Food Chemistry 229 (2017) 417–424

Contents lists available at ScienceDirect

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

Protein carbonylation sites in bovine raw milk and processed milk

products
Sanja Milkovska-Stamenova, Ruzanna Mnatsakanyan, Ralf Hoffmann ⇑
Institute of Bioanalytical Chemistry, Faculty of Chemistry and Mineralogy, Universität Leipzig, Leipzig, Germany
Center for Biotechnology and Biomedicine, Universität Leipzig, Leipzig, Germany

a r t i c l e i n f o a b s t r a c t

Article history: During thermal treatment of milk, proteins are oxidized, which may reduce the nutritional value of milk,
Received 1 January 2017 abolish protein functions supporting human health, especially important for newborns, and yield poten-
Received in revised form 20 February 2017 tially harmful products. The side chains of several amino acids can be oxidized to reactive carbonyls,
Accepted 20 February 2017
which are often used to monitor oxidative stress in organisms. Here we mapped protein carbonylation
Available online 22 February 2017
sites in raw milk and different brands of pasteurized, ultra high temperature (UHT) treated milk, and
infant formulas (IFs) after digesting the precipitated proteins with trypsin. Reactive carbonyls were
Keywords:
derivatized with O-(biotinylcarbazoylmethyl)hydroxylamine to enrich the modified peptides by avidin-
Aldehyde reactive probe
Glyoxal
biotin affinity chromatography and analyze them by nanoRP-UPLC-ESI-MS. Overall, 53 unique carbony-
Infant formula lated peptides (37 carbonylation sites, 15 proteins) were identified. Most carbonyls were derived from
Milk proteomics dicarbonyls (mainly glyoxal). The number of carbonylation sites increased with the harsher processing
Oxidation from raw milk (4) to pasteurized (16) and UHT milk (16) and to IF (24).
Protein carbonylation Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction during Maillard reactions or by oxidation of sugars and lipids, are

Thermal treatment is applied during milk processing to ensure its
microbiological safety and to extend its shelf-life. However, high
temperatures can also induce diverse reactions modifying proteins
to different degrees forming currently unknown products with
indefinite complexity, such as oxidations and Maillard reactions,
i.e., the reaction between reducing sugars and amino groups. Oxida-
tion products can be formed by different reactions affecting for
example lysine, tryptophan, methionine, cysteine, proline, histidine,
arginine, and tyrosine residues (Guy & Fenaille, 2006). Besides oxi-
dation of thiols (cysteine) and thioethers (methionine) the irre-
versible formation of alcohols, aldehydes, and ketones are most
common. Hydroxyl groups are most likely unproblematic modifica-
tions that will not affect human health, whereas both aldehydes and
ketones are reactive and are thus often termed ‘‘reactive carbonyls”
or ‘‘protein carbonyls” (Stadtman, 1995). This non-enzymatic pro-
tein modification is widely accepted to monitor oxidative stress in
cells or organisms (Fedorova, Bollineni, & Hoffmann, 2014;
Nystrom, 2005). In milk, proteins can be carbonylated by three dif-
ferent pathways. First, dicarbonyl compounds, such as glyoxal
(GO), methylglyoxal (MGO), and 3-deoxyglucosone (3-DG), formed
⇑ Corresponding author at: Biotechnologisch-Biomedizinisches
Deutscher Platz 5, 04103 Leipzig, Germany.
E-mail address: bioanaly@rz.uni-leipzig.de (R. Hoffmann).
highly reactive and can modify the side chains of lysine, arginine,
and cysteine residues (Aldini et al., 2015; Thornalley, Langborg, &
Minhas, 1999; Wolff & Dean, 1987, 1988). Second, the same residues
can be modified by lipid peroxidation products (LPPs), such as acro-
lein, malondialdehyde, 4-hydroxy-2-nonenal (HNE), 4-oxo-2-
nonenal (ONE), 4-hydroxy-2-hexanal (HHE), and 4-oxo-2-hexanal
(OHE) (Aldini, Orioli, & Carini, 2011; Aldini et al., 2015; Fedorova
et al., 2014; Guy & Fenaille, 2006; Loidl-Stahlhofen, Hannemann, &
Spiteller, 1994; Trnkova, Drsata, & Bousova, 2015). Third, heavy
metal ions (e.g., iron) can oxidize amino acid residues, such as lysine,
arginine, proline, and threonine, in the presence of oxidants
(Stadtman & Levine, 2003).
Protein carbonylation sites have been intensively studied as
markers of oxidative stress in the context of diabetes, Alzheimer’s
disease, atherosclerosis, and other diseases (Dalle-Donne et al.,
2005, 2006; Fedorova et al., 2014; Madian & Regnier, 2010), while
only a few studies have analyzed them in food. For example, the
formation of protein carbonyls can irreversibly block essential
amino acids, which also reduces protein digestibility, and thereby
decreases the nutritional value of milk. These modifications may
also change the protein structure, trigger aggregation, and initiate
or prevent allergenic reactions by forming or masking epitopes.
Zentrum, The content of oxidized proteins increases in total abundance
when heating milk (Meltretter, Wust, & Pischetsrieder, 2013,

