019,9, 1365. [CrossRef]
5.
Lundqvist, M.; Stigler, J.; Cedervall, T.; Berggård, T.; Flanagan, M.B.; Michelle, B.F.; Lynch, I.; Elia, G.; Dawson, K.A. The Evolution
of the Protein Corona around Nanoparticles: A Test Study. ACS Nano 2011,5, 7503–7509. [CrossRef] [PubMed]
6. Lynch, I.; Dawson, K.A. Protein-Nanoparticle Interactions. Nano Today 2008,3, 40–47. [CrossRef]
7.
Kelly, P.M.; Åberg, C.; Polo, E.; O’Connell, A.; Cookman, J.; Fallon, J.; Krpeti´c, Ž.; Dawson, K.A. Mapping Protein Binding Sites on
the Biomolecular Corona of Nanoparticles. Nat. Nanotechnol. 2015,10, 472–479. [CrossRef] [PubMed]
8.
Cedervall, T.; Lynch, I.; Lindman, S.; Berggård, T.; Thulin, E.; Nilsson, H.; Dawson, K.A.; Linse, S. Understanding the Nanoparticle-
Protein Corona Using Methods to Quantify Exchange Rates and Affinities of Proteins for Nanoparticles. Proc. Natl. Acad. Sci.
USA 2007,104, 2050–2055. [CrossRef] [PubMed]
9.
Wang, X.; Zhang, W. The Janus of Protein Corona on Nanoparticles for Tumor Targeting, Immunotherapy and Diagnosis. J.
Control. Release 2022,345, 832–850. [CrossRef]
10.
Xu, X.; Mao, X.; Wang, Y.; Li, D.; Du, Z.; Wu, W.; Jiang, L.; Yang, J.; Li, J. Study on the Interaction of Graphene Oxide-silver
Nanocomposites with Bovine Serum Albumin and the Formation of Nanoparticle-Protein Corona. Int. J. Biol. Macromol.
2018
,
116, 492–501. [CrossRef]
11.
Ke, P.C.; Lin, S.; Parak, W.J.; Davis, T.P.; Caruso, F. A Decade of the Protein Corona. ACS Nano
2017
,11, 11773–11776. [CrossRef]
[PubMed]
12.
Dell’Orco, D.; Lundqvist, M.; Oslakovic, C.; Cedervall, T.; Linse, S. Modeling the Time Evolution of the Nanoparticle-Protein
Corona in a Body Fluid. PLoS ONE 2010,5, e10949. [CrossRef] [PubMed]
13.
Walczyk, D.; Bombelli, F.B.; Monopoli, M.P.; Lynch, I.; Dawson, K.A. What the Cell “Sees” in Bionanoscience. J. Am. Chem. Soc.
2010,132, 5761–5768. [CrossRef] [PubMed]
14.
Tenzer, S.; Docter, D.; Kuharev, J.; Musyanovych, A.; Fetz, V.; Hecht, R.; Schlenk, F.; Fischer, D.; Kiouptsi, K.; Reinhardt, C.; et al.
Rapid Formation of Plasma Protein Corona Critically Affects Nanoparticle Pathophysiology. Nat. Nanotechnol.
2013
,8, 772–781.
[CrossRef] [PubMed]
15.
Mout, R.; Moyano, D.F.; Rana, S.; Rotello, V.M. Surface Functionalization of Nanoparticles for Nanomedicine. Chem. Soc. Rev.
2012,41, 2539. [CrossRef]
16.
Kopac, T. Protein Corona, Understanding the Nanoparticle–Protein Interactions and Future Perspectives: A Critical Review. Int. J.
Biol. Macromol. 2021,169, 290–301. [CrossRef]
17.
Walkey, C.D.; Olsen, J.B.; Song, F.; Liu, R.; Guo, H.; Guo, H.; Olsen, D.W.H.; Cohen, Y.; Emili, A.; Chan, W.C.W. Protein Corona
Fingerprinting Predicts the Cellular Interaction of Gold and Silver Nanoparticles. ACS Nano 2014,8, 2439–2455. [CrossRef]
18.
