J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 35 4 6 35 5 9

Available online at www.sciencedirect.com

www.elsevier.com/locate/jprot

Review

Mass spectrometry for nutritional peptidomics: How to analyze

food bioactives and their health effects

Alexandre Panchaud a, 1 , Michael Affolter a, 1 , Martin Kussmann b, c,

a
Functional Genomics Group, Nestl Research Centre, Lausanne, Switzerland
b
Proteomics and Metabonomics Core, Nestl Institute of Health Sciences, Lausanne, Switzerland
c
Faculty of Science, Aarhus University, Aarhus, Denmark

AR TIC LE I N FO ABS TR ACT

Article history: We describe nutritional peptidomics for discovery and validation of bioactive food peptide
Received 5 August 2011 and their health effects. Understanding nature and bioactivity of nutritional peptides
Accepted 14 December 2011 means comprehending an important level of environmental regulation of the human
Available online 30 December 2011 genome, because diet is the environmental factor with the most profound life-long
influence on health.
Keywords: We approach the theme from three angles, namely the analysis, the discovery and the
Mass spectrometry biology perspective. Food peptides derive from parent food proteins via in vitro hydrolysis
Peptidomics (processing) or in vivo digestion by various unspecific and specific proteases, as opposed
Peptide to the tryptic peptides typically generated in biomarker proteomics. A food bioactive
Bioactive peptide may be rare or unique in terms of sequence and modification, and many food
Bioavailability genomes are less well annotated than e.g. the human genome.
Nutrition Bioactive peptides can be discovered either empirically or by prediction: we explain both the
classical hydrolysis strategy and the bioinformatics-driven reversed genome engineering. In
order to exert bioactivity, food peptides must be either ingested and then reach the intestine in
their intact form or be liberated in situ from their parent proteins to act locally, that is in the
gut, or even systemically, i.e. through the blood stream. This article is part of a Special Section en-
titled: Understanding genome regulation and genetic diversity by mass spectrometry.
2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3547
1.1. Proteomic technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3547
1.2. Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3547
1.3. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3547
2. Technological challenges related to bioactive peptide discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3548

This article is part of a Special Section entitled: Understanding genome regulation and genetic diversity by mass spectrometry.
Corresponding author at: Proteomics & Metabonomics Core, Nestl Institute of Health Sciences, CH-1000 Lausanne, Switzerland. Tel.: +41
21 924 64 01.
E-mail address: martin.kussmann@nestle.com (M. Kussmann).
1
Equally contributed to the work.

1874-3919/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.jprot.2011.12.022
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 35 4 6 3 55 9 3547

2.1. Medium peptides (725 AAs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3549

2.2. Large peptides (>25 AAs). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3550
2.3. Small peptides (< 7 AAs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3550
2.4. Common issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3551
3. Status of bioactive peptide discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3551
3.1. Functional characterization of bioactive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3551
3.2. Classical screening vs. reverse genome engineering for bioactive peptide discovery . . . . . . . . . . . . . . 3554
3.3. Site of action and availability of bioactive peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3555
4. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3556
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3556

1. Introduction rise to a wide range of peptide sizes and terminal amino

Next to sequencing, mass spectrometry is the platform to
molecularly assess virtually all levels of genomic expression,
regulation and variability. The present issue on mass spec-
trometry in genomics discusses mass spectrometric tools for
the analysis of genome variability and regulation at those bio-
molecular levels that are directly encoded by the genome, i.e.
mass spectrometry for DNA, RNA, protein and peptide analy-
sis: we review the ensemble of genetics [Stange], epigenetics
[Sheppard; Gluckman], transcript analysis [Kirpekar; Lim-
bach], protein interactomics [Kster; Urlaub], protein/peptide
biomarker validation [Borchers; Aebersold] plus peptidomics
for bioactives [this article]. We do neither cover classical dis-
covery proteomics nor transcriptomics (today chip- or se-
quencing based) or metabonomics (MS or NMR-based). This
has been accomplished extensively elsewhere by various
groups including ours [14] and it would expand way beyond
the scope of the present and distinct mass spectrometric
and -omic angle on genomics.
This last chapter of the present issue focuses on nutri-
tional peptidomics for discovery, quantification and charac-
terization of food-derived peptide bioactives and, to some
extent, their health effects. Analyzing and understanding na-
ture and bioactivity of nutritional peptides, typically liberated
from parent food proteins, means comprehending an impor-
tant level of environmental regulation of the human genome:
food and drinks are the most important physical matter we
take into our body and diet is the environmental factor with
the most profound life-long influence on our health [5].
Including nutritional peptidomics into this scope reflects at
the same time also our understanding of nutritional genomics
as a metagenomic approach [6]: we need to consider the host,
the food and the gut microbial genomes to understand the
interplay between nutrition, environment and host health.
We approach the theme of mass spectrometry and peptide
bioactives in three ways, namely from the proteomic technol-
ogy, the bioinformatic or classical discovery and the function-
al biology angle.
1.1. Proteomic technology
The food peptidome poses a formidable challenge to be ana-
lyzed in the food matrix on the one hand and to be followed
up in vivo on the other hand: food peptides derive from parent
food proteins by in vitro hydrolysis (processing) or in vivo
digestion by various unspecific and specific proteases giving
acids, as opposed to the tryptic peptides typically generated
in biomarker proteomics. We describe adapted and tailored
approaches to cope with and manage this complexity. In addi-
tion, each food bioactive peptide may be rare or unique in
terms of sequence and modification and one may therefore
have only very few shots for its identification. Last but not
least, many food genomes are less well annotated than e.g.
the human genome, which may generate the necessity for
de novo sequencing by mass spectrometry.
1.2. Discovery
Bioactive peptides can be discovered by either an empirical or
a more innovative predictive approach: we briefly explain the
classical hydrolysis and activity testing/screening strategy as
increasingly complemented by the food genome-rooted,
peptide sequence-based bioinformatics method, also termed
reversed genome engineering [7].
1.3. Biology
Bioactive peptides, be they derived from food or other sources,
have been compiled and annotated in databases that provide
information on e.g. their activity in vivo, with the most nu-
merous cases being attributed to antioxidant and anti-hyper/
hypotensive effects. In order to exert such bioactivity, food
peptides must be either ingested and then reach the intestine
in their intact form, or be liberated in situ, i.e. in the intestine,
form their parent proteins to act locally, that is in the gut, or
even systemically, meaning through the blood stream. Apart
from the effective degradation of proteins to di- and tripep-
tides and their subsequent efficient uptake by specific trans-
porters [8] from the gut lumen into the blood stream (mainly
to provide the body with building blocks), ingested food pep-
tides rarely make it into the gut and across its wall as intact
species. Rather, proteins are degraded to these very short pep-
tides or completely hydrolyzed to amino acids [9]; or, rather
few, proteins are transported intact across the gut wall
[10,11]. The promising route for delivery of specific bioactivity
through food protein is in situ peptide release (by host or gut
microbial enzymes) and local action in the gut.
Following these three avenues we aim at providing an
integrated view of the hostfood genome interaction with
the emphasis on proteomics and mass spectrometry for the
assessment of protein nutrition beyond macronutrient sup-
ply, namely for in vivo delivery of healthy bioactivity. With
3548 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 35 4 6 35 5 9
such perspective, we argue that food peptidomics should be
increasingly combined and integrated with health biomarker
science, in nutrition and beyond.
2. Technological challenges related to
bioactive peptide discovery
Unlike biomarker proteomics, nutritional peptidomics deals
with the identification and quantification of nutritionally
relevant peptides that may individually or in combination
exert bioactivity. While for biomarkers, identification and
quantification relies on several peptides that can unambigu-
ously be inferred back to one parent protein sequence
accounting for the key biological activity, bioactive peptides
are the active molecules per se. Therefore, identification relies
on the detection of possibly very few copies of this one par-
ticular sequence in its full length. This imposes a challenge as
one cannot select so called proteotypic peptides for the
identification as usually performed in the case of protein
biomarkers [12].
If one considers two important parameters to represent
the analytical peptide environment, namely peptide length
(up to 500 amino acids) and dynamic range (e.g. twelve orders
of magnitude in human blood plasma [13,14], both fields, pro-
teomics (biomarkers) and peptidomics (bioactives), for which
identification and validation is based on the peptide level, do
cover different analytical spaces. While proteomics comprise
usually peptide molecular weights of approx. 700 to 3000 Da
with a dynamic range of twelve orders of magnitude, peptido-
mics certainly spans a greater peptide length distribution and
P5 P4 P3 P2 P1
-5 -4 -3 -2 -1
A B
Fig. 1 Enzyme specificity profiling. (A) Trypsin specificity which cleaves C-terminal of arginine (R) and lysine (K), (B) a less
specific enzyme cocktail with some trypsin/chymotrypsin activity detectable and (C) unspecific proteolytic enzyme activity
with non-detectable specificity.
possibly also a similar dynamic range. The major reason for
this greater analytical complexity in nutritional peptidomics
is the diverse selectivity and specificity at the food protein
processing and digestion level, conferred by the multiple in
vitro and in vivo proteases.
Specific enzymes of high purity, with trypsin being the
flagship, are usually applied in protein biomarker identifica-
tion at peptide level. As a consequence, generated peptides
share similar properties, e.g. charge state or length, which
reduces the analytical range to be covered. In the case of
bioactives, peptides are usually either released through
processes such as fermentation or enzymatic hydrolysis to
enrich a fraction or extract for specific bioactive peptides or
their precursors [15]; or proteins are left intact up to the final
product and the peptide bioactives are liberated in situ by
the host digestive system or gut microbial enzymes or a com-
bination of both [16]. Whether pre-digested or not, in both
cases enzymatic release becomes a very complex and unspe-
cific mechanism in which several enzymes, be they from
food processing (e.g. Alcalase, Protamex, Flavorzyme) or the
digestive system (e.g. pepsin, trypsin, chymotrypsin, elastase)
[17] with various activities are involved. Fig. 1 shows enzyme
specificity profiles of a whey protein isolate digested with
three different enzyme(s): sequencing grade trypsin (A), a
mixture of trypsin and chymotrypsin (B) and an enzyme cock-
tail (C). Position P5 to P5 from all cleavage sites are plotted
using the tool WebLogo [18] which measures sequence con-
servation by calculating the difference between the maximum
possible entropy (log2 20= 4.32 for 20 amino acids) and the en-
tropy of the observed amino acid distribution for each position.
The result is a graph documenting the specificity of a particular
P1 P2 P3 P4 P5
0 1 2 3 4
C
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 35 4 6 3 55 9 3549