http://dx.doi.org/10.1016/j.foodchem.2017.02.102
0308-8146/Ó 2017 Elsevier Ltd. All rights reserved.
418 S. Milkovska-Stamenova et al. / Food Chemistry 229 (2017) 417–424
2014; Meyer, Baum, Vollmer, & Pischetsrieder, 2012). However,
these studies focused on the overall protein oxidation content
using amino acid analysis of protein hydrolysates or studied oxida-
tion of a single or a few milk proteins incubated in vitro. Thereby,
oxidized methionine, cysteine, and tryptophan residues were iden-
tified in heated b-lactoglobulin (b-LG) and commercial milk prod-
ucts (Meltretter et al., 2013, 2014) or in b-LG and lactotransferrin
heated for different times (Dyer et al., 2016). Additionally, in pro-
teins isolated from commercial milk products and separated by
gel electrophoresis, His146 of b-LG was identified as a HNE-
carbonylation site using MALDI-TOF-MS (Fenaille, Parisod, Tabet,
& Guy, 2005). Despite many discussions about protein oxidation
and its potential health risk in food (Estevez & Luna, 2016),
detailed studies on milk products characterizing the modifications,
protein targets, modification sites and types, and their concentra-
tions in milk are missing. In order to better understand the impact
of protein-bound carbonyls on food quality and human health, the
first crucial step is to identify protein residues modified during
industrial processing.
Here, protein oxidation was analyzed in raw and commercial
bovine milk (different brands of pasteurized milk, UHT milk, and
IFs) using targeted bottom-up proteomics. After derivatization of
the carbonyl-groups with O-(biotinylcarbazoylmethyl)hydroxyla
mine, also known as aldehyde reactive probe (ARP), peptides were
enriched with avidin and analyzed on a nanoRP-UPLC-ESI-
Orbitrap-MS. Thus, 53 unique carbonylated peptides representing
37 carbonylation sites in 15 proteins were identified in total. Heat
treatment significantly increased the numbers of protein carbonyl
sites derived from dicarbonyl compounds, mainly GO, relative to
raw milk. The data clearly indicate that protein carbonylation is
more pronounced in infant formula than in pasteurized and UHT-
treated milk.
2. Materials and methods
2.1. Reagents and milk samples
AppliChem GmbH (Darmstadt Germany): Iodoacetamide (IAA)
(99%) and Tris (99.9%). Biosolve GmbH (Valkenswaard, Nether-
lands): Acetonitrile (ULC-MS grade, 99.97%), formic acid (ULC-MS
grade, 99%), and methanol (ULC-MS grade, 99.98%). Carl Roth
GmbH (Karlsruhe, Germany): Methanol (HPLC grade, 99.9%),
ethanol (HPLC grade, 99.8%), urea (99.5% p.a.), sodium dodecyl
sulfate (SDS, 99.5%), glycerol (99.5%), and dithiothreitol (DTT)
(99%). Kmf Laborchemie Handels GmbH (Lohmar, Germany):
Ammonia solution (25% p.a.). Promega GmbH (Mannheim, Ger-
many): sequencing grade modified trypsin. Merck KGaA (Darm-
stadt, Germany): Chloroform (99.8%). Riedel-de Haën
(Steinheim, Germany): Bromophenol blue sodium salt. SERVA Elec-
trophoresis GmbH (Heidelberg, Germany): Bovine serum albumin
(BSA) (>98%), ammonium persulfate (>99%), acrylamide/bis solu-
tion (30% w/v), tetramethylene diamine (98.5%), Coomassie Bril-
liant Blue G 250, glycine (>98.5), and CHAPS (98%). Sigma-Aldrich
Chemie GmbH (Steinheim, Germany): thiourea (99%), ammo-
nium bicarbonate (99.5%), acetic acid (LC–MS grade), b-
mercaptoethanol (99%), D-biotin (99%), sodium chloride
(99.5%), sodium deoxycholate (97%), sodium dihydrogen phos-
phate (99%), sodium hydroxide (>98%), tris-(2-carboxyethyl)
phosphine (TCEP) (98%). Cayman (Michigan, U.S.A.): O-(biotinyl
carbazoylmethyl)-hydroxylamine (aldehyde reactive probe, ARP)
(97.9%), Perbio Science Deutschland GmbH (Bonn, Germany):
Pierce monomeric avidin agarose.
Pasteurized milk (P) and UHT-treated milk from three different
companies and first stage IFs from five different companies were
bought in a local supermarket. Raw colostrum (RC) and raw milk
