Food Chemistry 365 (2021) 130456

Contents lists available at ScienceDirect

Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Review

Mass spectrometry-based protein and peptide profiling for food frauds,

traceability and authenticity assessment
Mariangela Valletta a, Sara Ragucci a, Nicola Landi a, Antimo Di Maro a, Paolo Vincenzo Pedone a,
Rosita Russo a, *, 1, Angela Chambery a, *, 1
a
Department of Environmental, Biological and Pharmaceutical Sciences and Technologies, University of Campania “Luigi Vanvitelli”, 81100 Caserta, Italy

A R T I C L E I N F O A B S T R A C T

Keywords: The ever-growing use of mass spectrometry (MS) methodologies in food authentication and traceability origi­
Mass spectrometry nates from their unrivalled specificity, accuracy and sensitivity. Such features are crucial for setting up analytical
Matrix-assisted laser desorption ionization strategies for detecting food frauds and adulterations by monitoring selected components within food matrices.
mass spectrometry (MALDI-TOF MS)
Among MS approaches, protein and peptide profiling has become increasingly consolidated.
Liquid-chromatography-electrospray ionization
tandem mass spectrometry (LC-ESI MS/MS)
This review explores the current knowledge on recent MS techniques using protein and peptide biomarkers for
Protein and peptide marker assessing food traceability and authenticity, with a specific focus on their use for unmasking potential frauds and
Food authenticity adulterations. We provide a survey of the current state-of-the-art instrumentation including the most reliable and
Food traceability sensitive acquisition modes highlighting advantages and limitations. Finally, we summarize the recent appli­
cations of MS to protein/peptide analyses in food matrices and examine their potential in ensuring the quality of
agro-food products.

1. Introduction
Food fraud is a broad term used to encompass any intentional vio­
lations of the agri-food chain legislation. These actions, often motivated
by economic gain, include “the deliberate and intentional substitution,
addition, tampering, or misrepresentation of food, food ingredients, or
food packaging”. Additional criteria for defining frauds entails some
forms of deception of the consumers including the use of false or
misleading labelling, mystifying the product quality or the nature of a
product, such as the declaration of false species, varieties or origin to
reduce importation costs or false statements on production processes.
The most common food frauds are summarized in Supplementary ma­
terial, Fig. S1.
Food fraud is a practice perpetrated since antiquity as witnessed by
the findings of counterfeit Roman seals on amphorae containing fraud­
ulent olive oil and wine. Nowadays, the need to compete with powerful
businesses and manufacturers in a globalized market is among the
driving forces for food frauds, together with the growing complexity of
the global food supply system that has substantially increased the dis­
tance between the food production site and consumers (Aung & Chang,
2014; Manning, 2016). Thus, since the raw materials come from several
countries, it is increasingly difficult to trace potential sources of unin­
tentional contamination. Besides intentional product frauds are not
easily detected in highly processed foods due to complex ingredients
often provided by multiple suppliers.
Food labelling systems are constantly evolving in parallel with
legislation to support and guarantee food quality and authenticity.
Indeed, besides food intrinsic properties, some “value descriptors” such
as geographic origin, production methods, safety claims and environ­
mental welfare standards are increasingly used as quality indicators. In
Europe, the certification and the protection of origin is one of the main
authenticity issues concerning foodstuffs. In particular, these legisla­
tions include the protected designation of origin (PDO) that indicates
products entirely manufactured in a defined geographical area. In
addition, the protected geographical indication (PGI) label correlates
products to a geographical area where at least one production step

Abbreviations: HRMS, high-resolution mass spectrometry; IRMS, isotope-ratio mass spectrometry; IT, ion trap analyser; FT-ICR, Fourier-transform ion cyclotron
resonance; MALDI, matrix-assisted laser desorption ionization; MS, mass spectrometry; MS/MS, tandem mass spectrometry; LC, liquid-chromatography; LTQ, linear
trap quadrupole analyser; Q, quadrupole analyser; QqQ, triple quadrupole analyser; QT, quadrupole-trap analyser; Q-TOF, quadrupole-time of flight analyser.
* Corresponding authors.
E-mail addresses: rosita.russo@unicampania.it (R. Russo), angela.chambery@unicampania.it (A. Chambery).
1
These authors share equal senior authorship.