enzymatic step. In the frame of the present article, results from
a biomarker discovery experiment are of type A, while
experiments conducted during bioactive discovery would be of
type B or C.
While this difference in proteolytic characteristics between
biomarker and bioactive discovery sounds obvious, its down-
stream impact on each step of an LCMS/MS-based peptide
identification/quantification experiment is significant. Fig. 2
summarizes all different steps related to such experiment
and highlights in color scale both for the biomarker and bioac-
tive discovery workflow the technology progress for each sin-
gle step. In the following paragraphs, each of the steps
following protein hydrolysis will be discussed in more details
with a particular focus given to the status and issues related
to peptide size (low, medium or large).
2.1. Medium peptides (725 AAs)
Because of the specificity of the enzymes used in biomarker
discovery, i.e. mainly trypsin, by far the largest pool of
peptides falls within 7 to 25 amino acids (approx. 800 to
2500 Da). Moreover, among all peptides generated for one
protein biomarker, only a few are necessary to uniquely
identify the protein and are most likely present within the
mentioned mass range. Therefore, peptide separation and its
optimization focus on these kinds of peptides. C18 reversed-
phase liquid chromatography is the method of choice for
separation of such peptides before their introduction into
the mass spectrometer. Introduction and ionization of the
peptides in the mass spectrometer is achieved through soft
techniques such as MALDI and ESI [19,20] ion sources. Peptide
detection is based on precise measurement of the mass of the
peptide precursor as well as of its related product ions.
Peptide identification is achieved through fragmentation
by various techniques such CID [21], HCD [22], ETD [23], ECD
[24] or IRMPD [25]. CID is by far the most common fragmenta-
tion method used with all the other techniques being increa-
singly complementary and even competitive. In the case of a
tryptic digest all peptides bear an amino group at the
N-terminus and most an additional one at the C-terminus
(on the side chain of the C-terminal arginine or lysine resi-
due). Corresponding fragmentation mechanisms have been
extensively studied and fragment ions can be predicted
depending on the type of instrument. Therefore, several
tools have been developed to analyze and identify such pep-
tide product ion spectra in an automated fashion by

Established Improvement needed Development needed

Biomarker discovery Bioactive discovery

Protein Unspecific hydrolysis

Specific hydrolysis
(Alcalase, Trypsin, industrial
HYDROLYSIS (Trypsin, ArgC, LyC)
process)

Peptide Small size (2-6 AAs) or large

Medium size (7-25 AAs)
size (>25 Aas)
SEPARATION IEF, SCX, RP
SEC

Peptide
LC-ESI-MSMS LC-ESI-MSMS
DETECTION

Peptide Against known protein

Many unknown sequences
sequences
SEARCH (theoretical vs experimental)
De novo prediction