(RM) were obtained from a farm. IFs were prepared according to
the manufacturers’ manuals. Milk samples were immediately ali-
quoted and stored at 80 °C.
Water was purified in-house (resistance >18 mX/cm; total
organic content <10 ppb) on a PureLab Ultra Analytic System (ELGA
Lab Water, Celle, Germany).
2.2. Protein preparation
Proteins were precipitated from milk (50 lL) using Folch
extraction (methanol/chloroform/water) (Milkovska-Stamenova &
Hoffmann, 2016a). The organic and aqueous phases were discarded
after centrifugation (10 min, 10,000g, 4 °C), the remaining protein
pellets were immediately dried under vacuum, and dissolved in
lysis buffer (50 mmol/L Tris-HCl, pH 7.5, 7 mol/L urea, 2 mol/L
thiourea, 2% w/v CHAPS).
Proteins were quantified by a colorimetric Bradford assay using
BSA as standard and confirmed by the band intensities obtained
after SDS-PAGE (15% T, 7 cm  8 cm; Mini Protean III cell, Bio-
Rad Laboratories, Munich, Germany) loading always 3 lg of pro-
tein per lane (diluted in sample buffer: 62.5 mmol/L Tris-HCl, pH
6.8, 50 mmol/L DTT, 2% w/v SDS, 20% w/v glycerol, 0.2% w/v bro-
mophenol blue) (Milkovska-Stamenova & Hoffmann, 2016a). Pro-
teins were stained with Coomassie Brilliant Blue G 250, the gel
imaged on a Gel DocTM EZ Imager (Bio-Rad), and the band intensi-
ties compared using ImageLab (Bio-Rad).
2.3. Tryptic digestion
Aliquots of the protein samples (0.1 mg protein) were diluted
with ammonium bicarbonate (25 mmol/L) to obtain protein con-
centrations of 1 mg/mL before sodium deoxycholate was added
(1% w/v) to denature the proteins. Proteins were reduced with
TCEP (5 mmol/L, 60 °C, 30 min, 550 rpm), alkylated with iodoac-
etamide (10 mmol/L, 37 °C, 30 min, darkness, 550 rpm), and excess
iodoacetamide was quenched with DTT (10 mmol/L, 37 °C, 30 min,
550 rpm) (Bollineni, Fedorova, Bluher, & Hoffmann, 2014). Trypsin
(2 lg) was added (37 °C, overnight, 550 rpm), the digest stopped
with formic acid (0.5% v/v), precipitated sodium deoxycholate
removed by centrifugation (10 min, 12,000 rpm), and supernatants
desalted by solid phase extraction (SPE; Oasis HLB 1 cc, 10 mg,
Waters GmbH, Eschborn, Germany). Briefly, cartridges were
washed with methanol and equilibrated with water (1 mL). Sam-
ples were loaded, the stationary phase washed with aqueous ace-
tonitrile (1 mL, 7%, v/v) containing formic acid (0.1%, v/v) three
times before the peptides were eluted (0.5 mL) with aqueous ace-
tonitrile (70%, v/v) containing formic acid (0.5%, v/v). Peptide frac-
tions were dried under vacuum.
2.4. Derivatization and affinity chromatography
Derivatization and affinity enrichment of carbonylated peptides
were performed as described previously (Bollineni et al., 2014). In
detail, tryptic digests were reconstituted in aqueous acetonitrile
(150 lL, 7%, v/v) containing formic acid (0.1%, v/v) and an aqueous
ARP solution (25 mmol/L, 0.1 mL) was added. After two hours
(room temperature, 550 rpm) the samples were purified by SPE,
as described in the previous paragraph. The dried samples were
reconstituted in phosphate buffered saline (PBS, 20 mmol/L NaH2-
PO4, 0.3 mol/L NaCl, pH 7.4, 0.1 mL). Mini-spin columns packed
with Pierce monomeric avidin agarose (0.1 mL) were connected
via a Luer-lock to a syringe and washed with phosphate buffer
(10 mmol/L NaH2PO4, pH 7.4, 1.5 mL). Irreversible binding sites
were blocked with D-biotin (2 mmol/L, 0.3 mL), washed with
glycine-HCl solution (pH 2.8, 0.1 mmol/L, 0.5 mL), and equilibrated
with PBS (2 mL). ARP-labelled tryptic digests (0.1 mL) were loaded,
 S. Milkovska-Stamenova et al. / Food Chemistry 229 (2017) 417–424
the column washed after 15 min with PBS (1 mL), phosphate buffer
(1 mL), and ammonium bicarbonate (2 mL, 50 mmol/L) containing
methanol (20%, v/v), and equilibrated with water (1 mL). Peptides
