MINIREVIEW
Applications of diagonal chromatography for proteome-
wide characterization of protein modifications and
activity-based analyses
Kris Gevaert
1,2
, Francis Impens
1,2
, Petra Van Damme
1,2
, Bart Ghesquie
`
re
1,2
, Xavier Hanoulle
3
and Joe
¨
l Vandekerckhove
1,2
1 Department of Medical Protein Research, VIB, Ghent, Belgium
2 Department of Biochemistry, Ghent University, Belgium
3 UMR 8576 CNRS ) University of Sciences and Technologies of Lille, Structural and Functional Glycobiology Unit, Villeneuve d’Ascq,
France
Introduction
Proteomics refers to a qualitative, differential and
quantitative estimation of a proteome. Proteomes can
be extremely complex, often encompassing more than
10 000 different components per cell. Two-dimensional
gel electrophoresis [1] followed by electroblotting and
microsequencing [2–4] or in-gel digestion combined

few years, introducing a move from proteins to peptides as bits of informa-
tion in qualitative and quantitative proteome studies. Many shotgun pro-
teomics techniques randomly sample thousands of peptides in a qualitative
and quantitative manner but overlook the vast majority of protein modifi-
cations that are often crucial for proper protein structure and function.
Peptide-based proteomic approaches have thus been developed to profile a
diverse set of modifications including, but not at all limited, to phosphory-
lation, glycosylation and ubiquitination. Typical here is that each modifica-
tion needs a specific, tailor-made analytical procedure. In this minireview,
we discuss how one technique ) diagonal reverse-phase chromatogra-
phy ) is applied to study two different types of protein modification: pro-
tein processing and protein N-glycosylation. Additionally, we discuss an
activity-based proteome study in which purine-binding proteins were pro-
filed by diagonal chromatography.
Abbreviations
ABP, activity-based probe; COFRADIC, combined fractional diagonal chromatography; FSBA, 5¢-p-fluorosulfonylbenzoyladenosine; FSBG,
5¢-p-fluorosulfonylbenzoylguanosine; iTRAQ, isobaric tags for relative and absolute quantification; MudPIT, multidimensional protein
identification technology; PNGaseF, peptide N-glycosidase F; SB, sulfobenzoyl; SILAC, stable isotope labeling by amino acids in cell culture;
Nbs
2
, 2,4,6-trinitrobenzenesulfonic acid.
FEBS Journal 274 (2007) 6277–6289 ª 2007 The Authors Journal compilation ª 2007 FEBS 6277
large number of species, and it now suffices to generate
partial protein sequence information with which to
access entire (predicted) protein sequences stored in
expressed sequence tag, gene and protein sequence
databases.
This brought the dawn of novel strategies for pro-
tein identification. Measured masses of peptides pro-
duced by cleaving a protein with a protease with

[21]). MudPIT has since then been used in several
studies and has demonstrated its value, but it still
suffers from undersampling [19].
Selecting a lower number of peptides representative
of each protein originally present in the mixture may
alleviate this problem. These so-called signature pep-
tides [22] are then the only analyzed components, and
in this way a less complex peptide mixture is presented
to the mass spectrometer. The first reports using this
strategy were selective for cysteinyl peptides, allowed
quantification (differential analysis), and used biotin
tagging for consecutive capture by immobilized avidin
[23]. Later on, affinity selection was used to isolate, for
instance, phosphopeptides [24], N-glycosylated peptides
[25], ubiquitinated peptides [26], and N-terminal pep-
tides [27].
COFRADIC as a peptide-sorting tool
Our peptide-centric proteome approach [28,29] sorts
signature peptides and selects the part of a proteome
containing the information of biological interest. Our
technique is based on diagonal chromatography [30,31]
consisting of two repeated, identical peptide separa-
tions with a specific modification reaction (sorting
step) in between. Peptides that remain unchanged elute
at the same position in the two chromatographic runs,
whereas peptides that acquire a modification segregate
from the unchanged peptides either in earlier or in
later fractions. To reduce the number of repetitive
chromatographic runs, several fractions from the pri-
mary run can be combined and subjected to the sort-