Peptide
False discovery rate False discovery rate
VALIDATION

Peptide Relative or absolute Relative or absolute

QUANTIFICATION Label-free or label-based Label-free or label-based

Fig. 2 Current technology status for both the biomarker and the bioactive discovery workflow. Major steps of an LCMS/MS
based peptide identification/quantification workflow are highlighted and technology status is assessed using a
green-via-yellow-to-red color gradient ranging from established via improvement needed to development needed.
While the biomarker workflow shows that improvement can mainly be achieved within the peptide detection step, i.e. mass
spectrometric instrumentation and data acquisition, and to some extent within peptide search and validation steps, the
bioactive workflow requires development needs all across the different analytical levels.
3550 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 35 4 6 35 5 9
comparing them to theoretical peptide spectra resulting from
in silico digestion of a target protein database and thereby
classifying best candidates using cross-correlation or probabi-
listic scores [2629]. Analysis and validation of proteomic data
generated by tandem mass spectrometry has been recently
reviewed in detail by Nesvizhskii et al. [30]. In order to auto-
matically validate such database search result, several tools
have been developed to automatically assess false positive
rates and allow to validate a list of identified peptides or
proteins using a determined error rate [3133].
Protein quantification can be performed in a relative man-
ner by comparing peptide abundance between conditions,
summing up peptide information for each protein and finally
calculating a protein ratio [2]. Alternatively, proteins can be
absolutely quantified in each condition by spiking known
amounts of reference peptides for each protein [34]. Either
way, many automatic tools are available to perform such
computationally intensive tasks [3537]. As shown in Fig. 2,
most of the steps related to a biomarker discovery workflow
are well advanced with the largest improvement still needed
in the peptide detection step to cope with the tremendous
complexity and dynamic range of biological fluids such as
for example blood plasma. While improvements and new
developments are always desired and being pursued today's
biomarker discovery workflows also referred to as classical
proteomics enable already highly mature and robust
platforms that deliver a large body of mechanistic insights
and candidate biomarkers in life sciences [14,38,39].
However, as previously mentioned, bioactive peptide
measurement typically relies on a few copies of one molecular
entity with a low, medium or large molecular weight.
Therefore, for medium size peptides the previously described
techniques apply well, but they do not so for large or small
peptides. As a consequence, an adapted, wider panel of
methodologies has to be developed and deployed.
2.2. Large peptides (> 25 AAs)
Recently a significant effort has been brought to characterize
large polypeptides (proteins) in high throughput by: (i) a com-
bination of liquid chromatography using capillary columns
packed with silica-based solid support e.g. C4, C8 or polymeric
media e.g. PLRP-S; and (ii) high-resolution and high-accuracy
mass spectrometers to achieve unambiguous identification
(top-down tandem mass spectrometry) [4044]. Typically,
such approach is preceded by pre-fractionation using
solution-phase isoelectric focusing (sIEF) [45] or gel-eluted
liquid fraction entrapment electrophoresis (GELFrEE) [46].
Separated polypeptides (proteins) are fragmented using clas-
sical CID or ECD techniques and product ions are measured
at high mass accuracy and resolution. More recently, nozzle-
skimmer dissociation (NSD) has been shown to be powerful
for the identification of proteins [47]. A few software tools
such as ProSightPTM or PIITA have been developed to auto-
matically interrogate such data with the possibility of esti-
mating error rates [44,48]. Protein visualization is very
important and tools such as THRASH [49] can deconvolute
such complex data which can further be used to generate
2-D heatmaps of protein abundance similar to the real
images of 2-D gels. One can obtain quantitative information
from mapping tools such as PTMcRAWLER [40] that also look
at particular patterns possibly deriving from post-
translational modifications (PTMs). This latter strategy could
be used to analyze intact modified proteins as occurring
during food processing such as the Maillard reaction for
example [50].
While separation and detection of such large peptides is
feasible, their identification remains more challenging for
bioactive peptides such as hormones, neuropeptides or
simply large bioactive polypeptides produced through mild
hydrolysis of a particular protein ingredient. Classical peptide
identification tools are based on a sequence-tag approach i.e.
on the presence of the tag plus its precursor mass as mea-
sured by the MS instrument. While allowing for a reasonable
mass deviation tolerance, an accurately curated database is
necessary that does not simply list precursor proteins but
also their active forms as well all known post translational
modifications. This is available in the case of sequenced or-
ganisms with ample analytical evidence. A database such as
UniProtKB offers extensive information on post-translational
processing, e.g. signal peptides, phosphorylation, glycosyla-
tion and pro-hormones. In the case of an unsequenced ge-
nome databases of known peptides measured by means of
analytical chemistry tools are available (e.g. venoms of differ-
ent snakes and spiders [51]) and can be used with such strate-
gy. However, in the case of a discovery approach in which for
example a particular bioactivity is measured in a peptide frac-
tion of which the composition is unknown, such approach is
less adapted: one could use rather standard search engines
which apply enzyme specificity to predict potential peptides;
but, unfortunately, specific hydrolysis is rather the exception
than the rule and, therefore, searches would have to be per-
formed ideally without any enzyme specificity, which would
then blow up the search space and thereby the number of pos-
sible candidates. Thus, scoring and validation is likely to be
compromised.
2.3. Small peptides (< 7 AAs)
In the case of biomarkers, this category is usually not
addressed. The main reason apart from the technological
challenge of measuring them as discussed later in the para-
graph being that with such short sequence length (6 AAs or
smaller), identified peptides are not unique and can belong
to hundreds of protein sequences within the same organism.
Therefore, their detection does not bring any value to protein
identification but rather confuses the outcome especially in
terms of quantification where the signal is multiplexed. How-
ever, for bioactives, the potency of the administered peptide(s)
decreases as the chain length increases [52] making this
peptide category one of the highest interest. For very small
peptides and particularly di-, tri- and tetra-peptides, the
analytical task is very difficult with the challenges mainly
manifesting at the interface between peptide and small
molecule analysis. While small peptides containing some hy-
drophobic amino acids are usually retained on classical C18
columns, small peptides composed of only hydrophilic
amino acids are not. Therefore, their prior separation before
injection to the mass spectrometer relies on a combination
of different separation techniques such as reversed phase
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 35 4 6 3 55 9
(e.g. C18 or C30) or normal phase (e.g. HILIC or amide). If
retention and separation is achieved, peptides are further
analyzed by mass spectrometry and their characterization is
by far not trivial. At this level, two parameters are critical:
(i) high mass accuracy; and (ii) collision energy used for frag-
mentation. In order to illustrate such statement, a calculation
example for all di- to tetra-peptides (168,400 sequences) is
given. Depending on the mass accuracy achieved by the in-
strument, 240 (1 ppm), 324 (10 ppm), 1020 (100 ppm) or 1360
(1000 ppm) peptides have to be considered as isobaric or not
distinguishable by their sole mass measurement. While this
holds true also for larger peptides, fragmentation here
makes a major difference. In the case of larger peptides, frag-
ment ion information is much richer and complex and usually
allows distinguishing between potential precursors. However,
in the case of small peptides, fragmentation is usually of poor
quality and less informative resulting in ambiguous identifi-
cation especially when dealing with low energy collision frag-
mentation [53]. Selectivity can be increased by using high
collision energy fragmentation such as available on Q-TOF in-
strument for example. In that case, more ions are obtained
such as signature immonium ions or ions related to side
chains of the different amino acids present in the sequence
(e.g. 43 for isoleucine or leucine, 57 for valine) with the relative
intensity of each of these signature ions playing a critical role
(e.g. distinguish between IV or VI) [53,54]. Taken all together,
these parameters (retention time, precursor mass and specific
high collision energy fragments) enable to specifically distin-
guish such peptides with high certainty in the case of manual
interpretation. However, no automatic and powerful tool is
available to date to perform computer based validation. A da-
tabase search approach such as used for medium size peptide is
not conceivable as scoring functions are not adapted to such
short sequences plus the fact that the searches have to be per-
formed without any enzyme specificity. Therefore scoring and
further statistical validation models do not succeed. As current-
ly on-going for some organisms at the biomarker level [55], the
future avenue for the automated identification of small pep-
tides (at least for di- to tetra-peptides) will be through the design
of specific SRM/MRM assays after the validation of each peptide
on the basis of libraries of synthetic peptides.
2.4. Common issues
Apart from molecular weight typical analytical challenges of
bioactive food-derived peptides, there are a few issues related
to all categories of peptides of either small, medium or larger
size. First and foremost, if the organism from which the
protein is derived has not been sequenced, none of the tools
described hereto can be used. Rather, de novo sequencing is
the only open avenue to follow because it solely relies on the
computer-based interpretation of a product ion spectrum
[56]. Potential sequences or sequence tags that can be
extracted can then be used to interrogate or blast genetically
similar organisms for which the genome is available. An ex-
cellent example of such tools is the commercial software
PEAKS which incorporates all the necessary modules to per-
form de novo search, database search, PTM search as well as
quantification [57]. Second, all quantitative tools are made
for building protein ratios based on all unique peptides
3551
identified for that particular protein. While in the case of
biomarkers it is expected that peptides do share similar ratios
among the same protein, this is not the case for bioactive
peptides. Indeed, such peptides are usually produced using
enzyme cocktails which have different kinetics for particular
amino acids. Therefore, peptides released from the same pro-
tein can have different ratios. As previously mentioned, bio-
marker quantification resides at protein level with the sum
of all peptides being the desired information, bioactive quan-
tification resides at the level of each individual peptide.
While most quantification tools allow exporting quantitative
information for each individual peptide, there is a lack of visual-
ization tools to better represent quantitative bioactive changes.
Fig. 3A illustrates peptides identified within six different whey
protein hydrolysates. An in-house tool that is frequently used
in our laboratory represents identified peptides within the se-
quence of alpha-lactalbumin with a heatmap on the left giving
information on the abundance of each peptide within the differ-
ent hydrolysates tested. We do believe such tools are very use-
ful in the context of bioactives to better help visualize complex
quantitative information.
Finally, we and others [58,59] have recently shown that
peptide size exclusion chromatography (SEC) is an interesting
alternative to the more classical pre-fractionation methods
usually applied in biomarker or bioactive discovery (e.g. ion
exchange or reversed phase chromatography). As illustrated
in Fig. 3B, SEC produces very informative data on peptide
size composition of a particular sample and the degree of
hydrolysis achieved. Moreover, while of low resolution, it
can still be used to fractionate samples based on peptide size
offering an additional orthogonal separation (Fig. 3C). Finally,
it helps isolating peptides from the sample (e.g. removal of
salts) by means of size, a process that shares high similarity
with the fractionation of proteins using SDS-PAGE [60,61] in
the biomarker discovery field.
3. Status of bioactive peptide discovery
3.1. Functional characterization of bioactive peptides
Identification and characterization of bioactive peptides has
gained a lot of interest in the past ten years. More than 150 sci-
entific papers with the words bioactive peptide(s) in their
title or abstract are published nowadays per year versus
roughly 50 only ten years ago (data extracted from PubMed
search). This clearly highlights the large interest given to bio-
active peptides recently. The aim of this section is to review
some useful resources when seeking for information about
known bioactive peptides as well as give a general overview
of the different activities that have been described so far.
Many bioactive peptide databases have been designed to
store information such as sequence, molecular weight, activi-
ty, reference to published work, EC50, source and more. Here
we will briefly list some of these main databases that are
available online:
BioPep [62] is a database containing information on bioac-
tive peptides (n = 2594), major allergens and their epitopes
(n = 135) as well as sensory peptides including amino
3552 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 35 4 6 35 5 9