were eluted with aqueous acetonitrile (30%, v/v) containing formic
acid (0.4%, v/v, 0.5 mL), dried under vacuum, and stored at 20 °C.
2.5. nanoRP-UPLC-MS
The enriched and desalted tryptic digests were dissolved in
aqueous acetonitrile (3%, v/v; 50 lL) containing formic acid (0.1%,
v/v). Aliquots of two replicates of RM and RC as well as from differ-
ent brands of pasteurized milk (P, n = 3), UHT milk (n = 3), and IF
(n = 5) were mixed to prepare pooled samples of RM, RC, P, UHT,
and IF. Pooled or individual samples were separated on a nano-
Acquity UPLC (Waters GmbH, Eschborn, Germany) coupled on-
line to a LTQ Orbitrap XL ETD mass spectrometer equipped with
a nano-ESI source (Thermo Fisher Scientific, Bremen, Germany).
Peptide samples (4 lL) were trapped (nanoAcquity Symmetry
C18-column, internal diameter (ID) 180 lm, length 20 mm, particle
diameter 5 lm) at a flow rate of 10 lL/min (3% eluent B) and sep-
arated on a BEH 130 column (C18-phase, ID 75 lm, length 100 mm,
particle diameter 1.7 lm; 30 °C) using a flow rate of 0.4 lL/min.
Eluents A and B were water and acetonitrile both containing formic
acid (0.1%, v/v). Peptides were eluted by consecutive linear gradi-
ents from 3% to 9% (2.1 min), 9.9% (1.9 min), 17.1% (10 min), 18%
(0.5 min), 20.7% (0.2 min), 22.5% (3.1 min), 25.6% (3 min), 30.6%
(5 min), 37.8% (2.8 min), 45% (3 min) and finally to 81% eluent B
(2 min) (Bollineni et al., 2014).
The transfer capillary temperature was set to 200 °C and an ion
spray voltage of 1.5 kV was applied to a PicoTipTM on-line nano-ESI
emitter (New Objective, Berlin, Germany). Mass spectra were
recorded for an m/z range from 400 to 2000 in the orbitrap mass
analyzer at a resolution of 60,000 at m/z 400. CID tandem mass
spectra (isolation width 2, activation Q 0.25, normalized collision
energy 35%, activation time 30 ms) were acquired using data
dependent acquisition (DDA) for the six most intense signals with
a dynamic exclusion window of 60 s. Data were acquired with
Xcalibur (Version 2.0.7; Thermo Fisher Scientific).
2.6. Data analysis
Acquired tandem mass spectra were searched against bovine
proteins present in the SwissProt database (release 2014_01) using
Sequest (Proteome Discoverer 1.4, Thermo Fisher). Parameter set-
tings were a precursor mass tolerance of 10 ppm, fragment mass
tolerance of 0.8 Da, trypsin as protease, and up to two missed
cleavage sites. As Proteome Discoverer allows only a maximum
of six variable modifications per search, the 16 targeted carbonyl
modifications were distributed in four different templates with
each template containing also carbamidomethylation of cysteine
and oxidation of methionine as variable modifications: i) ARP-
derivatized carbonylated lysine (mass shift of 312.08 m/z units),
arginine (270.06 m/z units), threonine (311.10 m/z units), and pro-
line (329.11 m/z units) residues, ii) ARP-derivatized malondialde-
hyde adducts (367.13 m/z units) at lysine and arginine residues
as well as acrolein (369.14 m/z units), pentenal (397.17 m/z units),
and crotonaldehyde (383.16 m/z units) adducts of cysteine, his-
tidine, and lysine residues, iii) ARP-derivatized adducts of HNE
(469.23 m/z units), HHE (427.18 m/z units), ONE (467.22 m/z units),
and OHE (425.17 m/z units) present at cysteine, histidine, and
lysine residues, and iv) MGO (385.14 m/z units) and GO (371.12
m/z units) modifications at arginine, cysteine, and lysine residues
as well as 3-DG (457.16 m/z units) and GO (353.11 m/z units)
adducts of arginine and lysine residues. Consequently each sample
was searched four times using a different template each time.
Modified peptides identified with high and medium confidence
419
considering charge state specific Xcorr scores (2 for z = 2, 2.25
for z = 3, 2.5 for z = 4, and 2.75 for z = 5) and ranked in position
1 were considered for further analysis.