covalently linked to a target protein and the corre-
sponding modified tryptic peptide is then sorted using
the principles of diagonal chromatography. The exam-
ple given here is a global activity-based proteome
COFRADIC and protein modifications K. Gevaert et al.
6278 FEBS Journal 274 (2007) 6277–6289 ª 2007 The Authors Journal compilation ª 2007 FEBS
analysis of purine-binding proteins in a total lysate of
human Jurkat T-cells [40].
COFRADIC analysis of protein
processing ) protease degradomics
Protein processing introduces novel protein fragments
that may be visualized on 2D polyacrylamide gels. For
example, Canals et al. used the fluorescent 2D differ-
ence gel electrophoresis technique [41] to catalog quan-
titative differences in the protein composition of
conditioned media of cells either expressing the metal-
loproteinase ADAMTS1 at physiological levels or
overexpressing it [42]. The latter scenario led to an
increase of fragments of proteins shed by ADAMTS1
into the medium that were picked by difference gel
electrophoresis and identified by MS. In fact, this
study led to the identification of five potential ADAM-
TS1 substrates, two of which (nidogens 1 and 2) were
further validated. Gel-free proteomic approaches have
been introduced for ‘degradomics’ [43] research as well.
The group of Overall used isotope-coded affinity tag
[23] combined with LC-MS ⁄ MS to quantify the levels
of secreted extracellular matrix proteins in breast carcin-
oma cell cultures overexpressing a membrane type 1
matrix metalloproteinase [44] and, more recently, they

0
200
400
600
800
1000
1200
1400
primary separation
combine primary fractions
COFRADIC sorting reaction
LC-MS/MS analysis
min
20 30 40 50 60 70 80
mAU
0
100
200
300
400
500
600
700
secondary separation
Fig. 1. The COFRADIC peptide sorting scheme. A peptide mixture
is first separated by RP-HPLC (the primary COFRADIC separation).
Here, the UV absorbance profile at 214 nm of a tryptic digest of a
proteome preparation from human Jurkat T-cells is shown. Primary
fractions (indicated in light gray boxes) are combined ) here, four
primary fractions (each 1 min wide) that are separated by a 13 min

number of primary fractions. Then, internal peptides
present in each fraction are reacted with 2,4,6-trinitro-
benzenesulfonic acid, which is known to efficiently and
quantitatively modify primary amines [49]. Internal
peptides thereby acquire a trinitrophenyl group at their
N-terminus and thus become very hydrophobic. Run-
ning such TNBS-modified primary fractions a second
time on the same column and under identical chro-
matographic conditions will now segregate TNBS-non-
reactive N-terminal peptides (all their amino groups
were already blocked) from TNBS-reacted internal
peptides, which underwent a very strong hydrophobic
shift (Table 1). Following metabolic or postmetabolic
labeling, N-terminal peptides of two (or more) proteo-
mes can be weighed against each other and, impor-
tantly, neo-N-termini originating from protein
processing are readily distinguished [34,36].
The characterization of protease substrates by such
a differential N-terminal COFRADIC approach is
illustrated in Fig. 2. In an ongoing project, host cell
substrates of the HIV-1 protease are catalogued in
human Jurkat T-cells grown in stable isotope labeling
by amino acids in cell culture (SILAC) medium supple-
mented with either natural, light
12
C
6
-arginine or
heavy
13