A Bovine Alpha-lactalbumin

123456

B C
Absorbance at 215 nm

Absorbance at 215 nm
86'398
41'838
21'203
11'245
6'242
3'626
2'204
1'402
934
651
475
362
289
242
212
194
186
186
195
214

86'398
41'838
21'203
11'245
6'242
3'626
2'204
1'402
934
651
475
362
289
242
212
194
186
186
195
214
Molecular weight [Da] Molecular weight[Da]

Fig. 3 Examples of tools useful for bioactive discovery. (A) Example of a qualitative and quantitative mapping of peptides
belonging to alpha-lactalbumin. An in-house tool written in Perl has been developed to read a list of identified peptides plus
their respective abundances in different conditions determined by a label-free approach and map it to a user selected protein
by positioning the peptide along the protein sequence and adding quantitative information in form of a heat map. Such tools
allow to rapidly compare peptide composition and abundance variations occurring, for example, during food processing such
as hydrolysis or fermentation; (B) Peptide size exclusion chromatography profiles of partially (blue) or extensively (red)
hydrolyzed milk proteins using two different enzyme preparations. Hydrolysates were injected on a Superdex Peptide column
(GE Healthcare, optimal SEC separation range is 10010,000 Da) using an isocratic flow of 30% acetonitrile (v/v) in 0.1% TFA (v/v)
over 60 min. UV signal was recorded at 215 nm; (C) Preparative fractionation of specific peptide mass ranges for further
characterization using the same Superdex Peptide column. The protein hydrolysate was fractionated every 2 min and each
fraction was re-injected to measure resolution and overlap between adjacent fractions. Such peptide size exclusion
chromatography is very useful to enrich or purify a particular molecular weight range that might contain peptide(s) of interest.