The tryptic digest of each pooled milk sample was analyzed by
nanoRP-UPLC-MS and the acquired data set searched against the
SwissProt database. All unmodified peptides identified with med-
ium to high confidence (Xcorr > 1.5) and ranked in position 1 were
used to generate an exclusion list for each pooled milk sample (RM,
RC, P, UHT or IF) considering both exact m/z values (10 ppm error)
and retention times (±1 min). The exclusion lists were used to
reanalyze each pooled milk sample again using the same instru-
mental settings as before. Peptides identified as carbonylated in
any of the ten analyses were used to create an inclusion list consid-
ering again the exact m/z values (10 ppm error). The signals were
grouped in segments of four minutes with an overlap among the
windows of 1 min. All individual milk samples (RM, RC, P1 to P3,
UHT1 to UHT3, and IF1 to IF5) were analyzed twice as independent
technical replicates (starting with Folch extraction) using the
instrumental settings described above. Only carbonylated peptides
confidently confirmed in two different analyses (Xcorr  2 for z = 2,
2.4 for z = 3, 3.3 for z = 4, and 4.2 for z = 5) and additionally
confirmed by manual interpretation of the tandem mass spectra
were considered as reliably identified.
3. Results
Despite the affinity enrichment, the signals of ARP-labelled pep-
tides were relatively weak compared to the still present unmodi-
fied sequences and thus typically missed by DDA, which
identified mostly unmodified peptides in the milk-product-
specific pooled samples. Thus, it was necessary to analyze each
sample a second time by excluding all signals corresponding to
unmodified sequences confidently identified in the first analysis
of each pooled sample by generating a timed exclusion list, which
contained 193–232 m/z values for the different samples along the
gradient with a maximum of about 90 values in a four minute win-
dow (Fig. S1). The confidence in the identified peptides was further
improved by combining all signals indicated by Sequest to be car-
bonylated in any of the five pooled milk samples in a retention
time-based inclusion list by combining all corresponding m/z val-
ues (10 ppm error) into 4 min segments with an overlap of 1 min
between consecutive segments. The confidence of the identifica-
tion was further improved by using relatively high charge state
dependent scores that had to be obtained in at least two different
analyses. Most importantly, the tandem mass spectra of all car-
bonylated peptides were confirmed by manual interpretation. They
typically contained intense signals and almost complete b- and y-
series (Fig. 1) and thus allowed a reliable identification of both
modification sites and carbonyl types. For example, peptides 2
and 3 (m/z 661.34 and 680.36, z = 3) both corresponding to resi-
dues 95 to 108 of a-lactalbumin (a-LA) that were carbonylated
on Lys98 (Fig. 1A and B). The mass shifts of 312.08 and 369.14
m/z units at lysine indicated by both the b- and y-series together
with ARP-specific signals identified these residues as derivatized
aminoadipic semialdehyde and acrolein adducts, respectively. Sim-
ilarly, peptide 29 (residues 149 to 162 of b-LG) was modified at
Cys160 by GO (Fig. 1C).
The analysis of all thirteen milk samples in two technical repli-
cates using the timed inclusion list identified 53 carbonylated pep-
tides with confident scores, which were all manually confirmed,
corresponding to 37 unique carbonylation sites in 15 proteins
(Tables 1 and S1). The peptides carried typically one and rarely
two carbonyl groups: GO, MGO or 3-DG adducts at lysine, cysteine,
and arginine residues, acrolein, pentenal, and OHE at lysine resi-
dues, and aminoadipic and glutamic semialdehyde at lysine and
420 S. Milkovska-Stamenova et al. / Food Chemistry 229 (2017) 417–424