peptide is identified as TEAPLNPKANR(106-116)
and constitutes a neo-N-terminus indicative of HIV-1-
mediated protein processing. Processing of b-actin by
the HIV-1 protease between Leu105 and Thr106 was
already identified in previous studies [51], thereby vali-
dating our findings.
Table 1. Overview of the different COFRADIC procedures that have been developed. The type of peptide, the sorting agent used in
between the two consecutive RP-HPLC separation steps and the type of evoked shift are indicated. References to our original papers, in
which full technical details can be found, are given.
Sorted peptide Sorting agent RP shift Reference
Methionyl peptide Hydrogen peroxide Hydrophilic [32]
Cysteinyl peptide Reduction of nitrothiobenzoic
acid-modified cysteine
Hydrophilic [80]
N-terminal peptide TNBS reaction on internal,
free a-amine peptides
Hydrophobic
(internal peptides)
[33]
Phosphorylated peptide Cocktail of phosphatases Hydrophobic [37]
N-glycosylated peptide PNGaseF Hydrophilic or
hydrophobic
[39]
ATP-binding peptide NaOH treatment Hydrophilic [40]
3-Nitrotyrosinyl peptide Reduction of NO group Hydrophilic [38]
COFRADIC and protein modifications K. Gevaert et al.
6280 FEBS Journal 274 (2007) 6277–6289 ª 2007 The Authors Journal compilation ª 2007 FEBS
A similar approach but now with postmetabolic,
trypsin-mediated
18

combine
N-terminal COFRADIC analysis
651.39
651.88
652.37
650.91
650 651 652 653 m/z
947.49
948.06
948.29
949.50
949.80
950.06
950.39
947 948 949 950 m/z
947.71
630.43; y5
744.46; y6
954.66; y8
200 400 600 800 1000 m/z
347.24; b3
444.41; b4
557.42; b5
671.37; b6
1012.66; b9
300 500 700 900 1100
Fig. 2. HIV-1 protease processes b-actin in vitro at Leu105. The experimental route is sketched in (A). Following N-terminal COFRADIC, two
different peptides from b-actin were identified. Its N-terminal peptide, DDDIAALVVDNGSGMCKAGFAGDDAPR(2–28) (N-terminus acetylated,
lysine trideuteroacetylated, methionine oxidized and cysteine carbamidomethylated) was present in both proteome digests [ion trap MS
spectrum of triply charged precursor in (B)], whereas a second peptide was only present in the proteome treated with the HIV-1 protease

ed proteins, several lectins were combined in multilec-
tin affinity chromatography [55,56]. Alternatively, the
lectins’ glycan bias was exploited in a serial lectin
approach separating N-glycosylated (concanavalin A)
from O-glycosylated peptides (Jacalin) [57]. Chemical
trapping and release of N-glycosylated peptides was
introduced by the group of Aebersold in 2003 [25]. In
their approach, aldehydes are first introduced into the
glycan by periodate oxidation. These aldehydes then
covalently bind to immobilized hydrazide groups by
which glycosylated proteins are retained and all non-
glycosylated proteins are removed. Immobilized gly-
cosylated proteins are then further trimmed by trypsin
such that only tryptic peptides carrying glycans
remained fixed. Such peptides are finally recovered by
peptide N-glycosidase F (PNGaseF), which efficiently
removes N-glycans from conjugated asparagines while
converting these to aspartic acids [58]. The potential of
this chemical trapping approach is evident from recent
studies [59–62]; however, it requires several chemical
and enzymatic modification steps, and it is therefore
more complex than lectin-affinity methods; this could
potentially obstruct its widespread introduction in
proteomics laboratories.
We recently showed that N-glycosylated peptides can
be isolated by diagonal chromatography [39]. In our
approach, a protein mixture containing N-glycosylated
proteins is digested with trypsin, and the resulting pep-
tide mixture is separated by RP-HPLC. N-glycosylated
peptides are then specifically targeted by PNGaseF and