acids (n = 326). It is well curated and usually contains are most relevant to you using text classification related
references from which the data has been extracted. (Link: to cancer, cardiovascular diseases, diabetes, apoptosis,
http://www.uwm.edu.pl/biochemia/index_en.php). angiogenesis and molecular imaging or peptides for
PepBank [63,64] is a database of peptides based on which binding data exist. (Link: http://pepbank.mgh.
sequence text mining and public peptide data sources harvard.edu).
(n = 21,691). Only peptides that are 20 amino acids or EROP-Moscow [65,66] is a knowledgebase of endogenous
shorter are stored within the database. The new heat regulatory oligopeptides (n = 10,229). It is a curated data-
map retrieval tool allows to quickly find the entries that base with sequences ranging from 2 to 50 amino acid
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 35 4 6 3 55 9
residues which strives to provide a high level of
annotations (such as descriptions of the structure of an
oligopeptide, its source and function, post-translational
modifications, etc.). (Link: http://erop.inbi.ras.ru).
BioPD is a resource of bioactive peptides (n = 1254). It in-
volves the basic and structure information of peptide, the
related gene information, the interactions between the
peptide and other proteins and information about diseases
and the peptide. (Link: http://biopd.bjmu.edu.cn).
PeptideDB is a database that assembles all naturally occur-
ring signaling peptides from animal source, which are de-
rived by cleavage from precursor proteins (n =20,027). It
includes cytokines and growth factors, peptide hormones,
antimicrobial peptides, toxins, and venom peptides, and
anti-freeze proteins. (Link: http://www.peptides.be).
Peptidome is a comprehensive fact-database for endoge-
nous peptides. The Peptidome project is aimed to establish
the intellectual database for medical, biological and
pharmaceutical research by accumulating data of the en-
dogenous peptides in cells, tissues and organisms. (Link:
http://www.peptidome.jp).
APD [67] is a database of natural antimicrobial peptides
with less than 100 amino acid residues (n = 1773) with the
following activities registered into this database: antiviral
peptides, antifungal peptides, anticancer/tumor peptides,
heparin membrane-active peptide
antibiotic binding opioid agonist
contracting
stimulating immunostimulating
different activities stimulating binding
regulating
anticancer
haemolytic
antiamnestic
antithrombotic
immunomodulating
neuropeptide
opioid
inhibitor
celiac toxic
Fig. 4 Classification of known peptides based on their reported activity in the BioPep database (n = 2594).
3553
antibacterial peptides, anti-HIV peptides, antiparasital
peptides, spermicidal peptides and insecticidal peptides.
(Link: http://aps.unmc.edu/AP/main.php).
In order to identify major bioactivities described in the lit-
erature to date, we have extracted all peptides present in the
BioPep database for which a classification is already
available. Fig. 4 represents in a pie chart the major activities
that are described in the literature. ACE inhibitors come first
with for example IPP and VPP being the most described ones
in the literature. Antibacterial peptides reflect the extensive
research that is deployed in the field of anti-microbial pep-
tides being the leading candidates to replace antibiotics
against which bacteria become more and more resistant.
Other key activities are antioxidant, celiac toxic, inhibitory
opiod, neuropeptides as well as immunomodulating peptides.
Finally, as previously mentioned, the potency of the adminis-
tered peptide(s) decreases as the chain length increases [52].
Interestingly, using the BioPep database again, if we look at
the peptide length distribution over the 2594 sequences pre-
sent, 42% are small peptides (26 AAs), another 46% percent
are of medium size (725 AAs) and only 12% are bigger than
25 AAs. These figures clearly emphasize the previous state-
ment on the inverse relationship between potency and pep-
tide length.
anti inflammatory
antifungal chemotactic
hypotensive reacting vasoconstrictor
ACE inhibitor
antibacterial
antioxidative
3554 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 35 4 6 35 5 9
3.2. Classical screening vs. reverse genome engineering for
bioactive peptide discovery
Increasing scientific evidence has been published describing
bioactive peptides released from food proteins that have
beneficial health effects. They are encrypted in the parent
protein and are released upon digestion, fermentation or
food processing and exert bioactivity either locally (in the GI
tract) or systemically (in the blood or organs) [6]. Bovine milk
and dairy products represent the most important source of
bioactive peptides but other animal and plant proteins such
as meat, fish, eggs, wheat maize, soy, rice, mushrooms,
pumpkin and sorghum have been described to contain
encrypted bioactive peptides which are released after enzy-
matic hydrolysis or fermentation [68].
Bioactive peptide discovery traditionally consists of a step-
wise approach, typically including (i) selection of an appropri-
ate protein source of plant or animal origin; (ii) proteolysis by
enzymatic processing, fermentation or gastrointestinal diges-
tion; (iii) initial in vitro screening for targeted bioactivity;
(iv) fractionation of the peptide pool into more defined
sub-fractions by chromatography or membrane separation;
(v) further bioactivity screening followed by identification of
the bioactive peptide(s) by mass spectrometry; and (vi)
production of synthetic peptide analogs to validate bioactivity
in vitro and in vivo [69].
In principle, any existing food protein can be considered as
a potential source of bioactive peptides. These latter may be
released during gastrointestinal digestion by the host or by
food processing using microbial digestive enzymes, including
fermentation. The choice of proteolytic enzymes or microor-
ganisms used for protein processing has a crucial impact on
the composition of the released peptides. Enzymatic hydroly-
sis of whole protein is the most common way to produce
bioactive peptides and many of the known food peptides are
also released by the action of gastrointestinal enzymes such
as pepsin, trypsin and chymotrypsin [70]. Microbial fermenta-
tion is another major process to generate bioactive peptides,
especially from milk proteins, because for example many
industrially used lactic acid bacteria are highly proteolytic.
Microbial intracellular and cell wall-bound proteases release
peptides during fermentation and produce dairy products
(i.e. yogurt, cheese) enriched in bioactive peptides [71,72].
The well known ACE-inhibitory peptides, VPP and IPP, were
identified in milk fermented with Lactobacillus helveticus
strains [15]. Lastly, in vitro and in vivo digestion systems are
developed to model the gastrointestinal digestion process
and the concomitant release of bioactive peptides in the gut
[7375].
Subsequent to protein hydrolysis, the complex peptide
mixtures are typically tested in vitro using bioassays for the
targeted activity, i.e. enzyme activity modulation; and
antioxidant, anti-hypertensive, anti-microbial or immuno-
regulatory activity [16]. A critical issue related to in vitro
testing is the translation into in vivo activity because of the
low stability of peptides in the GI tract caused by the presence
of large quantities of peptidases [17]. Ideally, in vitro studies
should only be used as pre-screening to evaluate the potential
of protein hydrolysates before confirmation in well-designed
in vivo studies. In order to identify active peptides in very
complex hydrolysates, extensive fractionation procedures
using standard chromatographic techniques such as size-
exclusion (SEC), ion-exchange (IEX), affinity or reversed-
phase HPLC are applied resulting in a simplified peptide pool
more amenable to peptide identification. Once the bioactive
sub-fractions have been prepared, mass spectrometry is the
method of choice to determine the peptide sequence and
thus bioactive identity, although amino acid analyzers and
protein sequencers are still traditional alternatives [69,76].
The typical mass spectrometry-based proteomic approach
used for protein identification in the context of biomarker
discovery needs to be adapted to the increased complexity of
food-derived peptides which can be smaller or larger than
the biomarker-typical tryptic peptides and which are released
by complex and less specific enzyme activities thus generat-
ing more heterogeneous peptide mixtures (see technology
section of this paper).
This traditional and rather empirical fractionate-and-
test peptide discovery strategy has proven to be very efficient
and most of today's known food-derived bioactive peptides
have been identified by this approach. Nevertheless, the
work requires substantial resources and even extensive
fractionation and purification not always enables
unambiguous identification of single bioactive peptides:
rather, one may end up with a highly purified fraction or
even compound that is, after all, no longer active, because
for food-derived bioactivity not only single active principles
matter but also the food matrix and other modulating factors
add to the overall activity
Thus, knowledge- and bioinformatics-driven approaches,
also called in silico prediction and discovery, have been
developed [7]. The use of computational methods to predict
functional peptides from genome sequence information has
received an ever increasing interest since the early 2000s,
also stimulated by the completion of the human genome
project [77,78]. These approaches enable in silico prediction
of (possibly new) functional peptides and their enzymatic