Fig. 1. CID tandem mass spectra recorded for precursor ions at m/z 661.34 (panel A), m/z 680.36 (panel B), and m/z 1015.45 (panel C) corresponding to a-LA(95–108) with
Lys98 either oxidized to aminoadipic semialdehyde or carbonylated by acrolein and b-LG(149–162) carrying GO-modified position Cys160, respectively. Proteins were
precipitated from IF-1, P-2, and UHT-1, respectively, digested, enriched with biotin-avidin affinity chromatography, and analyzed by targeted nanoRP-UPLC-ESI-MS/MS.
 S. Milkovska-Stamenova et al. / Food Chemistry 229 (2017) 417–424 421

Table 1
Carbonylation sites (CS) of 15 milk proteins identified in RM, RC, P, UHT, and IF. X indicates that a modification site was identified in at least one of the corresponding samples,
whereas blanks indicate that the CS was missed by targeted nanoRP-UPLC-ESI-MS/MS.

Protein/CS RM RC P UHT IF
Dicarbonyl-derived carbonylation sites
b-Lactoglobulin
K60 X X X
C66 X X
K69 X X X X
C106 X X
C160 X X
a-Lactalbumin
K94 X
C101 X
K114 X X X X
K122 X X
aS2-Casein
K41 X X X X
Bovine serum albumin
K76 X X X
C447 X X X X
R484 X
Lactotransferrin
C9 X X
C347 X X X
R428 X X X
b-2-Microglobulin
K19 X X
Polymeric immunoglobulin receptor
C366 X X X X
K478 X X
Butyrophilin subfamily 1 member A1
R101 X X X
K441 X
Protein MMS22-like
R308 X X
CD9 antigen
K190 X X
Phosphomannomutase 2
K109 X
Platelet glycoprotein 4
R272 X
K315 X X