https://doi.org/10.1016/j.foodchem.2021.130456
Received 23 March 2021; Received in revised form 22 June 2021; Accepted 22 June 2021
Available online 25 June 2021
0308-8146/© 2021 Elsevier Ltd. All rights reserved.
M. Valletta et al.
occurred, while traditional specialities guaranteed (TSG) label provide a
protection regime for traditional food products. Furthermore, optional
quality terms (OPT) have been recently defined as new types of quality
schemes to protect the reputation of regional foods and to promote good
practices in rural and agricultural activity. Some OPT as “mountain
product” and “product of island farming” were also defined to reinforce
the consumer perception of special quality attributed to mountain and
island product.
Food authentication is closely related to food quality and safety since
common adulteration may affect overall food characteristics and may
cause foodborne illnesses following consumption. Since food authenti­
cation, food quality and safety attracted public attention in recent years,
there is an increasing need for developing and applying more efficient
tools and methodologies to analyse food components. In this framework,
the set-up of innovative technological platforms to trace food authen­
tication and verify product compliance with label descriptions is also
desirable.
2. Mass spectrometry-based food authentication methodologies
Even though traditional methods are extensively used for food
traceability, innovative analytical techniques relying on molecular
markers are nowadays among the most reliable methodologies for
determining food authenticity. Because of intrinsic advantages and
disadvantages of each analytical approach, the most appropriate method
for foodstuff authentication should be selected for each application to
discriminate original food products from non-original counterparts.
In this topical review, we mainly focused on electrospray ionization
(ESI) and matrix-assisted laser desorption ionization (MALDI) mass
spectrometry (MS)-based approaches and related chromatographic
methodologies applied to protein/peptide analyses as markers for food
authentication. A brief overview is also given on other state-of-the-art
mass spectroscopy instrumentation currently utilized in food analysis.
2.1. Overview of chromatographic approaches in food analysis
Traditional chromatographic methods have been used for food
authentication and traceability to obtain the rapid and reliable identi­
fication or quantification of proteins and peptides in complex food
matrices (Esteki, Shahsavari, & Simal-Gandara, 2019; Fanali, Dugo, &
Mondello, 2016). These applications are challenging due to the high
dynamic range, complexity and heterogeneity of compounds within
food matrices. Nevertheless, despite the overall chemical complexity of
foodstuffs, the chromatographic separation principle of adsorption and/
or partition of molecules between a mobile and a stationary phase has
proved to be advantageous to obtain unique molecular fingerprints
useful to differentiate and authenticate foods. Chromatographic tech­
niques can be classified according to the properties of stationary and
mobile phases, the packaging of the stationary phase or the driving
forces of separation. Gas chromatography (GC) exploits separations
mediated by a solid stationary phase and a gas mobile phase. In this
technique, the introduction of the sample into the chromatographic
system is mainly responsible for the resolution depending on the specific
injection system (i.e. split/splitless injection, on-column, headspace). In
high-performance liquid chromatography (HPLC) systems, a high-
pressure pump delivers a liquid mobile phase through the column
packed with a solid stationary phase where compounds are separated on
the basis of their charge (ion-exchange chromatography), molecular
mass (size-exclusion chromatography) or polarity (reversed-phase
HPLC).
A more recent evolution of HPLC is represented by ultra-high-per­
formance liquid chromatography (UPLC) analytical systems. Although
based on the same principles of HPLC, UPLC achieve enhanced separa­
tion capabilities by essentially reducing the diameter of column material
particle size at a sub-2 μm level. Compared to traditional HPLC, the
separation and quantification in UPLC are performed under higher
2
Food Chemistry 365 (2021) 130456
pressures (up to 100 MPa) that improves the resolution and sensitivity of
the analysis while reducing separation times and solvent consumption.
Based on these advantages, the use of UPLC systems has become more
and more common allowing fast analyses of food matrices even in the
context of food authentication (Di Giuseppe, Giarretta, Lippert, Sever­
ino, & Di Maro, 2015). In addition, multidimensional-liquid-chroma­
tography systems (LC/LC) have been also developed to improve the
separation of compounds from very complex food matrices (Herrero,
Ibáñez, Cifuentes, & Bernal, 2009).
Chromatographic systems can be coupled with different types of
detectors. Thermal-conductivity and flame-ionization detectors are
typically integrated with GC while ultraviolet/visible light absorbance
detectors, diode arrays and electrochemical detectors are usually asso­
ciated with HPLC. Nevertheless, coupling of mass spectrometers to
chromatographic techniques has offered an unrivalled sensitivity,
reproducibility and specificity compared to other detectors. In food
analysis, hyphenated platforms based on LC-MS and GC–MS are the most
popular, also considering their high-throughput and automation capa­
bility (Cacciola, Donato, Beccaria, Dugo, & Mondello, 2012; Tranchida,
Dugo, & Mondello, 2012). Regarding applications, while GC–MS is the
primary technique used to analyse food volatile fractions, organic con­
taminants and fats, LC-MS is mainly used for the analysis of food pro­
teins, peptides, and carbohydrates. Several recent applications in the
field of food authentication of both HPLC and GC techniques, alone or in
combination with MS, have been accurately reviewed by Santos and
Oliveira (Santos & Oliveira, 2017).
2.2. Mass-spectrometry instrumentations for food protein and peptide
analysis
MS is an analytical technique that can provide both qualitative and
quantitative information on food proteins and peptides that are ionized
and sorted on the basis of their mass/charge ratio. Given its high
reproducibility, specificity, sensitivity and high-throughput features, MS
is emerging as a powerful tool in food research (Herrero, Simo, Garcia-
Canas, Ibanez, & Cifuentes, 2012; Wang, Wang, & Cai, 2013). Mass
spectrometry instruments consist of three parts: the ion source, the mass
analyser, and the detector. The ion source is responsible for the ioni­
zation of molecules in liquid, gas or dried form. The resulting ions are
then sorted and separated by electric and/or magnetic fields according
to their mass and charge in the mass analyser. Among ionization tech­
niques, the so-called “soft ionization” techniques such ESI and MALDI,
characterized by high sensitivity in ionization with minimal in-source
fragmentation, have evolved as methods of choice for food proteins
and peptides analysis.
In MALDI sources, samples are mixed with suitable matrix solutions
and placed on a stainless-steel plate. Upon evaporation of the solvents,
the matrix and molecules co-crystallize. Sinapinic acid and α-cyano-4-
hydroxycinnamic acid are well known suitable matrices for MALDI-MS
analysis of proteins and peptides. During the analysis, samples are
irradiated with laser beam shots (usually, nitrogen laser of 337 nm),
whose energy is mostly absorbed by the matrix that protects the bio­
molecules preventing their fragmentation. Afterwards, the matrix ions
transfer the energy to sample molecules triggering their desorption. The
resulting low charge protonated/deprotonated ions are subsequently
accelerated by an electric field and separated based on their mass/
charge (m/z) ratio typically using time-of-flight (TOF) analysers.
In ESI, high voltages are applied to a liquid flowing through an
emitter (usually a glass or metallic capillary). The charged droplets are
generated at the exit of the electrospray tip through a desolvation pro­
cess driven by gas flow (N2) and capillary heating (100–300 ◦ C). The
resulting multiply protonated ions are then focused toward the analyser
region of the mass spectrometer. ESI sources are the most sensitive,
robust and reliable for studying, at femto-mole levels in few micro-litre
sample volumes, non-volatile and thermally labile biomolecules that are
not responsive to analyses by other conventional MS techniques.
M. Valletta et al.
The mass analysers can be distinguished into quadrupole-based, ion
traps (IT) and TOF. Quadrupoles consists of four cylindrical rods, par­
allel to each other with oscillating electric fields applied, in which ions
are separated based on the stability of their trajectories in the rods. For a
given ratio of voltages, only ions of a certain m/z will reach the detector.
Other ions with unstable trajectories will collide with the rods. A linear
series of three quadrupoles is known as a triple quadrupole (QqQ) mass
analyser. The first (Q1) and third (Q3) quadrupoles act as mass filters,
and the middle (Q2) quadrupole is usually employed as a collision cell.
In particular, this mass analyser is widely used mainly to carry out
quantitative determinations related to food safety and quality issues
(Picó, 2015).
The IT mass analysers operate by storing ions in the trap and
manipulating them by using direct current and radio frequency electric
fields in a series of timed events. Among IT-based mass analysers, we can
distinguish “dynamic” traps (i.e. 3D IT) from “static” traps (i.e. linear
trap quadrupole, LTQ, or Fourier-transform ion cyclotron resonance, FT-
ICR). In a 3D IT, ions are dynamically stored in a three-dimensional
quadrupole in which RF and DC potentials can be scanned to eject
successive m/z from the trap into the detector. In an LTQ, ions are
confined radially by a two-dimensional RF field and axially by stopping
potentials applied to end electrodes. In FT-ICR mass analysers, ions
move in a circular path in a magnetic field. The cyclotron frequency of
the ion’s circular motion is mass-dependent. By measuring the cyclotron
frequency, the ion’s mass can be determined. More recent evolution of
ion traps includes the high-resolution Orbitrap analyser in which ions
are electrostatically trapped in an orbit around a central, spindle-shaped
electrode. The electrode confines the ions so that they orbit around the
central electrode and oscillate back and forth along the central elec­
trode’s long axis. The image current from the trapped ions is detected
and converted to a mass spectrum using the Fourier transform of the
frequency signal.
Different analysers are also combined in hybrid MS/MS instruments,
typically endowed with higher resolving power and measurement ac­
curacy with respect to single analysers instruments. For instance, LTQ-
Orbitrap instruments offer a wide range of fragmentation capabilities
through coupling a higher energy collisional dissociation (HCD) cell to
the C-trap (Makarov & Scigelova, 2010; Zubarev & Makarov, 2013).
Ions separated by different types of analysers finally reach detectors,
typically electron multipliers or microchannel plates, that record either
charge or current. Results are then displayed as spectra by plotting the
relative abundance of detected ions as a function of the m/z ratio.
2.3. Vibrational and fluorescence spectroscopy
In recent years, the combination of vibrational (i.e. near-infrared,
mid-infrared and Raman) and fluorescence spectroscopy analytical
techniques with multivariate methods has resulted in the development
of rapid approaches for the qualitative and quantitative analysis of
several food matrices (Karoui, Downey, & Blecker, 2010). By this
analytical technique, characteristic spectra can be evaluated with che­
mometric methods to provide information about food origin or pro­
cessing. Moreover, the non-destructive nature together with the minimal
sample preparation of both vibrational and fluorescence spectroscopies
make these methods promising for the rapid, efficient and reliable food
monitoring for protecting consumers from potential frauds.
Several applications of vibrational spectroscopy have been applied
for the assessment of the authenticity of meat (Prieto, Pawluczyk,
Dugan, & Aalhus, 2017), fish and seafood (Cozzolino & Murray, 2012),
honey (Cozzolino, Corbella, & Smyth, 2011), wine (Basalekou, Pappas,
Tarantilis, Kotseridis, & Kallithraka, 2017) and cereal grains (Caporaso,
Whitworth, & Fisk, 2018). A variety of fluorescence spectroscopy-based
analytical techniques for food quality assessment has been also previ­
ously extensively reviewed (Karoui & Blecker, 2011).
Despite the advantages of applying vibrational spectroscopy as a
large-scale fingerprinting tool for food traceability, implementation of
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Food Chemistry 365 (2021) 130456
food spectra databases is needed. In addition, these methodologies also
require a combination with other analytical techniques to fully charac­
terize potential changes in food components at a molecular level.
2.4. Nuclear magnetic resonance
Nuclear magnetic resonance (NMR) represents a gold standard
methodology for metabolite food analysis providing valuable informa­
tion about quality, geographical origin and processing. Among several
NMR-based strategies for investigating foodstuffs metabolite composi­
tion (i.e. target analysis, metabolic profiling, metabolic fingerprinting,
and metabolomics), metabolomics is the most comprehensive approach
(Agapito-Tenfen, Guerra, Wikmark, & Nodari, 2013).
A schematic workflow of NMR-based metabolomics analyses is
shown in Fig. 1. At first, clear criteria should be defined for sample se­
lection to ensure the representation of whole populations and the
availability of related documentation about their properties. Sample
preparation typically includes an extraction step, where compounds of
interest are extracted from the food product using suitable solvents. For
solution-state NMR analyses, samples are resuspended in appropriate
NMR solvents which also contains a reference compound. Following the
NMR acquisition, the set of domain signals, called free induction decay
(FIDs), are processed and converted to spectra which are then used for
compound identification using multivariate statistical analyses of rele­
vant chemical shift values.
Given the chemical complexity of food matrices and the high dy­
namic range of metabolites, the sensitivity of NMR is a major limitation
of this technique, especially compared to mass spectrometry. Never­
theless, continuous developments of hardware technology enable the
analysis of even more small sample amounts through the use of the
cryogenically cooled NMR probes (cryoprobes) with a substantial in­
crease of sensitivity.
A comprehensive overview of recent applications in the field of
compositional analysis, food authentication, quality control, and human
nutrition has been recently proposed by Consonni and co-workers
(Consonni & Cagliani, 2019).
3. Isotope-ratio mass spectrometry
Among MS approaches, isotope-ratio mass spectrometry (IRMS) has
been widely applied to food authentication and traceability, providing
valuable information on food geographic, chemical, and biological ori­
gins by measuring relative isotopic ratios of their components (Camin,
Bontempo, Perini, & Piasentier, 2016; Katerinopoulou, Kontogeorgos,
Salmas, Patakas, & Ladavos, 2020). In IRMS approaches, rather than
specifically examining food protein and peptides, whole food products
or selected animal or plant tissues are usually analysed with high
quantitative accuracy.
In the present review, we focus on the application of protein and
peptide biomarkers-based MS methodologies to food analyses. Alterna­
tive approaches, such as IRMS have been discussed elsewhere (Camin,
Bontempo, Perini, & Piasentier, 2016; Katerinopoulou, Kontogeorgos,
Salmas, Patakas, & Ladavos, 2020) and are beyond the scope of this
review. Nevertheless, a summary of the most recent applications of
IRMS for the determination of food botanical and geographical origin as
well as other potential applications, including those in the field of food
authentication and traceability are reported in Supplementary material,
Tables S1–S6.
4. Proteomic workflows for protein and peptide-based
biomarker discovery for food profiling
Among the mass spectrometry and spectroscopy methodologies
currently utilized in food analysis, those based on protein and peptide
analyses by MS technologies are at the forefront as tools for food
authenticity assessment because of their sensitivity, specificity,
M. Valletta et al. Food Chemistry 365 (2021) 130456