Ashkarran, A.A.; Gharibi, H.; Voke, E.; Landry, M.P.; Saei, A.A.; Mahmoudi, M. Measurements of Heterogeneity in Proteomics
Analysis of the Nanoparticle Protein Corona across Core Facilities. Nat. Commun. 2022,13, 6610. [CrossRef]
Nanomaterials 2023,13, 1132 14 of 16
19.
Lesniak, A.; Fenaroli, F.; Monopoli, M.P.; Åberg, C.; Dawson, K.A.; Salvati, A. Effects of the Presence or Absence of a Protein
Corona on Silica Nanoparticle Uptake and Impact on Cells. ACS Nano 2012,6, 5845–5857. [CrossRef]
20.
Mishra, R.K.; Ahmad, A.; Vyawahare, A.; Alam, P.; Khan, T.H.; Khan, R. Biological Effects of Formation of Protein Corona onto
Nanoparticles. Int. J. Biol. Macromol. 2021,175, 1–18. [CrossRef]
21.
Ahmad, A.; Khan, F.; Mishra, R.K.; Khan, R. Precision Cancer Nanotherapy: Evolving Role of Multifunctional Nanoparticles for
Cancer Active Targeting. J. Med. Chem. 2019,62, 10475–10496. [CrossRef] [PubMed]
22.
Hühn, D.; Kantner, K.; Geidel, C.; Brandholt, S.; De Cock, I.; Soenen, S.J.; Gil, P.R.; Montenegro, J.-M.; Braeckmans, K.; Müllen, K.;
et al. Polymer-Coated Nanoparticles Interacting with Proteins and Cells: Focusing on the Sign of the Net Charge. ACS Nano
2013
,
7, 3253–3263. [CrossRef] [PubMed]
23.
Monopoli, M.P.; Pitek, A.S.; Lynch, I.; Dawson, K.A. Formation and Characterization of the Nanoparticle-Protein Corona. Methods
Mol. Biol. 2013,1025, 137–155. [CrossRef] [PubMed]
24.
Huang, D.E.; Zia, R.N. Sticky, Active Microrheology: Part 1. Linear-Response. J. Colloid Interface Sci.
2019
,554, 580–591. [CrossRef]
[PubMed]
25.
Galmarini, S.; Hanusch, U.; Giraud, M.; Cayla, N.; Chiappe, D.; Von Moos, N.R.; Hofmann, H.; Maurizi, L. Beyond Unpre-
dictability: The Importance of Reproducibility in Understanding the Protein Corona of Nanoparticles. Bioconjug. Chem.
2018
,29,
3385–3393. [CrossRef]
26.
Lynch, I.; Cedervall, T.; Lundqvist, M.; Cabaleiro-Lago, C.; Linse, S.; Dawson, K.A. The Nanoparticle–Protein Complex as a
Biological Entity; a Complex Fluids and Surface Science Challenge for the 21st Century. Adv. Colloid Interface Sci.
2007
,134–135,
167–174. [CrossRef]
27.
Moreira-Alvarez, B.; Cid-Barrio, L.; Ferreira, H.S.; Costa-Fernández, J.M.; Encinar, J.R. Integrated Analytical Platforms for the
Comprehensive Characterization of Bioconjugated Inorganic Nanomaterials Aiming at Biological Applications. J. Anal. At.
Spectrom. 2020,35, 1518–1529. [CrossRef]
28.
Schöttler, S.; Becker, G.; Winzen, S.; Steinbach, T.; Mohr, K.; Landfester, K.; Mailänder, V.; Wurm, F.R. Protein Adsorption Is
Required for Stealth Effect of Poly(Ethylene Glycol)- and Poly(Phosphoester)-Coated Nanocarriers. Nat. Nanotechol.
2016
,11,
372–377. [CrossRef]
29.
Bouzas-Ramos, D.; García-Alonso, J.I.; Costa-Fernández, J.M.; Ruiz Encinar, J. Quantitative Assessment of Individual Populations
Present in Nanoparticle–Antibody Conjugate Mixtures Using AF4-ICP-MS/MS. Anal. Chem. 2019,91, 3567–3574. [CrossRef]
30.
Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K.A. Nanoparticle Size and Surface Properties Determine the
Protein Corona with Possible Implications for Biological Impacts. Proc. Natl. Acad. Sci. USA 2008,105, 14265–14270. [CrossRef]
31.