MCO
a-Lactalbumin
K98 X
aS1-Casein
P5 X
K34 X
K132 X X X X
j-Casein
P36 X X
P70 X X X X
P84 X X
P101 X X X
P109 X X X

a,b-Unsaturated aldehydes(LPPs)
b-Lactoglobulin
K60 X
K69 X X X
a-Lactalbumin
K34 X X X
K98 X X
j-Casein
K46 X X
Nucleotide-binding oligomerization domain-containing protein 2
K843 X X
422 S. Milkovska-Stamenova et al. / Food Chemistry 229 (2017) 417–424

Fig. 2. Percentages of modified (unique) amino acid residues (panel A) and total numbers of residues modified by dicarbonyls (GO, MGO, and 3-DG), metal-catalyzed
oxidation (glutamic and aminoadipic semialdehydes), and a,b-unsaturated aldehydes (pentenal, OHE, and acrolein) (panel B).

Fig. 3. Percentages of amino acid residues modified by different carbonylation reactions (panel A) and total number of carbonylated residues derived from dicarbonyls
identified in raw milk (RM), raw colostrum (RC), pasteurized milk (P), ultra high temperature treated (UHT) milk, and infant formula (IF) by targeted nanoRP-UPLC-ESI-MS/MS
(panel B).

proline residues, respectively (Fig. S2). Among the modified pro-
teins, j-casein (j-CN) and whey proteins b-LG and a-LA contained
most carbonylation sites, while the other twelve milk proteins con-
tained one to three modification sites (Tables 1 and S2). Most mod-
ification sites were identified on lysine (57%), whereas the others
were equally distributed among cysteine (17%), proline (14%),
and arginine (12%) residues (Fig. 2A). The majority of lysine resi-
dues was modified by GO (Fig. 2B), which was also the main mod-
ification type of cysteine and arginine residues (Fig. 2B) accounting
for 90% of dicarbonyl-derived carbonylation sites. This appears to
be the main carbonylation route in processed milk products (70%,
26 unique modification sites in 12 milk proteins), followed by
metal-catalyzed oxidation (MCO; 24.3%, nine modification sites
in three proteins), and reaction products of a,b-unsaturated alde-
hydes (LPPs; 16.2%, six sites in four proteins) (Fig. 3A, Table 1).
The number of peptides containing dicarbonyl-derived modifica-
tions was typically two times higher in processed milk than in
raw milk, except for one infant formula with four times more car-
bonylated peptides observed (Fig. S3). The number of dicarbonyl-
derived carbonylation sites increased from raw milk (4) fourfold
to P and UHT milk (16) and further to IF (24) (Fig. 3B) indicating
that the overall degree of dicarbonyl-derived carbonylation may
increase in the same order in milk products.
Most peptides carrying carbonyl groups derived from MCO
were identified in aS1- and j-CN indicating that they might be
the main target of this carbonylation type (Table 1). For example,
aS1-CN contained aminoadipic semialdehyde at lysine residues
(positions 34 and 132) and glutamic semialdehyde at proline in
position 5. Interestingly, five of six proline residues oxidized to glu-
tamic semialdehydes were detected in j-CN. Additionally, Lys98 of
a-LA was also detected as aminoadipic semialdehyde. However,
the rather low numbers of MCO-generated carbonyls do not allow
any conclusions about the influence of thermal processing on this
carbonylation reaction in milk (Table 1). Six carbonylation sites
detected in four proteins originated from a,b-unsaturated aldehy-
des (lipid peroxidation products, Table 1), i.e., three from acrolein
(Lys34 in aS1-CN, Lys46 in j-CN and Lys98 in a-LA) (Table 1),
one from OHE (Lys60 in b-LG), and one from pentenal (Lys69 in