Fig. 1. NMR-based metabolomics workflow for food analysis. Critical steps, such as sample collection and preparation, spectra processing and data analysis
are depicted.

multiplexing capacity and high-throughput capability. Additional ad­
vantages such as the relative protein/peptide stability to food processing
also support the development of novel protein-based MS workflows for
food authenticity monitoring.
Specific “fit-for-purpose” proteomic approaches can be selected for
the qualitative identification of species-specific peptide markers useful
for food authentication. Many techniques are also available for quanti­
tative proteomic analyses (Panchaud, Affolter, Moreillon, & Kussmann,
2008). Sometimes specific markers quantification by LC-MS can be also
easily obtained by extracted ion chromatograms (XIC) by measuring
peak heights or integrating peak areas to determine peptides
abundances.
Untargeted methods might be better suited for the early stages of
food biomarker discovery workflows, while targeted approaches are
usually preferred for the later steps of validation and implementation.
4.1. Untargeted proteomic approach
The boost of research output in untargeted proteomic strategies in
food analysis during the last decade is tightly associated with the
availability of improved high-resolution mass spectrometry (HRMS)
instrumentation and advanced user-friendly software for data process­
ing. Untargeted approaches are strictly related to the “-omic” concept of
characterizing the whole proteome, defined as the set of all the proteins
expressed by a cell or a tissue in a defined moment. A data-dependent
analysis (DDA) is the most popular acquisition mode of untargeted MS
strategies. In DDA mode, MS/MS fragmentation spectra are acquired for
a fixed number of the most abundant ions detected during a survey MS
scan. Generally, given the high dynamic range of proteins in food-
related matrices, for untargeted proteomic profiling, a sample prepara­
tion step is required for the enrichment or partial purification of a spe­
cific subset of target proteins or the depletion of potential interfering
proteins (Martínez-Maqueda, Hernández-Ledesma, Amigo, Miralles, &
Gómez-Ruiz, 2013). Moreover, before MS analysis, a further separation
step typically based on gel electrophoresis (gel-based approach) and/or
liquid chromatography (gel-free approach) is performed at protein and/
or peptide level. The two typical workflows currently used for protein
identification and characterization using MS are based on the so-called
“bottom-up” and “top-down” approaches. In top-down proteomics,
intact proteins or large protein fragments are directly subjected to gas-
phase fragmentation for MS/MS analysis. By contrast, in bottom-up
approaches, widely used for protein identification by MS, complex
protein mixtures or purified proteins are subjected to proteolytic
cleavage and the resulting peptides are analysed by MS or MS/MS.
A schematic diagram of a bottom-up proteomic workflow is shown in
Fig. 2A. The first developed bottom-up strategy was introduced more
than 36 years ago by combining a two-dimensional polyacrylamide gel
electrophoresis (2-DE) with peptide mass fingerprinting (PMF) by
MALDI-TOF MS. Search engines such as Sequest and Mascot are
frequently used to match and score the peptide assignment to a protein
by comparing accurate experimental and theoretical peptide masses
from curated protein databases. Most algorithms also statistically anal­
yse data to find the best match on probabilistic bases. This can be ach­
ieved by calculating the probability that the observed match is not a
random event. Despite the fractionation step based on electrophoresis
(mono- or two-dimensional) is still used in bottom-up protocols, protein
identification by MALDI-TOF-based PMF has been now mostly replaced
by LC-ESI MS/MS methodologies. In the so-called “shot-gun” proteomic
approaches, protein enzymatic digestion is directly performed in solu­
tion on complex protein mixtures without a preliminary electrophoretic
fractionation step. The resulting peptides are then separated by RP-
HPLC or UPLC coupled on-line with the mass spectrometers for MS/
MS analyses. Differently from PMF, protein identification by MS/MS
analysis is also based on amino acid sequencing of the peptide deduced
from collision induced dissociation (CID) or higher-energy c-trap
dissociation (HCD) fragmentation.
In top-down strategies, ions from intact proteins generated usually
by electrospray ionization are subjected to CID, HCD and electron
transfer dissociation (ETD) fragmentation, yielding monoisotopic mo­
lecular weights of both the intact protein and its fragments (Fig. 2B).
This analysis can integrate and support information obtained from
bottom-up approaches, in particular regarding the presence and locali­
zation of post-translational modifications (PTM). However, the
complexity of multicharged isotopic clusters from intact proteins and
their related fragments still limits the wide use of top-down strategies for
intact proteins high-throughput identification.
5. Targeted proteomic approach
The limited sensitivity of shotgun proteomic approaches related to
the high dynamic range of complex food matrices together with the high
similarity in their chemical composition is one of the main limitations in
food analysis. Nevertheless, proteome profiling of food samples is usu­
ally the first step for the identification of suitable peptides for the

4
M. Valletta et al. Food Chemistry 365 (2021) 130456

Fig. 2. Schematic diagrams of the bottom-up (A) and top-down (B) MS-based proteomics pipelines commonly used in food research.

development of targeted methods aimed at monitoring or quantifying
species-specific biomarkers within food matrices.
Indeed, differently from untargeted approaches, that focus on the
simultaneous detection of many unknown potential biomarkers, tar­
geted proteomic analyses can be used to detect and quantify a limited
number of specific peptide ions. This selective monitoring is especially
useful for the quantification of low-abundance peptides usually hardly
detectable by untargeted approaches. In the acquisition modes known as
selected ion monitoring (SIM) or selected MS/MS ion monitoring
(SMIM), selected m/z values of precursor ions are detected or frag­
mented, respectively, in a given time range or along a whole chro­
matographic run. Moreover, selected reaction monitoring (SRM) is a
tandem acquisition mode in which both a precursor ion and an associ­
ated product ion are simultaneously monitored while in multiple-
reaction monitoring (MRM), multiple precursors and product ions can
be selected for a targeted analysis. By restricting the acquisition mass
range around the m/z value of the ion(s) of interest, compared to XICs
from full scans, targeted acquisition modes strongly increase the sensi­
tivity of detection since mass spectrometer is allowed to dwell for a
longer time over a small mass range.
The unique capabilities of triple quadrupole (QqQ) mass spectrom­
eters are extremely suitable for quantitative targeted analyses. In SRM
mode, the first and the third quadrupoles act as filters to select pre­
defined m/z values of precursor and fragment ions of a given peptide,
while the second quadrupole serves as a collision cell (Fig. 3A). Several
transitions (precursor/fragment ion pairs) can be monitored, yielding a
set of chromatographic traces in which co-eluting background ions are
filtered out increasing sensitivity up to five orders of magnitude with a

Fig. 3. Representative scheme of an SRM/MRM acquisition mode on a triple quadrupole mass spectrometer (A). A typical workflow of a targeted quantitative
analysis by mass spectrometry-based on proteotypic peptides monitoring (B).