Deng, Z.J.; Mortimer, G.; Schiller, T.; Musumeci, A.; Martin, D.; Minchin, R.F. Differential Plasma Protein Binding to Metal Oxide
Nanoparticles. Nanotechnology 2009,20, 455101. [CrossRef] [PubMed]
32.
Sharifi, S.; Mahmoud, N.N.; Voke, E.; Landry, M.P.; Mahmoudi, M. Importance of Standardizing Analytical Characterization
Methodology for Improved Reliability of the Nanomedicine Literature. Nano Micro Lett. 2022,14, 172. [CrossRef]
33.
Nandakumar, A.; Wei, W.; Siddiqui, G.; Tang, H.; Li, Y.; Kakinen, A.; Wan, X.; Koppel, K.; Hoffmann, M.R.; Lin, S.; et al. Dynamic
Protein Corona of Gold Nanoparticles with an Evolving Morphology. ACS Appl. Mater. Interfaces
2021
,13, 58238–58251. [CrossRef]
[PubMed]
34.
Casals, E.; Pfaller, T.; Duschl, A.; Oostingh, G.J.; Puntes, V.F. Time Evolution of the Nanoparticle Protein Corona. ACS Nano
2010
,
4, 3623–3632. [CrossRef] [PubMed]
35.
Monopoli, M.P.; Åberg, C.; Salvati, A.; Dawson, K.A. Biomolecular Coronas Provide the Biological Identity of Nanosized Materials.
Nat. Nanotechol. 2012,7, 779–786. [CrossRef]
36.
Mahmoudi, M. The Need for Improved Methodology in Protein Corona Analysis. Nat. Commun.
2022
,13, 49. [CrossRef]
[PubMed]
37.
Carrillo-Carrion, C.; Carril, M.; Parak, W.J. Techniques for the Experimental Investigation of the Protein Corona. Curr. Opin.
Biotechnol. 2017,46, 106–113. [CrossRef]
38.
Olson, B.J.S.C.; Markwell, J. Assays for Determination of Protein Concentration. Curr. Protoc. Protein Sci.
2007
,48, 3.4.1–3.4.29.
[CrossRef]
39.
Benetti, F.; Fedel, M.; Minati, L.; Speranza, G.; Migliaresi, C. Gold Nanoparticles: Role of Size and Surface Chemistry on Blood
Protein Adsorption. J. Nanopartic. Res. 2013,15, 1694. [CrossRef]
40.
Sheibani, S.; Basu, K.; Farnudi, A.; Ashkarran, A.; Ichikawa, M.; Presley, J.F.; Bui, K.H.; Ejtehadi, M.R.; Vali, H.; Mahmoudi, M.
Nanoscale Characterization of the Biomolecular Corona by Cryo-Electron Microscopy, Cryo-Electron Tomography, and Image
Simulation. Nat. Commun. 2021,12, 573. [CrossRef]
41.
Calderón-Celis, F.; Encinar, J.R.; Sanz-Medel, A. Standardization Approaches in Absolute Quantitative Proteomics with Mass
Spectrometry. Mass Spec. Rev. 2018,37, 715–737. [CrossRef] [PubMed]
42.
Rampado, R.; Crotti, S.; Caliceti, P.; Pucciarelli, S.; Agostini, M. Recent Advances in Understanding the Protein Corona of
Nanoparticles and in the Formulation of “Stealthy” Nanomaterials. Front. Bioeng. Biotechnol. 2020,8, 166. [CrossRef] [PubMed]
43.
Langevin, D.; Lozano, O.; Salvati, A.; Kestens, V.; Monopoli, M.; Raspaud, E.; Mariot, S.; Salonen, A.; Thomas, S.; Driessen,
M.; et al. Inter-Laboratory Comparison of Nanoparticle Size Measurements Using Dynamic Light Scattering and Differential
Centrifugal Sedimentation. NanoImpact 2018,10, 97–107. [CrossRef]
Nanomaterials 2023,13, 1132 15 of 16
44.
Faria, M.; Björnmalm, M.; Thurecht, K.J.; Kent, S.J.; Parton, R.G.; Kavallaris, M.; Johnston, A.P.R.; Gooding, J.J.; Corrie, S.R.; Boyd,
B.J.; et al. Minimum Information Reporting in Bio-Nano Experimental Literature. Nat. Nanotechnol.