ing vasoactive and inflammatory peptides containing
C-terminal arginine or lysine [66]. The large subunit of
this complex binds and stabilizes the catalytic subunit
and thereby keeps the complex in circulation. In silico
predictions indicate that this protein potentially has
nine different targets for N-glycosylation, six of which
were identified in our study: the asparagines at posi-
tions 74, 111, 119, 348, 359 and 367 (Table 2). Three
other asparagines at positions 266, 311 and 520 were
missed and, as is evident from the annotations in the
UniProtKB ⁄ Swiss-Prot database, have hitherto not
been experimentally characterized. A closer look at the
sequences of the tryptic peptides harboring the poten-
tial glycosylation sites at positions 266 and 311 clearly
indicates that these peptides are very large (66 and 36
amino acids long, respectively). Therefore, they could
have been missed either because they are insoluble or
because our mass spectrometers, which have an empiri-
cal upper mass limit close to 3000 Da for producing
COFRADIC and protein modifications K. Gevaert et al.
6282 FEBS Journal 274 (2007) 6277–6289 ª 2007 The Authors Journal compilation ª 2007 FEBS
MS ⁄ MS spectra that are unambiguously identified by
mascot [67], could not detect them. One obvious way
to overcome this is by using proteases with nontryptic
specificities such as proteinase K that generally pro-
duce smaller peptides [68] and thus increase the chance
that more glycopeptides will be finally identified.
However, such protease digests sharply augment the
complexity of the analyte mixture.
Activity-based proteome-wide profiling

we profiled purine-binding proteins on a proteome
scale [40]. For this purpose, we used 5¢- p-fluoro-
sulfonylbenzoyladenosine (FSBA) (Fig. 3B), a known
reactive homolog of ATP (Fig. 3A) that binds proteins
in their nucleotide-binding region and then covalently
modifies nucleophilic amino acids (especially tyrosine
and lysine) in its proximity [75]. In the past, FSBA
was mainly used to profile the ATP-binding features of
selected, individual proteins. However, in 2004, Moore
et al. published a study in which FSBA and 5¢-p-fluoro-
sulfonylbenzoylguanosine (FSBG) were used to profile
ATP-binding and GTP-binding proteins, respectively,
in the proteomes of different lymphoid cells [76]. In
their approach, proteins were labeled with FSBA or
FSBG in cell extracts, separated by 2D PAGE and
electrotransferred onto a poly(vinylidene difuoride)
membrane. Subsequent treatment of sulfobenzoyl
adenosine ⁄ sulfobenzoyl guanosine (SBA ⁄ SBG)-labeled
proteins with NaOH hydrolyzed the ester bond
between the adenosine or guanosine and the sulfo-
benzoyl (SB) group and exposed the latter. Antibodies
to SB were then used to immunodetect FSBA-targeted
or FSBG-targeted proteins. Overlaying an image of
the immunoblot with the 2D pattern of silver-stained
proteins pointed to candidate ATP-binding or GTP-
binding proteins that were selected from the 2D gel
and identified by MS. In this way, 12 different proteins
could be identified as FSBA-labeled proteins.
Given the fact that a mild alkaline treatment as used
by Moore et al. [76] hydrolyzes the rather unstable

bors the site known to be involved in the catalytic
transfer of c-phosphate from ATP to protein kinase A
substrates [77].
When this sorting procedure was applied to a whole
proteome ) here, a human Jurkat T-cell proteome
depleted of small compounds like ATP and GTP,
which could compete with FSBA ) 185 sites in 132
proteins were identified. Clearly, this is a significantly
higher number of proteins than were detected in
the previous gel-based study [76]. Therefore, our
COFRADIC technique allows the functional interpre-
tation of a larger part of a sampled proteome. More
importantly, our approach directly points to the actual
site that was modified and might thereby aid in inter-
preting structural features of ATP-binding proteins.
As expected, the majority of FSBA-labeled Jurkat
proteins were known binders of small nucleotides,
cofactors, or DNA and RNA molecules. However,
several proteins and sites were not readily explained by
the known affinity of FSBA for purine-binding pock-
ets. Closer inspection revealed that at least 23 of such
unexplainable sites were previously characterized as
tyrosine phosphorylation sites. Therefore, we assume
that when FSBA is recognized by an ATP-binding site,
there are two options for SBA labeling: either the
fluorosulfonyl group reacts with a target side chain
located on the protein carrying the ATP-binding site
(homo-reaction), or, through lack of a suitable reac-
tion partner, it may react with a side chain present on
proteins that interact with the protein carrying the