5
M. Valletta et al.
linear response over a wide dynamic range in highly complex mixtures
such as foods.
The crucial step of targeted experiments is the selection and the
optimization of transitions subsequently used for quantitation purposes
(Fig. 3B). At first, starting from a protein of interest, a set of peptides,
named proteotypic peptides, is selected based on their good MS re­
sponses and the unique assignment to the target protein. Then, instru­
mental parameters such as ionization and fragmentation parameters are
optimized for each transition. Validation steps, including the determi­
nation of the limits of detection (LOD) and quantification (LOQ), are
required before quantifying the targeted protein within samples.
Besides canonical SRM experiments performed on triple quadrupole
mass spectrometers, acquisition methods known as “pseudo SRM” are
conducted on linear ion traps or quadrupole-time of flight (Q-TOF) in­
struments. This scan mode is based on the acquisition, upon fragmen­
tation of a precursor ion, of MS/MS data on a restricted mass range
including a fragment ion of interest. Although resembling an SRM
experiment, can be essentially viewed as a SIM focused on selected
fragment ions.
Overall, targeted scanning modes are the most suitable to detect
known peptides in complex protein mixtures and already proved to be
effective tools for food product authentication (Ortea, Cañas, & Gal­
lardo, 2011).
6. Application of protein-based mass spectrometry analyses to
food authentication
Food authentication is essential to verify that a given product is
complying with its label description according to specific compositional
claims. Although specific regulations may differ among countries, for
many foods more stringent requirements must be met concerning the
species of origin or the variety of the organism. As a consequence, one of
the main authenticity issues is the need to monitor whether food prod­
ucts from one species have been mixed with similar material derived
from a cheaper species. Protein-based mass spectrometry analyses are
being increasingly applied to food authentication (Mamone, Picariello,
Caira, Addeo, & Ferranti, 2009; Mamone, Picariello, Nitride, Addeo, &
Ferranti, 2013; Samperi, Capriotti, Cavaliere, Colapicchioni, Chiozzi, &
Laganà, 2015).
We therefore reviewed significant literature studies indexed within
the Scopus database and published on different food matrices in the last
ten years using peptides and proteins as markers for food authenticity
and traceability by mass spectrometry strategies.
6.1. Milk and dairy products
Milk and dairy products represent important diet components being
sources of nutrients for both children and adults. Based on their wide
consumption, milk and dairy products are among the top foods sus­
ceptible to adulteration with financial gains for unscrupulous producers.
In recent years, MALDI-TOF and LC-MS protein analyses have been
widely used for the detection of fraudulent practices in dairy industry
(Table 1). In particular, MALDI-TOF analysis of low molecular weight
intact proteins was used to reveal potential adulterations of donkey and
goat milk with cow, ewe and buffalo milk (Di Girolamo, Masotti, Sal­
vatori, Scapaticci, Muraca, & Putignani, 2014). Moreover, the addition
of frozen buffalo milk to fresh buffalo milk was detected through the
analysis of phosphorylated peptides (Arena, Salzano, & Scaloni, 2016).
Peptide-based MALDI-TOF MS approaches proved to be efficient also to
reveal the adulteration of foods endowed with PDO, such as buffalo
mozzarella (Caira, Pinto, Nicolai, Chianese, & Addeo, 2016) and buffalo
ricotta (Russo, Rega, & Chambery, 2016).
Regarding milk processing, since the European Union Directives (92/
46 CEE and 94/71 CEE) set specific rules for the production of heat-
treated milk and milk-based products such as the use of specific tem­
peratures/times combinations for pasteurized and ultra-high
6
Food Chemistry 365 (2021) 130456
temperature (UHT) milk, several studies were focused on the analysis of
milk thermal treatments. For example, the lactosylated counterparts of
some casein phosphopeptides were found to be specific to intensely
heated kinds of milk by MALDI-TOF MS and proved to be useful to
discriminate raw and pasteurized from UHT milk (Pinto, Caira, Cuollo,
Fierro, Nicolai, Chianese, et al., 2012). Similarly, the identification of a
PyroQ modified β-casein peptide marker allows the differentiation of the
UHT milk from mildly heated milk (Dalabasmaz, Ebner, & Pischets­
rieder, 2017). Other peptide markers and specific markers related to
thermal treatments were also identified by Sassi and co-workers for
bovine, water buffalo, ovine and goat milk (Sassi, Arena, & Scaloni,
2015).
Casein tryptic peptides were used for identifying and quantifying
milk adulteration by MALDI-TOF and LC-ESI-Q MS analyses, allowing
the detection of as low as 0.5% of bovine, ovine, buffalo, and caprine
milk in milk mixtures (Cuollo, Caira, Fierro, Pinto, Picariello, & Addeo,
2010). A similar approach combining MALDI-TOF with LC-ESI-Q-TOF
MS was used to verify the authenticity of 27 kinds of cheese of the
Czech food market (Kuckova, Zitkova, Novotny, & Smirnova, 2019).
Calvano et al. described a method based on in-solution digestion of
whole milk samples followed by MALDI-TOF MS analysis of the tryptic
digests to detect cow and goat milk adulteration up to 5% (Calvano, De
Ceglie, Monopoli, & Zambonin, 2012). 2-DE in association with MALDI-
TOF MS was also successfully used to unmask powder milk in liquid milk
(Calvano, Monopoli, Loizzo, Faccia, & Zambonin, 2013) and to detect
plant protein adulterants in milk (Yang, Ghosh, & Beaudry, 2018).
Similar adulterations were also evaluated by LC-ESI-Q-TOF MS (Lu, Liu,
Gao, Lv, & Yu, 2017) and high-resolution nano-LC-ESI-Orbitrap MS/MS
(Yang, Zheng, Soyeurt, Yang, & Wang, 2019). Moreover, the detection of
powdered milk in liquid whole milk was also performed by analysing
both intact proteins and peptides through a sensitive and robust
approach using LC-ESI-Q-TOF MS analysis combined with chemometric
tools and data fusion technologies (Du, Lu, Zhang, Gao, & Yu, 2020).
Although these MS1-based methods are valuable tools for analysing
simple matrices, the extra specificity of MS/MS-based approaches is
more accurate in preventing false positives/negatives in more complex
mixtures by providing amino acid sequence information. Species-
specific peptides from bovine β-lactoglobulin and α-s1-casein were
identified by nano-LC-ESI-IT MS/MS as suitable markers for the assess­
ment of goat milk adulteration with cheaper bovine milk (Nardiello,
Natale, Palermo, Quinto, & Centonze, 2018). Recently, high-resolution
nano-LC-tandem MS analyses on a Q-Exactive Orbitrap instrument
were applied for the first time to the characterization of lactic acid
bacteria within buffalo and bovine whey starter cultures used for
cheeses production (Russo, Valletta, Rega, Marasco, Muscariello,
Pedone, et al., 2019). In this study, bacteria identification was per­
formed at genus, species and sub-species level by using the sequence
regions 33–52 and 72–82 of the S16 ribosomal protein as proteotypic
peptide markers. Regarding finished products, Guarino et al. (Guarino,
Fuselli, La Mantia, Longo, Faberi, & Marianella, 2010) developed an LC-
ESI-IT MS/MS methodology for the detection and quantification of
sheep’s milk in goat’s and cow’s cheeses. Following plasmin digestion of
the casein fraction extracted from cheese samples, a peptide specific for
sheep species was at first selected. Then, a pseudo-selected reaction
monitoring (pSRM) method targeting this marker peptide was optimized
and calibration curves were built using cheeses containing different
percentages of the three milk types. By using this approach, the authors
reported the detection of 2% of sheep’s milk in goat’s and cow’s cheeses.
A similar strategy has been applied to the detection of PDO buffalo
mozzarella adulteration with cow milk through a targeted LC-ESI-QqQ
MRM-based approach aimed at monitoring a proteotypic phosphory­
lated β-casein tryptic peptide (Russo, Severino, Mendez, Lliberia,
Parente, & Chambery, 2012). The limit of detection was three orders of
magnitude lower than that declared by the methodology officially
recognized by the European Commission (Reg. CE N. 273/ 2008).
An LC-ESI-quadrupole-trap (QT) pSRM-based approach was also
M. Valletta et al. Food Chemistry 365 (2021) 130456