2018
,13, 777–785. [CrossRef]
45.
Dupree, E.J.; Jayathirtha, M.; Yorkey, H.; Mihasan, M.; Petre, B.A.; Darie, C.C. A Critical Review of Bottom-Up Proteomics: The
Good, the Bad, and the Future of This Field. Proteomes 2020,8, 14. [CrossRef] [PubMed]
46.
Ignjatovic, V.; Geyer, P.E.; Palaniappan, K.K.; Chaaban, J.E.; Omenn, G.S.; Baker, M.S.; Deutsch, E.W.; Schwenk, J.M. Mass
Spectrometry-Based Plasma Proteomics: Considerations from Sample Collection to Achieving Translational Data. J. Proteome Res.
2019,18, 4085–4097. [CrossRef]
47.
Pernemalm, M.; Sandberg, A.; Zhu, Y.; Boekel, J.; Tamburro, D.; Schwenk, J.M.; Björk, A.; Wahren-Herlenius, M.; Åmark, H.;
Östenson, C.-G.; et al. In-Depth Human Plasma Proteome Analysis Captures Tissue Proteins and Transfer of Protein Variants
across the Placenta. eLife 2019,8, e41608. [CrossRef]
48.
Meermann, B.; Nischwitz, V. ICP-MS for the Analysis at the Nanoscale—A Tutorial Review. J. Anal. At. Spectrom.
2018
,33,
1432–1468. [CrossRef]
49.
Olesik, J.W.; Gray, P.J. Considerations for Measurement of Individual Nanoparticles or Microparticles by ICP-MS: Determination
of the Number of Particles and the Analyte Mass in Each Particle. J. Anal. At. Spectrom. 2012,27, 1143. [CrossRef]
50.
Cid-Barrio, L.; Calderón-Celis, F.; Costa-Fernández, J.M.; Encinar, J.R. Assessment of the Potential and Limitations of Elemental
Mass Spectrometry in Life Sciences for Absolute Quantification of Biomolecules Using Generic Standards. Anal. Chem.
2020
,92,
13500–13508. [CrossRef]
51.
Kuznetsova, O.V.; Mokhodoeva, O.B.; Maksimova, V.V.; Dzhenloda, R.K.; Jarosz, M.; Shkinev, V.M.; Timerbaev, A.R. High-
Resolution ICP-MS Approach for Characterization of Magnetic Nanoparticles for Biomedical Applications. J. Pharm. Biomed.
Anal. 2020,189, 113479. [CrossRef]
52.
Kuznetsova, O.V.; Jarosz, M.; Keppler, B.K.; Timerbaev, A.R. Toward a Deeper and Simpler Understanding of Serum Protein-
Mediated Transformations of Magnetic Nanoparticles by ICP-MS. Talanta 2021,229, 122287. [CrossRef]
53.
Pitkänen, L.; Striegel, A.M. Size-Exclusion Chromatography of Metal Nanoparticles and Quantum Dots. TrAC Trends Anal. Chem.
2016,80, 311–320. [CrossRef] [PubMed]
54.
Trapiella-Alfonso, L.; Montoro Bustos, A.R.; Encinar, J.R.; Costa-Fernández, J.M.; Pereiro, R.; Sanz-Medel, A. New Integrated
Elemental and Molecular Strategies as a Diagnostic Tool for the Quality of Water Soluble Quantum Dots and Their Bioconjugates.
Nanoscale 2011,3, 954. [CrossRef] [PubMed]
55.
Menendez-Miranda, M.; Fernandez-Arguelles, M.T.; Costa-Fernandez, J.M.; Encinar, J.R.; Sanz-Medel, A. Elemental Ratios for
Characterization of Quantum-Dots Populations in Complex Mixtures by Asymmetrical Flow Field-Flow Fractionation on-Line
Coupled to Fluorescence and Inductively Coupled Plasma Mass Spectrometry. Anal. Chim. Acta
2014
,839, 8–13. [CrossRef]
[PubMed]
56.