O
S
O
O
PEPTIDE
A
C
B
N
N
N
N
NH
2
OH
O
S
O
O
PEPTIDE
N
N
N
N
NH
2
O
HO OH
O
P

biological problem under consideration, thereby reduc-
ing the complexity of the problem without losing
much information. Unlike targeted peptide-centric
approaches such as isotope-coded affinity tag [23],
COFRADIC is not based on affinity procedures,
which are limited at two levels: first, the chemistries
used to convert sets of peptides into affinity probes;
and second, the limitations of mass transfer that are
inherently to liquid–solid state chemistries [79]. At the
first level, COFRADIC has a fundamental advantage
because its chemistries do not need to create different
affinity labels. For instance, an affinity tag specific for
methionyl peptides is extremely difficult to establish; in
contrast, a simple oxidation step by hydrogen peroxide
will specifically produce methionyl-sulfoxide derivatives
showing significant hydrophilic shifts in diagonal
reverse-phase chromatography [32]. At the second
level, affinity-based experiments [23,27,48] have limita-
tions either at the level of incomplete or variable incor-
poration of the tag (for example, linking a biotinyl
group to a specific set of peptides can be incomplete
and partly unspecific) or at the level of interactions of
tagged peptides with the affinity resin, where the high-
est affinities and avidities are not always reached. In
contrast, using COFRADIC, we select subsets of pep-
tides related to the biological question under investiga-
tion. For instance, for the study of the oxidation of
protein methionines during oxidative stress, cells can
be differentially labeled with [
13

Table 1, the chemical or enzymatic modification
reactions that have been used successfully in a
COFRADIC-based approach are listed. They cover
sorting methods varying from modifications to spe-
cific side chains, such as cysteinyl [80] and methionyl
[32] moieties, to post-translational modifications by
phosphatase [37] and PNGaseF [39] treatments. In
another application, peptides located at the N-termini
of proteins or of their fragments are sorted [33]. In
this way, we have successfully analyzed protein pro-
cessing in highly complex proteomes by the target
proteins and identified the exact cleavage site(s), cre-
ating the basis for fundamental protease degradomics
[34,36].
As mentioned above, it is also possible to set up spe-
cific covalent interactions between proteins and small
molecules such as drugs or mimetic molecules of natu-
ral metabolites such that chromatographic shifts can
be evoked, thus allowing sorting of the conjugated
peptides by COFRADIC. The example shown relates
to a study with an ATP analog [40]; however, it can,
in principle, be extended to drugs that covalently inter-
act with their target protein, either directly or after
being metabolized in the tissue or organism to form
reactive products.
One of the drawbacks of COFRADIC relates to the
segmentation of the peptide separation flow during the
primary run: many peptides may end up in two con-
secutive fractions for their secondary analyses. When
the same separation is repeated a second time or fur-

only those peptides that can be separated by RP-
HPLC, ionize well in mass spectrometers and yield
informative MS ⁄ MS spectra can be identified. An
interesting, recent development is top-down protein
sequencing, which enables researchers to focus on an
increasing set of protein modifications [81,82]. Such
top-down techniques focus on complete proteins, allow
detection of normally labile protein modifications, and
avoid several problems associated with signature pep-
tides (see above). However, proteins of interest need to
be rather pure (the number of contaminating proteins
should be low), which may currently hinder the routine
applicability of such approaches.
This review has shown that the COFRADIC tech-
nology is extremely versatile and flexible and provides
profound insights into biological questions, often
much more than what could be obtained by alterna-
tive proteomics procedures such as 2D gels or shotgun
proteomics. Its strong point is its high flexibility in
selecting specific chemistries or enzymatic modifica-
tions oriented towards the biological question(s) under
consideration. It should be clear from the supporting
concepts that the repertoire of applications can only
be expected to grow in the future through the develop-
ment of specific chemical or enzymatic sorting reac-
tions that alter the chemical nature of a predetermined
set of peptides.
Acknowledgements
F. Impens is a research assistant of the Fund for
Scientific Research ) Flanders (Belgium). The work in

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