Table 1
Studies published in the last ten years reporting applications of protein and peptide-based mass spectrometry analyses to milk and dairy products, meat, fish and
seafood.
MS technique Research aim Peptide/protein biomarker* Reference

Milk and dairy MALDI-TOF Adulteration of donkey/goat milk with cow, ewe Low MW proteins (less than25 kDa) Di Girolamo et al., 2014
products and buffalo milk
Adulteration of ovine, caprine and buffalo milk with CNs and whey proteins/peptides Sassi et al., 2015
bovine milk
Adulteration of cheese with bovine, ovine, buffalo α-s1-CN peptides Cuollo et al., 2010
and caprine milk
Adulteration of sheep and goat milk with bovine CNs and β-LGB peptides Calvano et al., 2012
milk
Addition of frozen buffalo milk to fresh buffalo milk CNs; α-LAC, β-LGB peptides Arena et al., 2016
Adulteration of buffalo Mozzarella milk/curd with β-CN and α-s1-CN peptides Caira et al., 2016
bovine milk
Effects of thermal treatments on milk α-s2-CN and β-CN peptides Pinto et al., 2012
Effects of thermal treatments on milk PyroQ modified β-CN peptide Dalabasmaz et al., 2017
Adulteration of PDO buffalo ricotta with bovine milk β-LGB peptide Russo et al., 2016
2-DE MALDI-TOF Adulteration of milk with plant proteins Soy, pea and wheat proteins Yang et al., 2018
Adulteration of fresh milk with powdered milk Whey proteins and/or CNs peptides Calvano et al., 2013
LC-MS Adulteration of milk with plant proteins Soy and pea proteins Lu et al., 2017
Detection of milk powder in liquid whole milk Caseins and whey milk proteins Du et al., 2020
Cheese authenticity CNs peptides Kuckova et al., 2019
LC-MS/MS Adulteration of milk with plant proteins Soy, pea, wheat and rice proteins Yang et al., 2019
Milk authenticity β-LGB and α-s1-CN peptides Nardiello et al., 2018
Characterization of buffalo and bovine whey starter Ribosomal proteins of lactic acid Russo et al., 2019
cultures bacteria
Adulteration of goat and sheep cheeses with sheep Peptides from plasmin digestion of Guarino et al., 2010
and cow milk CNs
Detection of milk allergens in foods α-CN, β-CN, α-LAC and β-LGB Ansari et al., 2011
Detection of milk allergens in foods β-LGB, β-CN, α-s2-CN and κ-CN Lutter et al., 2011
Adulteration of PDO buffalo mozzarella with bovine β-CN Russo et al., 2012
milk
Adulteration of milk adulteration with cheese whey Caseinomacropeptide Motta et al., 2014
Meat 2-DE MALDI-TOF Detection of cattle, pork, chicken, turkey, duck and Myosin light chains Montowska et al., 2012;
goose meats 2013
Detection of buffalo, sheep and goat meats Myosin light chains peptides Naveena et al., 2018
LC-MS/MS Detection of chicken meat Myosin light chains peptides Sentandreu et al., 2010
Detection of non-meat proteins in beef burgers Soy, pea, beetroot and milk peptides Mikołajczak et al., 2018
Detection of allergenic proteins in sausages, Soy, milk and egg white peptides Montowsa & Fornal,
frankfurters and pâtés 2018
Detection of pork, beef, chicken, duck and goose Peptide markers from meat Montowska & Fornal,
meats 2017
Detection of horse, pork and beef meats Myosin and myoglobin peptides Montowska & Spychaj,
2018
Detection of beef, horse, pork and lamb meats Myosin, myoglobin, haemoglobin Orduna et al., 2015;
peptides 2017
Detection of chicken, pork, beef, duck, goose meats Meat, soy, milk and egg white Montowska & Fornal,
and allergens peptides 2019
Detection of allergenic proteins in sausages Barley, maize, oats, rice, rye Jira & Munch, 2019
peptides
Detection of allergenic proteins in sausages Lupine, pea and soy peptides Hoffmann et al., 2017
LC-MS/MS Detection of soy, milk and egg white in meat Meat, soy, milk and egg peptides Montowska et al., 2019
products
Detection of meat species in Bolognese sauce Peptide markers from meat proteins Prandi et al., 2019
Detection of horse and pork meats in halal beef Peptide markers from meat proteins Von Bargen et al., 2013;
2014
Detection of pork in beef, chevron and chicken meats Peptide markers from meat proteins Sarah et al., 2016
Detection of beef, pork, horse, and lamb meats Myoglobin peptides Watson et al., 2015
Detection of pork, beef, lamb, chicken, duck meats Soy, peanut, pea and meat peptides Li et al., 2018
and allergens
Detection of pork, chicken, duck, beef, sheep meats Peptide markers from meat proteins Wang et al., 2018
Detection of duck, goose, chicken, pork and beef Peptide markers from meat proteins Fornal & Montowska,
meats 2019
Authentication of raw pork cuts and processed Myoglobin peptides Nalazek-Rudnicka et al.,
products 2019
Fish and seafood IEF + LC-MS/MS Identification of closely related shrimp and prawn Sarcoplasmic calcium-binding Ortea et al., 2010
species proteins
2-DE MALDI-TOF and/or LC- Discrimination between Sperata seenghala and Peptides from TIM Barik et al., 2013
MS/MS Sperata aor
Differentiation of Pandalus borealis from other Peptides from arginine kinase Pascoal et al., 2012
crustaceans
Differentiation of tuna species Muscle proteins Pepe et al., 2010; 2012
Detection of pufferfish Takifugu rubripes Muscle proteins Lu et al., 2010
Discrimination of two scallop populations Mantle proteins Artigaud et al., 2014
MS coupled to spectral library Differentiation of shrimp species Muscle proteins Salla & Murray, 2013
matching Identification of scallop species Muscle proteins Stephan et al., 2014
(continued on next page)