Bouzas-Ramos, D.; Cigales Canga, J.; Mayo, J.C.; Sainz, R.M.; Ruiz Encinar, J.; Costa-Fernandez, J.M. Carbon Quantum Dots
Codoped with Nitrogen and Lanthanides for Multimodal Imaging. Adv. Funct. Mater. 2019,29, 1903884. [CrossRef]
57.
Garcia-Cortes, M.; Sotelo González, E.; Fernández-Argüelles, M.T.; Encinar, J.R.; Costa-Fernández, J.M.; Sanz-Medel, A. Capping
of Mn-Doped ZnS Quantum Dots with DHLA for Their Stabilization in Aqueous Media: Determination of the Nanoparticle
Number Concentration and Surface Ligand Density. Langmuir 2017,33, 6333–6341. [CrossRef]
58.
Bouzas-Ramos, D.; García-Cortes, M.; Sanz-Medel, A.; Encinar, J.R.; Costa-Fernández, J.M. Assessment of the Removal of Side
Nanoparticulated Populations Generated during One-Pot Synthesis by Asymmetric Flow Field-Flow Fractionation Coupled to
Elemental Mass Spectrometry. J. Chromatogr. A 2017,1519, 156–161. [CrossRef]
59.
Degueldre, C.; Favarger, P.-Y. Colloid Analysis by Single Particle Inductively Coupled Plasma-Mass Spectroscopy: A Feasibility
Study. Colloids Surf. A Physicochem. Eng. Asp. 2003,217, 137–142. [CrossRef]
60.
Mozhayeva, D.; Engelhard, C. A Critical Review of Single Particle Inductively Coupled Plasma Mass Spectrometry—A Step
towards an Ideal Method for Nanomaterial Characterization. J. Anal. At. Spectrom. 2020,35, 1740–1783. [CrossRef]
61.
Lee, S.; Bi, X.; Reed, R.B.; Ranville, J.F.; Herckes, P.; Westerhoff, P. Nanoparticle Size Detection Limits by Single Particle ICP-MS
for 40 Elements. Environ. Sci. Technol. 2014,48, 10291–10300. [CrossRef] [PubMed]
62.
Peters, R.; Herrera-Rivera, Z.; Undas, A.; van der Lee, M.; Marvin, H.; Bouwmeester, H.; Weigel, S. Single Particle ICP-MS
Combined with a Data Evaluation Tool as a Routine Technique for the Analysis of Nanoparticles in Complex Matrices. J. Anal. At.
Spectrom. 2015,30, 1274–1285. [CrossRef]
63.
Bustos, A.R.M.; Purushotham, K.P.; Possolo, A.; Farkas, N.; Vladár, A.E.; Murphy, K.E.; Winchester, M.R. Validation of Single
Particle ICP-MS for Routine Measurements of Nanoparticle Size and Number Size Distribution. Anal. Chem.
2018
,90, 14376–14386.
[CrossRef]
64.
Cuello-Nuñez, S.; Abad-Álvaro, I.; Bartczak, D.; del Castillo Busto, M.E.; Ramsay, D.A.; Pellegrino, F.; Goenaga-Infante, H. The
Accurate Determination of Number Concentration of Inorganic Nanoparticles Using SpICP-MS with the Dynamic Mass Flow
Approach. J. Anal. At. Spectrom. 2020,35, 1832–1839. [CrossRef]
65.
Laborda, F.; Abad-Álvaro, I.; Jiménez, M.S.; Bolea, E. Catching Particles by Atomic Spectrometry: Benefits and Limitations
of Single Particle—Inductively Coupled Plasma Mass Spectrometry. Spectrochim. Acta Part B At. Spectrosc.
2023
,199, 106570.
[CrossRef]
Nanomaterials 2023,13, 1132 16 of 16
66.
Pace, H.E.; Rogers, N.J.; Jarolimek, C.; Coleman, V.A.; Higgins, C.P.; Ranville, J.F. Determining Transport Efficiency for the
Purpose of Counting and Sizing Nanoparticles via Single Particle Inductively Coupled Plasma Mass Spectrometry. Anal. Chem.
2011,83, 9361–9369. [CrossRef] [PubMed]
67.
Lin, F.; Miyashita, S.; Inagaki, K.; Liu, Y.-H.; Hsu, I.-H. Evaluation of Three Different Sample Introduction Systems for Single-
Particle Inductively Coupled Plasma Mass Spectrometry (SpICP-MS) Applications. J. Anal. At. Spectrom.