release in a targeted fashion thereby reducing the number of
bioactivity tests needed to select nutritionally most relevant
and accessible candidates.
Computational tools in combination with protein and
peptide sequence databases provide the basic analytical
platform to explore food genomes in the animal and plant
world. A typical in silico approach, also termed reverse-
genome engineering [7], can be divided into two distinct
principles to evaluate the biological activity of a given peptide:
first, peptides are screened directly by molecular similarities
and chemoinformatic methods [79]; and second, binding of
the peptide to a specific receptor is modeled to estimate
biological activity [80]. While this strategy has successfully
been applied to a multitude of different biopharmaceutical
products, its application to food-derived peptide discovery
has only begun to be leveraged [7].
A large number of human digestive enzymes involved in
protein degradation have been described including their
cleavage site specificity [17] that unlocks in silico prediction
of peptide fragments produced by gastrointestinal digestion.
With the availability of plant and animal genomes (i.e. rice
[81], maize [82], soy [83], bovine [84,85]) as well as the genomes
of important symbiotic intestinal bacteria [86], it may be
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 35 4 6 3 55 9
possible to predict the proteolysis of dietary proteins based on
the specific enzyme activity of a selected bacterial strain. The
same holds true for purified technical enzymes commonly
used in food processing as their enzyme specificities can be
precisely determined.
Pripp and colleagues recently reviewed a comprehensive
approach to model and predict peptide and protein structures
with high biological activity, called quantitative structure
activity relationship (QSAR) [87]. Peptide QSAR modeling was
particular efficient in prediction of antimicrobial, ACE-
inhibitory and bitter tasting peptide activity [8890]. Using
physicochemical descriptors on a dataset of peptides derived
from milk proteins, a QSAR model was developed indicating
that side chain hydrophobicity in the C-terminal position and
side chain size in the penultimate amino acid were critical
for the ACE-inhibitory potential [89]. As the QSAR methodolo-
gy can help elucidate structureactivity relationships, future
exploration of this approach should enable to extend the pre-
diction of novel food protein derived peptide bioactivity.
With the availability of increasing amounts of data and
software tools, only structured organization according to
clear criteria permits efficient application [91]. As discussed
previously, food peptidomics requires specific methods and
tools to tackle the challenge of processing highly complex
and heterogeneous peptide information based on very short
amino acid sequences. A very efficient collection of computa-
tional tools and databases was developed by Minkiwicz and
colleagues to construct profiles of potentially active protein
fragments using peptide bioactivity information stored in the
BIOPEP database [92]. The database currently contains more
than 700 proteins and almost 2600 bioactive peptides (status
June 2011) which are classified according to thirty-seven
types of bioactivities. This collection of peptide-centric com-
putational tools represents an important and highly useful
starting point for in silico based bioactive peptide discovery.
New computational approaches in combination with ever in-
creasing amounts of genomic data from any kind of food related
species (animal, plant, microorganism, fungi) unlock new possi-
bilities and approaches to predict, analyze, and evaluate novel
bioactive peptides. The dissection of the genomes using bioin-
formatic tools will provide a cost-effective strategy for novel
peptide discovery. Compared to classical fractionation-
identification approaches, computational methods originating
from pharmaceutical research started to be successfully ex-
plored in the field of nutritional research and will further revolu-
tionize the discovery of functional peptides with more adapted
in silico tools for food-related peptides.
3.3. Site of action and availability of bioactive peptides
Given the many challenges of defining and releasing bioactive
peptides from food proteins as described in the previous
sections, the final step remains delivering the peptides to
the site of action while maintaining its bioactivity. Although
many peptides derived from food proteins have been detected
in the stomach or in the small intestine [93,94], or even in the
cardiovascular system [95], the presence alone of these
peptides is not sufficient to establish its bioavailability.
The biggest threat to any bioactive peptide lies i) in the
lumen of the small intestine which contains large quantities
3555
of peptidases secreted by the pancreas and cellular peptidases
from mucosal cells and ii) in the brush border membrane of
the epithelial cells which contains more than a dozen pepti-
dases [17,96]. Bioactive peptides can only survive this massive
proteolytic armada when their sequence resists degradation
(i.e. very short peptides, containing proline residues [97]). An
alternative strategy is to reach the site of action quickly after
digestive release in situ thus targeting receptors located in
the small intestine.
There is evidence that intact proteins can cross the gastro-
intestinal barrier, although not significant in terms of amino-
nitrogen source, but relevant for potential functional implica-
tions. Normal absorption probably occurs predominantly by
transcellular endocytosis with some possible contribution by
a route between cells [10]. Animal studies have demonstrated
that larger peptides (1051 amino acids long) generated from
the diet can be absorbed intact through the intestinal tract
and produce biologic effects at the tissue level. The potency
of bioactive peptides decreases as the chain length increases
[52]. Interestingly, small di- and tripeptides are absorbed
more rapidly from the small intestine than free amino acids
as they are transported by two independent systems [98].
Those very small peptides are transported into intestinal
cells by the proton-coupled di- and tripeptide transporter
PepT1 and similarly into renal epithelial cells by PepT2 [8,99].
After intracellular uptake most of the di- and tripeptides are
rapidly hydrolyzed except very resistant proline-containing
peptides that reach the portal venous system intact [9].
Peptides that escape the normal process of digestion and
absorption can be taken up intact across the intestinal muco-
sa by two possible mechanisms, a paracellular and transcellu-
lar route. The former permits passage of large hydrophilic
peptides via tight junctions between cells, the latter favors
passage of hydrophobic peptides through the brush border
and basolateral membranes of enterocytes [16]. Ziv and
colleagues have shown that insulin was transported by
internalization through invaginations of the luminal plasma
membrane and that the vesicles released intact peptides
into the interstitial space to reach the circulation [100].
Bioactive peptides generated from food proteins and even
intact proteins can be absorbed in the intestine and induce
biological functions in the gut or at tissue level. For example,
many reports have described a role for food-derived peptides
in increasing gut secretory and absorptive capacity or gut
tissue growth. An effect on satiety has also been described
as well as stimulation of hormone secretion. Finally, many
peptides do exhibit opioid activity either in agonist or
antagonist mode. For a detailed review on bioactive peptides
and their influence on gut function, readers are referred to
the review from Moughan and colleagues [101]. In relation to
milk, this has particular importance in delivering bioactive
proteins and peptides to infants whose mucosal barrier in
the gastrointestinal tract is delayed in maturation. Human
milk contains not only antibodies and immune cells but
many other substances that can interfere with bacterial
colonization and prevent antigen penetration [102104]. The
immaturity of the digestive system which limits proteolysis
of milk proteins represents a second key element to maintain
and modulate beneficial activities of milk proteins and
peptides in infants.
3556 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 35 4 6 35 5 9
In conclusion, the primary goal of our digestive system is
to degrade food proteins to free amino acids which are
absorbed primarily in the small intestine and re-used in the
body for protein synthesis. Nevertheless, proteins may resist
to digestion and can be transported intact across the gut but
more likely, partial digestion releases bioactive peptides
hidden in the parent sequence. Larger peptides can be taken
up via different routes but the most studied absorption process
involves the transport of di- and tripeptides through the
proton-coupled peptide transporter PepT1. Small peptides
having a C-terminal proline residue, i.e. VPP or IPP, have been
shown to remain active during digestion and are able to reach
the cardiovascular system in an active form [105]. Although
important progress has been realized in the field of peptide
availability and transport, the increasing interest in bioactive
peptides released from food proteins will certainly stimulate
further research.
4. Concluding remarks
Proteins represent one of the main classes of macronutrients,
beside carbohydrates and lipids, that are consumed in our
daily diet and thus are an environmental factor with profound
life-long influence on health. Intact proteins in form of
antibodies, growth factors, hormones, etc. provided in the