7
M. Valletta et al.
Table 1 (continued )
MS technique Research aim
Identification of fish species
Differentiation of closely related flatfish
LC-MS/MS Differentiation of shrimp species
Differentiation of hake species
Differentiation of wild and farmed gilthead bream
DART-MS/MS Differentiation of wild and farmed salmon
* CN, Casein; LAC, lactalbumin; LGB, lactoglobulin; TIM, triose phosphate isomerase.
applied for the quantification of milk traces in chocolate with a limit of
detection of 1 ng/mL in the food samples under investigation (Ansari,
Stoppacher, Rudolf, Schuhmacher, & Baumgartner, 2011). Moreover, a
LC-ESI-QqQ analysis in SRM mode was applied to the determination of
milk in protein-rich infant cereals with a detection limit of 0.2–0.5 mg/
kg (Lutter, Parisod, & Weymuth, 2011). The quantitation of milk adul­
terations with cheese whey was also accomplished by monitoring spe­
cific fragments originating from caseinomacropeptide digestion with
pepsin with a limit of detection of 1 µg/mL (Motta, Hoff, Barreto,
Andrade, Lorenzini, Meneghini, et al., 2014).
6.2. Meat and animal ingredients in foods
The potential of mass spectrometry analysis of intact proteins for
meat speciation has long been recognized since the first study reporting
the determination of the molecular weights of haemoglobin and
myoglobin from different animal species by ESI-MS (Taylor, Linforth,
Weir, Hutton, & Green, 1993). Subsequently, the evidence that the
assessment of protein mass differences could be a valuable strategy for
meat speciation was supported by the development of ESI-MS/MS ap­
proaches based on the use of protein fragments (Ponce-Alquicira &
Taylor, 2000). More recently, advances in mass spectrometry instru­
mental technologies allowed to overcome the limitations of these pio­
neering attempts (Table 1).
A combined approach exploiting 2-DE and MALDI-TOF MS has been
used to unravel differences in protein patterns between cattle, pig,
chicken, turkey, duck and goose meats and their processed products. In
this work, myosin light chain isoforms, together with some regulatory
proteins and metabolic enzymes, were identified as proteins markers for
meat authentication (Montowska & Pospiech, 2012, 2013). Based on
these findings, Naveena et al. also utilized both two-dimensional and
OFFGEL electrophoresis coupled to MALDI-TOF MS for the authentica­
tion of raw and cooked buffalo, sheep and goat meats and their mixtures
by monitoring species-specific peptide markers derived from myosin
light chains (Naveena, Jagadeesh, Kamuni, Muthukumar, Kulkarni,
Kiran, et al., 2018).
A proteomic technique has been developed for the detection of
chicken meat within mixed meat preparations by using OFFGEL iso­
electric focusing coupled to ESI-LC-IT MS/MS (Sentandreu, Fraser,
Halket, Patel, & Bramley, 2010). Starting from selected species-specific
biomarkers, the same study reported the development of a quantitative
approach allowing the detection of up to 0.5% contaminating chicken
meat in pork meat by using the absolute quantification (AQUA™) stable
isotope peptides (Sentandreu, Fraser, Halket, Patel, & Bramley, 2010).
High-resolution mass spectrometry was also shown to be suitable to
reveal vegetable proteins with allergenic potential in various types of
meat products. In particular, nano-LC-ESI-Q-TOF MS/MS analysis of
sausages, frankfurters and pâtés allowed the identification of allergenic
proteins from soy, milk and egg white (Montowska & Fornal, 2018).
Since eggs are widely used in the food industry a list of 16 potential
peptide biomarkers for the detection of eggs in other processed foods
was also recently reported (Gavage, Van Vlierberghe, Van Poucke, De
Loose, Gevaert, Dieu, et al., 2019). Hoffmann et al. monitored specific
marker peptides for the simultaneous detection of lupine, pea and soy in
sausages (Hoffmann, Münch, Schwägele, Neusüß, & Jira, 2017). After
8
Food Chemistry 365 (2021) 130456
Peptide/protein biomarker* Reference
Peptides from muscle proteins Wulff et al., 2013
Muscle proteins Nessen et al., 2016
Peptides from arginine kinase Ortea et al., 2011
Peptides from parvalbumin Carrera et al., 2011
Peptides from sarcoplasmic proteins Piovesana et al., 2016
Peptides from muscle proteins Fiorino et al., 2019
optimization of a targeted MRM methodology, the authors demon­
strated the capability of simultaneous detection of legumes lupine, pea,
and soy proteins in meat products with LODs in the range of 2–5 mg/kg
(Hoffmann, Münch, Schwägele, Neusüß, & Jira, 2017). Jira et al. simi­
larly investigated the presence of the six most important grain species (i.
e. barley, maize, oats, rice, rye and wheat) in sausages with detection
limits below 5 mg/kg (Jira & Munch, 2019). Moreover, LC-ESI-Q-TOF
MS/MS was applied to the analysis of ready-to-cook commercially
produced burgers with the aim to detect soy, pea, milk and beetroot
proteins, including known allergenic proteins from soy (glycinin and
β-conglycinin), pea (vicilin and provicilin) and milk (αS1-casein and
β-lactoglobulin) (Mikołajczak, Fornal, & Montowska, 2018). High-
resolution LC-ESI-Orbitrap MS/MS proved also to be efficient for the
detection of beef, horse, pork, and lamb meat in meat mixtures by
monitoring tryptic peptides from myosin, myoglobin and haemoglobin
(Ruiz Orduna, Husby, Yang, Ghosh, & Beaudry, 2015; 2017).
In the context of high-resolution MS-based quantitative techniques,
label-free approaches further represent a reliable, versatile and cheaper
alternative MS technique for meat authentication. In particular, nano-
LC-ESI-Q-TOF MS/MS enabled to discriminate between pork, beef,
chicken, duck and goose meats, as well as to quantify relative changes in
protein abundance in various commercially processed meat products by
using 20 heat-stable peptide markers (Montowska & Fornal, 2017). The
method allowed the detection of 1% (w/w) of chicken and 1% (w/w)
pork in meat mixtures of the three species, as well as of 0.8% (w/w) of
beef proteins in commercial poultry frankfurters. A similar approach has
been proposed to authenticate industrially processed sausages made
with horse meat, pork and beef with a LOD of 5% (w/w) for pork and
beef and 1% (w/w) for horse meat in the matrix containing the three-
components (Montowska & Spychaj, 2018). The same authors also re­
ported the application of the AQUA strategy (Montowska & Fornal,
2019) and an LC-ESI-QqQ analysis in MRM mode (Montowska, Fornal,
Piątek, & Krzywdzińska-Bartkowiak, 2019) for the simultaneous quan­
tification of meat and allergenic protein additives in meat products by
using peptide markers identified in previous studies (Fornal & Mon­
towska, 2019; Montowska & Fornal, 2017, 2018). Indeed, by MRM
analysis, a LOD of 0.14% (w/w) for milk proteins and of 0.45% (w/w)
for soy and egg white proteins was achieved in final meat products
(Montowska, Fornal, Piątek, & Krzywdzińska-Bartkowiak, 2019).
Von Bargen et al. developed an MRM method for the detection of
horse and pork in halal raw beef using species-specific tryptic marker
peptides with a LOD of 0.55% (w/w). By using MRM3 mode on a QT
system, the authors were able to enhance sensitivity and to detect pork
contamination in beef down to 0.13% (w/w) (von Bargen, Dojahn,
Waidelich, Humpf, & Brockmeyer, 2013). A similar experimental pro­
tocol allowed the analysis of processed/highly processed food samples
with a LOD down to 0.24% for horse or pork meat in a beef matrix (von
Bargen, Brockmeyer, & Humpf, 2014).
Other quantitative MRM approaches have been developed for the
rapid and reliable detection of meat species in food products. Among
them, myoglobin peptides were successfully used as protein markers for
the quantitative determination of meat species (i.e. beef, pork, horse and
lamb) (Watson, Gunning, Rigby, Philo, & Kemsley, 2015) and the
authentication of commercially raw pork cuts and other meat products
(Nalazek-Rudnicka, Klosowska-Chomiczewska, Wasik, & Macierzanka,
M. Valletta et al.
2019). MRM was also used for the identification, at a qualitative level, of
porcine-specific peptide markers in thermally processed meat to differ­
entiate pork from beef, chevon and chicken meats (Sarah, Faradalila,
Salwani, Amin, Karsani, & Sazili, 2016). Moreover, heat-stable specific
peptides were used to identify and simultaneously quantify eight
different species (i.e. pork, beef, lamb, chicken, duck, soy, peanut, and
pea), allowing the detection of up to 0.5% (w/w) contamination of any
of the eight species in self-made food mixtures (Li, Zhang, Li, Zhao, Guo,
& Wang, 2018). Other heat-stable peptide biomarkers were also used for
the MRM-based detection of five animal species (i.e. pork, chicken,
duck, beef, and sheep) in cocked meats (Wang, Zhou, Ren, Xu, Yang,
Guo, et al., 2018). On whole finished products, proteotypic peptides
from eight different meat species (i.e. duck, rabbit, chicken, turkey,
buffalo, equine, deer and sheep) were identified within Bolognese sauce
with LODs ranging from 0.2% to 0.8% (w/w) (Prandi, Varani, Faccini,
Lambertini, Suman, Leporati, et al., 2019).
Regarding animal ingredients, gelatin is one of the most widely used
in the food industry as a foam stabilizer, gelling, clarifying or binding
agent and emulsifier. Gelatin is obtained by partial denaturation of
collagen extracted from skins, bones and connective tissues of animals
such as bovine and porcine. An accurate and sensitive nano-LC-ESI Q-
TOF MS/MS approach has been developed to determine the origin of
species in commercial food gelatines, meat mixtures and extracts pre­
pared from meats (Grundy, Reece, Buckley, Solazzo, Dowle, Ashford,
et al., 2016). Yang and co-workers also set up a targeted strategy for
gelatin speciation (Yang, Ghosh, & Beaudry, 2018). In this work,
species-specific peptide biomarkers were identified, characterized and
quantified by using extracted ion chromatograms by LC-ESI-Orbitrap
MS/MS analysis. Then, the method was tested on gelatin used in char­
cuterie meats obtained from grocery stores, fruit-snacks and gelatin
capsules allowing the detection down to 0.1% (w/w) of pork gelatin
(Yang, Ghosh, & Beaudry, 2018). Furthermore, a single porcine marker
peptide from gelatin was selected to develop a sensitive MRM-based
approach to identify porcine materials within both in-house-made and
commercial products down to 0.04% (w/w) (Guo, Xu, Zhou, & Huang,
2018). Yilmaz and co-workers developed a nano-UPLC-ESI-Q-TOF MSE
(i.e. elevated energy MS) workflow based on porcine and bovine marker
peptides to identify the animal origin of gelatin in dairy products such as
yoghurt, cheese and ice cream (Yilmaz, Kesmen, Baykal, Sagdic, Kulen,
Kacar, et al., 2013). The MSE tandem MS data acquisition method is
based on alternating low-energy collision-induced dissociation and
high-energy collision-induced dissociation, where the former is used to
obtain precursor ion accurate mass and intensity data for quantification
and the latter is used to obtain product ion accurate mass.
6.3. Fish and seafood
The high phenotypic similarity between related marine species
makes intentional or accidental adulteration of seafood products,
including shellfish and fish, one of the most common food frauds. As a
consequence, global commercial requirements on labelling and trace­
ability are needed to guarantee the authenticity and traceability of
seafood trading. In the last ten years, the development of reliable
analytical methodologies aimed at identifying closely related marine
species represents an issue of primary relevance for the seafood industry
to prevent mislabelling and adulteration (Table 1).
One of the first approaches proposed for fish authentication was
focused on the assessment of intra-species variability of fourteen shrimp
species of commercial interest by native isoelectrofocusing (IEF)
coupled to LC-ESI-IT MS/MS analysis (Ortea, Cañas, Calo-Mata, Barros-
Velázquez, & Gallardo, 2010). To date, this study represents the most
comprehensive electrophoretic characterization of closely related
shrimp species. The same authors further identified several species-
specific peptides from Pandalus borealis potentially useful for the un­
equivocal identification of this shrimp species by coupling a genomic
approach based on PCR-RFLP (restriction fragment length
9
Food Chemistry 365 (2021) 130456
polymorphism) assays and MALDI-TOF and LC-IT MS/MS analyses of
protein spots from 2-DE (Pascoal, Ortea, Gallardo, Canas, Barros-
Velazquez, & Calo-Mata, 2012). Other combined proteomic strategies
integrating 2-DE and mass spectrometry analyses have been extensively
used in the field of fish authentication (Barik, Banerjee, Bhattacharjee,
Das Gupta, Mohanty, & Mohanty, 2013; Mazzeo & Siciliano, 2016).
Specifically, MALDI-TOF and LC-QT MS/MS analyses were both applied
to the characterisation of electrophoretically separated sarcoplasmic
proteins of tuna species (i.e. Thunnus thynnus, Thunnus alalunga, Thunnus
albacares, and Thunnus obesus) (Pepe, Ceruso, Carpentieri, Ventrone,
Amoresano, & Anastasio, 2010; Pepe, Ceruso, Carpentieri, Ventrone,
Amoresano, Anastasio, et al., 2012). Skeletal muscle proteins profiling of
the pufferfish Takifugu rubripes was also performed to investigate their
use as potential marker proteins for monitoring and controlling the
quality of Takifugu rubripes meat (Lu, Zheng, Liu, Li, Chen, & Chen,
2010). 2-DE in combination with MALDI-TOF/TOF MS analysis has been
also used for the identification of potential protein biomarkers for
discriminating between two populations of the great scallop Pecten
maximus (Artigaud, Lavaud, Thebault, Jean, Strand, Strohmeier, et al.,
2014).
To overcome the limited availability of genome sequences, fish
species authentication by MALDI-TOF MS and spectral library matching
without the electrophoretic separation step was also proposed as a
robust and reliable alternative for shrimps (Salla & Murray, 2013) and
scallop (Stephan, Johler, Oesterle, Näumann, Vogel, & Pflüger, 2014)
identification. Alternatively, protein digests can be used in proteome-
wide MS/MS approaches to identify different fish species by counting
the number of shared tandem mass spectra between the investigated
sample and reference spectral libraries (Nessen, van der Zwaan, Grevers,
Dalebout, Staats, Kok, et al., 2016; Wulff, Nielsen, Deelder, Jessen, &
Palmblad, 2013).
Selected ion monitoring of parvalbumin peptides by ESI-IT tandem
mass spectrometry was shown to be suitable for the identification of
relevant commercial fish species belonging to the Merlucciidae family in
less than 2 h (Carrera, Canas, Lopez-Ferrer, Pineiro, Vazquez, & Gal­
lardo, 2011). By applying a similar methodology, seven commercial
closely related species of Decapoda shrimps were classified by using
proteotypic peptides of arginine kinase proteins (Ortea, Cañas, & Gal­
lardo, 2011). A shotgun method was developed for the proteome char­
acterization of gilthead sea bream (Sparus aurata) muscle samples
(Piovesana, Capriotti, Caruso, Cavaliere, La Barbera, Zenezini Chiozzi,
et al., 2016). More recently, high-resolution MS analyses on an Orbitrap
analyser coupled to a direct analysis in real time (DART) source allowed
the discrimination of wild-type from farmed salmons belonging to Salmo
salar species (Fiorino, Losito, De Angelis, Arlorio, Logrieco, & Monaci,
2019).
6.4. Cereals
Cereals, including rice, barley, and wheat are the most widely har­
vested crops in the world. Given the increasing demand in the global
market, cereals production is exposed to a growing number of frauds. In
the last decade, the improvements in MS technologies together with the
increasing availability of genomic database, enabled the use of MS-
based approaches for the analysis of cereal proteins (Table 2). Most ef­
forts are finalized to identify marker peptides suitable for discriminating
different species of high economic interests or even those representing a
risk for consumers with allergies and/or intolerances.
Different durum wheat cultivars were analysed by Prandi and co-
workers to determine the content of the amylase/trypsin inhibitor
CM3, a common wheat allergen, by using marker peptides derived from
the protein. The authors detected the CM3 protein at concentrations
ranging from 0.22 to 1.11 mg/g in wheat samples by a quantitative
analysis performed on a single quadrupole mass spectrometer by using
an isotopically labelled standard peptide (Prandi, Faccini, Tedeschi,
Galaverna, & Sforza, 2013).
M. Valletta et al. Food Chemistry 365 (2021) 130456