2019
,34, 401–406.
[CrossRef]
68.
Labied, L.; Rocchi, P.; Doussineau, T.; Randon, J.; Tillement, O.; Lux, F.; Hagège, A. Taylor Dispersion Analysis Coupled to
Inductively Coupled Plasma-Mass Spectrometry for Ultrasmall Nanoparticle Size Measurement: From Drug Product to Biological
Media Studies. Anal. Chem. 2021,93, 1254–1259. [CrossRef]
69.
Degasperi, A.; Labied, L.; Farre, C.; Moreau, E.; Martini, M.; Chaix, C.; Hagège, A. Probing the Protein Corona of Gold/Silica
Nanoparticles by Taylor Dispersion Analysis-ICP-MS. Talanta 2022,243, 123386. [CrossRef]
70.
Nosti, A.J.; Barrio, L.C.; Calderón Celis, F.; Soldado, A.; Encinar, J.R. Absolute Quantification of Proteins Using Element Mass
Spectrometry and Generic Standards. J. Proteom. 2022,256, 104499. [CrossRef]
71.
Calderón-Celis, F.; Encinar, J.R. A Reflection on the Role of ICP-MS in Proteomics: Update and Future Perspective. J. Proteom.
2019,198, 11–17. [CrossRef] [PubMed]
72.
Calderón-Celis, F.; Cid-Barrio, L.; Encinar, J.R.; Sanz-Medel, A.; Calvete, J.J. Absolute Venomics: Absolute Quantification of Intact
Venom Proteins through Elemental Mass Spectrometry. J. Proteom. 2017,164, 33–42. [CrossRef] [PubMed]
73.
Cid-Barrio, L.; Calderón-Celis, F.; Abásolo-Linares, P.; Fernández-Sánchez, M.L.; Costa-Fernández, J.M.; Encinar, J.R.; Sanz-Medel,
A. Advances in Absolute Protein Quantification and Quantitative Protein Mapping Using ICP-MS. TrAC Trends Anal. Chem.
2018
,
104, 148–159. [CrossRef]
74.
Liu, C.; Yu, D.; Ge, F.; Yang, L.; Wag, Q. Fluorescent and mass spectrometric evaluation of the phagocytic internalization of a
CD47-peptide modified drug-nanocarrier. Anal. Bioanal. Chem. 2019,411, 4193–4202. [CrossRef]
75.
Matczuk, M.; Legat, J.; Shtykov, S.N.; Jarosz, M.; Timerbaev, A.R. Characterization of the Protein Corona of Gold Nanoparticles by
an Advanced Treatment of CE-ICP-MS Data. Electrophoresis 2016,37, 2257–2259. [CrossRef]
76.
Fernández-Iglesias, N.; Bettmer, J. Complementary Mass Spectrometric Techniques for the Quantification of the Protein Corona:
A Case Study on Gold Nanoparticles and Human Serum Proteins. Nanoscale 2015,7, 14324–14331. [CrossRef]
77.
Legat, J.; Matczuk, M.; Timerbaev, A.; Jarosz, M. CE Separation and ICP-MS Detection of Gold Nanoparticles and Their Protein
Conjugates. Chromatographia 2017,80, 1695–1700. [CrossRef] [PubMed]
78.
Matczuk, M.; Anecka, K.; Scaletti, F.; Messori, L.; Keppler, B.K.; Timerbaev, A.R.; Jarosz, M. Speciation of Metal-Based Nanomate-
rials in Human Serum Characterized by Capillary Electrophoresis Coupled to ICP-MS: A Case Study of Gold Nanoparticles.
Metallomics 2015,7, 1364–1370. [CrossRef] [PubMed]
79.
López-Sanz, S.; Fariñas, N.R.; Martín-Doimeadios, R.d.C.R.; Ríos, Á. Analytical Strategy Based on Asymmetric Flow Field Flow
Fractionation Hyphenated to ICP-MS and Complementary Techniques to Study Gold Nanoparticles Transformations in Cell
Culture Medium. Anal. Chim. Acta 2019,1053, 178–185. [CrossRef]
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