diet can have direct biological activity, e.g. in infants due to
their still immature digestive system, but need to resist
digestion in order to exert their function. However, more
commonly the fate of food proteins during digestion is com-
plete degradation to free amino acids which are then absorbed
and re-used by the organism for the synthesis of new
proteins. During this process, bioactive peptides can be gener-
ated and absorbed to provide health beneficial activities.
Many activities have been identified in vitro and in vivo, and
products containing bioactive peptides, i.e. the ACE-
inhibitory peptides VPP and IPP, are commercially available.
As a general rule, the smaller the peptide size the higher the
chance to survive further proteolytic degradation and
therefore to exert its activity, either locally in the gut or even
systemically through the cardiovascular system at tissue
level.
The analysis of the food peptidome poses a formidable
challenge. In contrast to the tryptic peptides typically generat-
ed in biomarker proteomics, food peptides are released by in
vitro processing or in vivo digestion by various unspecific
and specific enzymes. This gives rise to a wide distribution
of peptide sizes which poses this particular analytical
challenge. Modern mass spectrometric techniques are being
developed enabling access to this small-size peptidome but
adapted software tools still remain to be developed and
optimized. Technological challenges are recognized and
many initiatives are evolving to address such points as
dynamic range and small non-tryptic peptide analysis.
Nevertheless, novel approaches and improved mass
spectrometric systems are still needed to further extend
detailed analysis of the food peptidome. This will help deepen
our knowledge on food-derived peptides and link it to the in-
creasing understanding of bioactive peptides that have a bene-
ficial effect on health.
REFERENCES
[1] Kussmann M, Panchaud A, Affolter M. Proteomics in
nutrition: status quo and outlook for biomarkers and
bioactives. J Proteome Res 2010;9:487687.
[2] Panchaud A, Affolter M, Moreillon P, Kussmann M.
Experimental and computational approaches to
quantitative proteomics: status quo and outlook.
J Proteomics 2008;71:1933.
[3] Kussmann M, Rezzi S, Daniel H. Profiling techniques in
nutrition and health research. Curr Opin Biotechnol 2008;19:
8399.
[4] Kussmann M, Raymond F, Affolter M. OMICS-driven
biomarker discovery in nutrition and health. J Biotechnol
2006;124:75887.
[5] Kussmann M, Fay LB. Nutrigenomics and personalized
nutrition: science and concept. Pers Med 2008;5:44755.
[6] Kussmann M, Van Bladeren PJ. The externded
nutrigenomics understanding the interplay between the
genomes of food, gut microbes, and human host. Front
Genet 2011;2:113.
[7] Grigorov MG, Van Bladeren PJ. Functional peptides by
genome reverse engineering. Curr Opin Drug Discov Devel
2007;10:3416.
[8] Daniel H, Kottra G. The proton oligopeptide cotransporter
family SLC15 in physiology and pharmacology. Pflugers Arch
2004;447:6108.
[9] Caspary WF. Physiology and pathophysiology of intestinal
absorption. Am J Clin Nutr 1992;55:299S308S.
[10] Gardner ML. Gastrointestinal absorption of intact proteins.
Annu Rev Nutr 1988;8:32950.
[11] Hemmings WA, Williams EW. Transport of large breakdown
products of dietary protein through the gut wall. Gut
1978;19:71523.
[12] Mallick P, Schirle M, Chen SS, Flory MR, Lee H, Martin D, et al.
Computational prediction of proteotypic peptides for
quantitative proteomics. Nat Biotechnol 2007;25:12531.
[13] Domon B, Aebersold R. Options and considerations when
selecting a quantitative proteomics strategy. Nat Biotechnol
2010;28:71021.
[14] Surinova S, Schiess R, Huttenhain R, Cerciello F, Wollscheid
B, Aebersold R. On the development of plasma protein
biomarkers. J Proteome Res 2011;10:516.
[15] Nakamura Y, Yamamoto N, Sakai K, Okubo A, Yamazaki S,
Takano T. Purification and characterization of angiotensin
I-converting enzyme inhibitors from sour milk. J Dairy Sci
1995;78:77783.
[16] De Leo F, Panarese S, Gallerani R, Ceci LR. Angiotensin
converting enzyme (ACE) inhibitory peptides: production
and implementation of functional food. Curr Pharm Des
2009;15:362243.
[17] Vlieghe P, Lisowski V, Martinez J, Khrestchatisky M.
Synthetic therapeutic peptides: science and market. Drug
Discov Today 2010;15:4056.
[18] Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: a
sequence logo generator. Genome Res 2004;14:118890.
[19] Stults JT. Matrix-assisted laser desorption/ionization mass
spectrometry (MALDI-MS). Curr Opin Struct Biol 1995;5:
6918.
[20] Fenn JB, Mann M, Meng CK, Wong SF, Whitehouse CM.
Electrospray ionization for mass spectrometry of large
biomolecules. Science 1989;246:6471.
[21] Wells JM, McLuckey SA. Collision-induced dissociation
(CID) of peptides and proteins. Methods Enzymol 2005;402:
14885.
[22] Olsen JV, Macek B, Lange O, Makarov A, Horning S, Mann M.
Higher-energy C-trap dissociation for peptide modification
analysis. Nat Methods 2007;4:70912.
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 35 4 6 3 55 9
[23] Syka JE, Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF.
Peptide and protein sequence analysis by electron transfer
dissociation mass spectrometry. Proc Natl Acad Sci U S A
2004;101:952833.
[24] Zubarev RA, Horn DM, Fridriksson EK, Kelleher NL, Kruger
NA, Lewis MA, et al. Electron capture dissociation for
structural characterization of multiply charged protein
cations. Anal Chem 2000;72:56373.
[25] Little DP, Speir JP, Senko MW, O'Connor PB, McLafferty FW.
Infrared multiphoton dissociation of large multiply charged
ions for biomolecule sequencing. Anal Chem 1994;66:280915.
[26] Yates III JR, Eng JK, McCormack AL, Schieltz D. Method to
correlate tandem mass spectra of modified peptides to
amino acid sequences in the protein database. Anal Chem
1995;67:142636.
[27] Perkins DN, Pappin DJ, Creasy DM, Cottrell JS.
Probability-based protein identification by searching
sequence databases using mass spectrometry data.
Electrophoresis 1999;20:355167.
[28] Geer LY, Markey SP, Kowalak JA, Wagner L, Xu M, Maynard
DM, et al. Open mass spectrometry search algorithm.
J Proteome Res 2004;3:95864.
[29] Craig R, Beavis RC. TANDEM: matching proteins with
tandem mass spectra. Bioinformatics 2004;20:14667.
[30] Nesvizhskii AI, Vitek O, Aebersold R. Analysis and validation
of proteomic data generated by tandem mass spectrometry.
Nat Methods 2007;4:78797.
[31] Keller A, Nesvizhskii AI, Kolker E, Aebersold R. Empirical
statistical model to estimate the accuracy of peptide
identifications made by MS/MS and database search. Anal
Chem 2002;74:538392.
[32] Elias JE, Gygi SP. Targetdecoy search strategy for increased
confidence in large-scale protein identifications by mass
spectrometry. Nat Methods 2007;4:20714.
[33] Kall L, Canterbury JD, Weston J, Noble WS, MacCoss MJ.
Semi-supervised learning for peptide identification from
shotgun proteomics datasets. Nat Methods 2007;4:9235.
[34] Lange V, Picotti P, Domon B, Aebersold R. Selected reaction
monitoring for quantitative proteomics: a tutorial. Mol Syst
Biol 2008;4:222.
[35] Mueller LN, Rinner O, Schmidt A, Letarte S, Bodenmiller B,
Brusniak MY, et al. SuperHirn a novel tool for high
resolution LCMS-based peptide/protein profiling.
Proteomics 2007;7:347080.
[36] Li XJ, Zhang H, Ranish JA, Aebersold R. Automated statistical
analysis of protein abundance ratios from data generated by
stable-isotope dilution and tandem mass spectrometry.
Anal Chem 2003;75:664857.
[37] Han DK, Eng J, Zhou H, Aebersold R. Quantitative profiling of
differentiation-induced microsomal proteins using
isotope-coded affinity tags and mass spectrometry.
Nat Biotechnol 2001;19:94651.
[38] Schiess R, Wollscheid B, Aebersold R. Targeted proteomic
strategy for clinical biomarker discovery. Mol Oncol 2009;3:
3344.
[39] Mallick P, Kuster B. Proteomics: a pragmatic perspective. Nat
Biotechnol 2010;28:695709.
[40] Durbin KR, Tran JC, Zamdborg L, Sweet SM, Catherman AD,
Lee JE, et al. Intact mass detection, interpretation, and
visualization to automate Top-Down proteomics on a large
scale. Proteomics 2010;10:358997.
[41] Capriotti AL, Cavaliere C, Foglia P, Samperi R, Lagana A.
Intact protein separation by chromatographic and/or
electrophoretic techniques for top-down proteomics.
J Chromatogr A 2011;1218:876076.
[42] Lee JE, Kellie JF, Tran JC, Tipton JD, Catherman AD, Thomas
HM, et al. A robust two-dimensional separation for
top-down tandem mass spectrometry of the low-mass
proteome. J Am Soc Mass Spectrom 2009;20:218391.
3557
[43] Tipton JD, Tran JC, Catherman AD, Ahlf DR, Durbin KR,
Kelleher NL. Analysis of intact protein isoforms by mass
spectrometry. J Biol Chem 2011;286:254518.
[44] Tsai YS, Scherl A, Shaw JL, MacKay CL, Shaffer SA,
Langridge-Smith PR, et al. Precursor ion independent
algorithm for top-down shotgun proteomics. J Am Soc Mass
Spectrom 2009;20:215466.
[45] Tran JC, Doucette AA. Rapid and effective focusing in a
carrier ampholyte solution isoelectric focusing system: a
proteome prefractionation tool. J Proteome Res 2008;7:
17616.
[46] Tran JC, Doucette AA. Gel-eluted liquid fraction entrapment
electrophoresis: an electrophoretic method for broad
molecular weight range proteome separation. Anal Chem
2008;80:156873.
[47] Vellaichamy A, Tran JC, Catherman AD, Lee JE, Kellie JF,
Sweet SM, et al. Size-sorting combined with improved
nanocapillary liquid chromatographymass spectrometry
for identification of intact proteins up to 80 kDa. Anal Chem
2010;82:123444.
[48] Zamdborg L, LeDuc RD, Glowacz KJ, Kim YB, Viswanathan V,
Spaulding IT, et al. ProSight PTM 2.0: improved protein
identification and characterization for top down mass