Table 2
Studies published in the last ten years reporting applications of protein and peptide-based mass spectrometry analyses to cereals, tree nuts and wine.
MS technique Aim of the analysis Peptide/protein biomarker Reference

Cereals LC-MS/MS Differentiation of durum wheat varieties Peptides from α-amylase inhibitor Prandi et al., 2013
CM3
Detection of common wheat in commercial durum wheat flour Peptides from α-gliadin Prandi et al., 2012
Detection of common wheat in commercial durum wheat flour and pasta Peptides from puroindoline-A Russo et al., 2014
Detection of wheat, rye and spelt in bread Peptides from gluten proteins Bönick et al., 2017
Detection of wheat gluten Peptides from gluten proteins Schalk et al., 2018
Detection of wheat in rye, millet, oats, sorghum, buckwheat and soy Peptides from gluten proteins Colgrave et al., 2015
flours
Detection of oats in processed foods Peptides from gluten proteins Dawson et al., 2018
Detection of barley in processed foods Peptides from gluten proteins Colgrave et al., 2016
Identification of wheat, rye, barley, oats, corn, soy and rice Peptides from gluten proteins Martínez-Esteso et al., 2016
Tree nuts LC-MS/MS Detection of hazelnut in foods Peptides from allergenic proteins Van Vlierberghe et al., 2020
Detection of peanut in processed foods Peptides from allergenic proteins Gavage et al., 2020
Detection of peanut and tree nuts in processed or unprocessed foods Peptides from allergenic proteins Sealey-Voyksner, et al.
2016
Detection of almond, cashew, hazelnut, peanut, pistachio, walnut in foods Peptides from allergenic proteins Korte et al., 2016
Detection of hazelnut in foods Peptides from allergenic proteins Ansari et al., 2012
Wine MALDI-TOF MS Classification of Croatian white wines Wine proteins and peptides Rešetar et al., 2016
LC-MS/MS Detection of fining agents in white wines Peptides from caseins Cereda et al., 2010
Detection of fining agents in red wines Caseins D’Amato et al., 2010
Authentication of Amaro Branzi Soluble liqueur proteins Lerma-García et al., 2014
Detection of fining agents in white wines Peptides from caseins Monaci et al., 2010; 2011
Detection of fining agents in white wines Peptides from milk and egg proteins Monaci et al., 2013

The determination of common wheat in durum wheat samples was
assessed by LC-ESI-QqQ MS/MS analysis, at both qualitative and
quantitative level, by using peptide markers from α-gliadin (Prandi,
Bencivenni, Tedeschi, Marchelli, Dossena, Galaverna, et al., 2012) and
puroindoline A (Russo, Cusano, Perissi, Ferron, Severino, Parente, et al.,
2014). In this work, by using an MRM-based approach, Russo and co-
workers were able to detect up to 0.01% of common wheat in raw ma­
terials (kernels) and processed products (pasta).
Several studies also applied MRM-based quantitative strategies to
cereal composition analysis within commercial food products. By
determining the total protein content and the gluten-enriched fractions
of sixteen cereal grains, including wheat, rye, barley and oats, Colgrave
and co-workers selected a pool of specific wheat markers for detecting
wheat contaminations in several commercially flours, with a limit of
detection down to 15 mg/kg in a deliberately contaminated soy flour
(Colgrave, Goswami, Byrne, Blundell, Howitt, & Tanner, 2015). Simi­
larly, by LC-MS/MS analysis of gluten-enriched extracts from barley and
oat cultivars, a panel of peptide markers allowed the detection of barley
and oat contamination in breakfast cereals (Colgrave, Byrne, Blundell, &
Howitt, 2016; Dawson, Mendoza-Porras, Byrne, Hooper, Howitt, &
Colgrave, 2018). Moreover, peptide markers from common wheat, spelt
and rye were used to detect and quantify contaminations of these crop
species in baked products with LOD values in the range 0.5%-1%
(Bönick, Huschek, & Rawel, 2017). Specific wheat gluten peptides
identified by LC-MS/MS were also proposed as specific markers in an
MRM-based screening for the quantification of gluten within gluten-
containing species (Martínez-Esteso, Nørgaard, Brohée, Haraszi,
Maquet, & O’Connor, 2016; Schalk, Koehler, & Scherf, 2018).
6.5. Tree nuts
Tree nuts, including almonds, Brazil nuts, cashews, hazelnuts, mac­
adamia nuts, pecans, pine nuts, pistachio nuts and walnuts, are common
allergen sources with a prevalence of up to 5% in the general population
(McWilliam, Koplin, Lodge, Tang, Dharmage, & Allen, 2015). A
remarkable increase in tree nuts-driven allergy incidence could be also
likely due to the large consumption justified by their nutritional benefits
and wide use in the food industry (Özenç & Bender Özenç, 2015).
Reliable MS-based analytical methods have been developed for the
detection and quantification of allergenic proteins from tree nuts
(Table 2) according to the EU Regulation N. 1169/2011 to establish
compliance of food ingredients indicated within foodstuff labels.
Marker peptides from the three major hazelnut allergenic proteins
(Cor a8, Cor a9 and Cor a11) have been used for the development of LC-
MS/MS methods for detecting hazelnut in foods. Indeed, an LC-ESI QT
MS/MS method in SRM mode was developed as a valid alternative to
immunological assays to determinate trace amounts of allergenic nut
proteins in processed foods (Ansari, Stoppacher, & Baumgartner, 2012).
In this study, synthetic peptides of eight tryptic fragments from Cor a8,
Cor a9 and Cor a11 proteins, were used to build a calibration curve
ranging between 3.1 and 4166.7 ng/mL (Ansari, Stoppacher, & Baum­
gartner, 2012). Furthermore, through a comprehensive LC-ESI-Q-TOF
MS/MS-based approach, additional marker peptides from the same
allergenic proteins were identified and their robustness for applications
in food processing was validated by analysing roasted and caramelized
nuts, chocolate and fermented nut milk (Van Vlierberghe, Gavage, Dieu,
Renard, Arnould, Gillard, et al., 2020). A similar approach was also
applied to the identification of highly conserved marker peptides for
peanuts and tree nuts detection in food matrices (Sealey-Voyksner,
Zweigenbaum, & Voyksner, 2016).
Untargeted high-resolution mass spectrometry also allowed the
identification and quantification of selected proteotypic peptides from
six allergenic nut species (almond, cashew, hazelnut, peanut, pistachio
and walnut) by a shotgun proteomic approach. By this strategy, trace nut
contaminations in processed foods were detected and quantified by LC-
QT MS. Furthermore, LODs below 10 μg/g, comparable with routinely
used ELISA and PCR methods, were obtained for several nuts by ana­
lysing spiked matrix samples (Korte, Lepski, & Brockmeyer, 2016).
Recently, by high-resolution LC-ESI- Q-TOF MS analysis, sixteen po­
tential peanut peptide biomarkers from Ara h1 and Ara h3 proteins, the
two most abundant peanut allergens, were identified in both raw and
processed food products, paving the way to further studies aimed at the
development of an MS-based quantitative approach for peanut detection
in complex food matrices (Gavage, Van Vlierberghe, Van Poucke, De
Loose, Gevaert, Dieu, et al., 2020).
6.6. Wine
In parallel with the growing attention to quality control in the wine
industry, there has been an increasing demand for analytical techniques
to assess wine authenticity and traceability. Regarding the development
and application of protein-based MS approaches for wine analysis, the
extraction and the enrichment of proteins present at low concentration
levels within finished products are the most critical steps. Nevertheless,

10
M. Valletta et al.
several MS-based applications were established to classify wines and to
detect and quantify non-wine proteins such as fining agents (Table 2).
MALDI-TOF fingerprinting of intact proteins followed by a statistical
interpretation of data using principal component and cluster analyses
was applied to the characterization of 33 Croatian white wines (Rešetar,
Marchetti-Deschmann, Allmaier, Katalinić, & Kraljević Pavelić, 2016).
Reliable methods enabling the detection of proteins added to wine as
fining agents to remove tannins, phenols and the residual of other pro­
teins are also required. Indeed, these protein agents, generally caseins
and lysozyme, must be declared on wine labels only if above 0.25 mg/L,
representing potential food allergens (Commission Implementing
Regulation EU N. 579/2012). Analytical approaches based on combi­
natorial peptide ligand libraries (CPLLs) coupled to nano-LC-ESI Q-TOF
were reported to be highly efficient in capturing traces of proteinaceous
additives in food matrices (Cereda, Kravchuk, D’Amato, Bachi, &
Righetti, 2010; D’Amato, Kravchuk, Bachi, & Righetti, 2010). By this
approach, traces of casein used as a fining agent in both white and red
wines were identified with detection limits of 1 µg/L and 3.8 µg/L,
respectively. Similarly, CPPLs were used for investigating the proteome
of a popular aperitif in Northern Italy, called “Amaro Branzi”, to assess
the genuineness of this commercial liqueur (Lerma-García, D’Amato,
Fasoli, Simó-Alfonso, & Righetti, 2014). In detail, nano-LC-ESI-IT MS/
MS analysis of proteins captured by CPPLs allowed the detection of
proteins used for the aperitif production and originating from honey,
orange peels, Angelica officinalis, Gentiana lutea (Lerma-García, D’Am­
ato, Fasoli, Simó-Alfonso, & Righetti, 2014). Monaci and co-workers
developed LC-ESI MS/MS methods on both Q-TOF and Orbitrap in­
struments for the detection of casein peptides in white wine (Monaci,
Losito, Palmisano, & Visconti, 2010, 2011). An LC-ESI-Orbitrap MS/MS
method for the simultaneous detection of casein, lysozyme and oval­
bumin in white wines has been proposed by the same authors, allowing
the multiplex determination of these fining agents with LODs ranging
from 0.4 to 1.1 μg/mL (Monaci, Losito, De Angelis, Pilolli, & Visconti,
2013).
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
This work was supported by funding from University of Campania
“Luigi Vanvitelli” through the VALERE program and MISE, project
NUTRABEST PON I&C 2014-2020 Prog. n. F/200050/01-03/X45.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.foodchem.2021.130456.
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