spectrometry. Nucleic Acids Res 2007;35:W7016.
[49] Horn DM, Zubarev RA, McLafferty FW. Automated reduction
and interpretation of high resolution electrospray mass
spectra of large molecules. J Am Soc Mass Spectrom 2000;11:
32032.
[50] Davidek T, Kraehenbuehl K, Devaud S, Robert F, Blank I.
Analysis of Amadori compounds by high-performance
cation exchange chromatography coupled to tandem mass
spectrometry. Anal Chem 2005;77:1407.
[51] Escoubas P, Quinton L, Nicholson GM. Venomics:
unravelling the complexity of animal venoms with mass
spectrometry. J Mass Spectrom 2008;43:27995.
[52] Roberts PR, Burney JD, Black KW, Zaloga GP. Effect of
chain length on absorption of biologically active
peptides from the gastrointestinal tract. Digestion 1999;60:
3327.
[53] Paizs B, Suhai S. Fragmentation pathways of protonated
peptides. Mass Spectrom Rev 2005;24:50848.
[54] Morifuji M, Koga J, Kawanaka K, Higuchi M. Branched-chain
amino acid-containing dipeptides, identified from whey
protein hydrolysates, stimulate glucose uptake rate in L6
myotubes and isolated skeletal muscles. J Nutr Sci Vitaminol
(Tokyo) 2009;55:816.
[55] Picotti P, Lam H, Campbell D, Deutsch EW, Mirzaei H, Ranish
J, et al. A database of mass spectrometric assays for the
yeast proteome. Nat Methods 2008;5:9134.
[56] Seidler J, Zinn N, Boehm ME, Lehmann WD. De novo
sequencing of peptides by MS/MS. Proteomics 2010;10:63449.
[57] Ma B, Zhang K, Hendrie C, Liang C, Li M, Doherty-Kirby A,
et al. PEAKS: powerful software for peptide de novo
sequencing by tandem mass spectrometry. Rapid Commun
Mass Spectrom 2003;17:233742.
[58] Tran BQ, Hernandez C, Waridel P, Potts A, Barblan J, Lisacek
F, et al. Addressing trypsin bias in large scale (phospho)
proteome analysis by size exclusion chromatography and
secondary digestion of large post-trypsin peptides.
J Proteome Res 2011;10:80011.
[59] Johns PW, Jacobs WA, Phillips RR, McKenna RJ, O'Kane KA,
McEwen JW. Characterisation of peptide molecular mass
distribution in commercial hydrolysates and
hydrolysate-based nutritional products. Food Chem
2011;125:104150.
[60] de Godoy LM, Olsen JV, Cox J, Nielsen ML, Hubner NC,
Frohlich F, et al. Comprehensive mass-spectrometry-based
proteome quantification of haploid versus diploid yeast.
Nature 2008;455:12514.
3558 J O U RN A L OF P ROT EO M IC S 7 5 ( 2 0 12 ) 35 4 6 35 5 9
[61] Laemmli UK. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature 1970;227:
6805.
[62] Iwaniak A, Dziuba J, Niklewicz M. The BIOPEP database a
tool for the in silico method of classification of food proteins
as the source of peptides with antihypertensive activity.
Acta Aliment 2005;34:41725.
[63] Shtatland T, Guettler D, Kossodo M, Pivovarov M, Weissleder
R. PepBanka database of peptides based on sequence text
mining and public peptide data sources. BMC Bioinformatics
2007;8:280.
[64] Duchrow T, Shtatland T, Guettler D, Pivovarov M, Kramer S,
Weissleder R. Enhancing navigation in biomedical
databases by community voting and database-driven text
classification. BMC Bioinformatics 2009;10:317.
[65] Zamyatnin AA. EROP-Moscow: specialized data bank for
endogenous regulatory oligopeptides. Protein Seq Data Anal
1991;4:4952.
[66] Zamyatnin AA, Borchikov AS, Vladimirov MG, Voronina OL.
The EROP-Moscow oligopeptide database. Nucleic Acids Res
2006;34:D2616.
[67] Wang Z, Wang G. APD: the antimicrobial peptide database.
Nucleic Acids Res 2004;32:D5902.
[68] Moller NP, Scholz-Ahrens KE, Roos N, Schrezenmeir J.
Bioactive peptides and proteins from foods: indication for
health effects. Eur J Nutr 2008;47:17182.
[69] Mamone G, Picariello G, Caira S, Addeo F, Ferranti P. Analysis
of food proteins and peptides by mass spectrometry-based
techniques. J Chromatogr A 2009;1216:713042.
[70] Korhonen H, Pihlanto A. Bioactive peptides: production and
functionality. Int Dairy J 2006;16:94560.
[71] Hayes M, Ross RP, Fitzgerald GF, Stanton C. Putting microbes
to work: dairy fermentation, cell factories and bioactive
peptides. Part I: overview. Biotechnol J 2007;2:42634.
[72] Hayes M, Stanton C, Fitzgerald GF, Ross RP. Putting microbes
to work: dairy fermentation, cell factories and bioactive
peptides. Part II: bioactive peptide functions. Biotechnol J
2007;2:43549.
[73] Majumder K, Wu J. Angiotensin I converting enzyme
inhibitory peptides from simulated in vitro gastrointestinal
digestion of cooked eggs. J Agric Food Chem 2009;57:4717.
[74] Bauchart C, Morzel M, Chambon C, Mirand PP, Reyns C,
Buffire C, et al. Peptides reproducibly released by in vivo
digestion of beef meat and trout flesh in pigs. Br J Nutr
2007;98:118795.
[75] Schmelzer CE, Schops R, Reynell L, Ulbrich-Hofmann R,
Neubert RH, Raith K. Peptic digestion of beta-casein. Time
course and fate of possible bioactive peptides. J Chromatogr
A 2007;1166:10815.
[76] Contreras MM, Lopez-Exposito I, Hernandez-Ledesma B,
Ramos M, Recio I. Application of mass spectrometry to the
characterization and quantification of food-derived
bioactive peptides. J AOAC Int 2008;91:98194.
[77] Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC,
Baldwin J, et al. Initial sequencing and analysis of the
human genome. Nature 2001;409:860921.
[78] Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG,
et al. The sequence of the human genome. Science 2001;291:
130451.
[79] Agrafiotis DK, Lobanov VS, Salemme FR. Combinatorial
informatics in the post-genomics ERA. Nat Rev Drug Discov
2002;1:33746.
[80] Lyne PD. Structure-based virtual screening: an overview.
Drug Discov Today 2002;7:104755.
[81] Goodman RM, Naylor R, Tefera H, Nelson R, Falcon W. The
rice genome and the minor grains. Science 2002;296:1801.
[82] Schnable PS, Ware D, Fulton RS, Stein JC, Wei F, Pasternak S,
et al. The B73 maize genome: complexity, diversity, and
dynamics. Science 2009;326:11125.
[83] Schmutz J, Cannon SB, Schlueter J, Ma J, Mitros T, Nelson W,
et al. Genome sequence of the palaeopolyploid soybean.
Nature 2010;463:17883.
[84] Elsik CG, Tellam RL, Worley KC, Gibbs RA, Muzny DM,
Weinstock GM, et al. The genome sequence of taurine cattle:
a window to ruminant biology and evolution. Science
2009;324:5228.
[85] Lemay DG, Lynn DJ, Martin WF, Neville MC, Casey TM,
Rincon G, et al. The bovine lactation genome: insights into
the evolution of mammalian milk. Genome Biol 2009;10:
R43.
[86] Schell MA, Karmirantzou M, Snel B, Vilanova D, Berger B,
Pessi G, et al. The genome sequence of Bifidobacterium longum
reflects its adaptation to the human gastrointestinal tract.
Proc Natl Acad Sci U S A 2002;99:144227.
[87] Pripp AH, Isaksson T, Stepaniak L, Sorhaug T, Ardoe Y.
Quantitative structure activity relationship modelling of
peptides and proteins as a tool in food science. Trends Food
Sci Technol 2005;16:48494.
[88] Nakai S, Chan JC, Li-Chan EC, Dou J, Ogawa M. Homology
similarity analysis of sequences of lactoferricin and its
derivatives. J Agric Food Chem 2003;51:121523.
[89] Pripp AH, Isaksson T, Stepaniak L, Sorhaug T. Quantitative
structureactivity relationship modelling of ACE-inhibitory
peptides derived from milk proteins. Eur Food Res Technol
2004;219:57983.
[90] Asao M, Iwamura H, Akamatsu M, Fujita T. Quantitative
structureactivity relationships of the bitter thresholds of
amino acids, peptides, and their derivatives. J Med Chem
1987;30:18739.
[91] Cannata N, Merelli E, Altman RB. Time to organize
the bioinformatics resourceome. PLoS Comput Biol 2005;1:
e76.
[92] Minkiewicz P, Dziuba J, Iwaniak A, Dziuba M, Darewicz M.
BIOPEP database and other programs for processing
bioactive peptide sequences. J AOAC Int 2008;91:96580.
[93] Svedberg J, de HJ, Leimenstoll G, Paul F, Teschemacher H.
Demonstration of beta-casomorphin immunoreactive
materials in in vitro digests of bovine milk and in small
intestine contents after bovine milk ingestion in adult
humans. Peptides 1985;6:82530.
[94] Kuwata H, Yip TT, Yip CL, Tomita M, Hutchens TW. Direct
detection and quantitative determination of bovine
lactoferricin and lactoferrin fragments in human gastric
contents by affinity mass spectrometry. Adv Exp Med Biol
1998;443:2332.
[95] Sato K, Iwai K, Aito-Inoue M. Identification of food-derived
bioactive peptides in blood and other biological samples.
J AOAC Int 2008;91:9951001.
[96] Woodley JF. Enzymatic barriers for GI peptide and
protein delivery. Crit Rev Ther Drug Carrier Syst 1994;11:
6195.
[97] Vanhoof G, Goossens F, De MI, Hendriks D, Scharpe S.
Proline motifs in peptides and their biological processing.
FASEB J 1995;9:73644.
[98] Webb KE. Intestinal absorption of protein hydrolysis
products: a review. J Anim Sci 1990;68:301122.
[99] Meredith D. Review. The mammalian proton-coupled
peptide cotransporter PepT1: sitting on the
transporter-channel fence? Philos Trans R Soc Lond B Biol
Sci 2009;364:2037.
[100] Ziv E, Bendayan M. Intestinal absorption of peptides through
the enterocytes. Microsc Res Tech 2000;49:34652.
[101] Moughan PJ, Fuller MF, Han KS, Kies AK, Miner-Williams W.
Food-derived bioactive peptides influence gut function. Int J
Sport Nutr Exerc Metab 2007(Suppl. 17):S5S22.
[102] Walker WA. Absorption of protein and protein fragments in
the developing intestine: role in immunologic/allergic
reactions. Pediatrics 1985;75:16771.
 J O U RN A L OF P ROT EO M I CS 7 5 ( 2 0 12 ) 35 4 6 3 55 9 3559

[103] Vidal K, Labeta MO, Schiffrin EJ, Donnet-Hughes A. Soluble
CD14 in human breast milk and its role in innate immune
responses. Acta Odontol Scand 2001;59:3304.
[104] Donnet-Hughes A, Duc N, Serrant P, Vidal K, Schiffrin EJ.
Bioactive molecules in milk and their role in health and
disease: the role of transforming growth factor-beta.
Immunol Cell Biol 2000;78:749.
[105] Vermeirssen V, Van CJ, Verstraete W. Bioavailability of
angiotensin I converting enzyme inhibitory peptides. Br J
Nutr 2004;92:35766.