b-LG, Table S1).
Interestingly, some carbonylation sites contained different car-
bonylation types, such as Lys98 in a-LA identified as aminoadipic
semialdehyde in peptide 2 and as acrolein-adduct in peptides 3
and 4 (Table S1). The same two modifications were present at
Lys34 in aS1-casein (aS1-CN; peptides 12 and 13 in Table S1). Fur-
thermore, in b-LG Lys60 was modified by GO and OHE (peptides
18–20) and Lys69 by GO and pentenal (peptides 22–27) (Table S1).
Although the current study intended to identify reactive car-
bonyls in milk proteins, the signal intensities allow quantitative con-
clusions for several intense GO-modified peptides. However, it
should be noted that the methods were not tested for dynamic
ranges, recovery rates, and limits of qunatification and thus have
to be taken with caution. GO-modified peptides of a-LA, b-LG, BSA,
and lactotransferrin (Fig. S4, panels A-D) showed significantly
higher peak areas in IFs than in the other milk samples. Some GO-
modified peptides were detected in pasteurized milk at slightly
higher levels than in UHT-treated milk (Fig. S4, panels A and B),
which partially contained slightly higher (Fig. S4, panel D) but
mostly similar quantities as raw milk. Thus, quantities partially fit
to the numbers of unique peptides identified in the different sam-
ples (Fig. S3), although more peptides have to be quantified to con-
firm this trend. Peptides modified by other carbonylation types
could not be quantified due to their considerably lower peak
intensities.
4. Discussion
The analysis of posttranslational modifications is always chal-
lenging due to their naturally low content in the proteome, which
is often less than 10% of the corresponding unmodified proteins.
 S. Milkovska-Stamenova et al. / Food Chemistry 229 (2017) 417–424
Thus, mass spectrometry typically misses modified peptides with
their very low signal intensities, which are often close to or even
hidden in the background noise. This limitation is independent of
the applied dissociation technique (e.g., collisions or electron
transfers) and also true for both data dependent and independent
acquisition modes (Szabo & Janaky, 2015). Thus, enrichment tech-
niques are essential to increase the contents of the targeted mod-
ifications relative to unmodified or differently modified sequences.
However, enrichment techniques are hampered by rather unspeci-
fic interactions limiting their efficacy and resulting often in signif-
icant sample losses, especially when derivatization or labelling
strategies are involved. Derivatization of reactive carbonyls with
ARP is highly efficient at the peptide level when using acidic con-
ditions, even in complex sample mixtures like human serum
(Bollineni et al., 2014). Importantly, ARP appears to possess a
rather broad activity range against aldehydes and ketones of differ-
ent structures. Moreover, the biotin group allows enriching the
derivatives using well established avidin affinity techniques, as
applied here. Thus, the set of carbonylation sites identified here
should closely resemble the carbonylation status of bovine milk
providing for the first time an overview of which proteins and sites
are affected during industrial processing.
Expectedly, the numbers of identified carbonylation sites
increased from raw milk to pasteurized milk and UHT milk and
were highest in infant formula confirming that harsher processing
favors oxidations. In all samples j-CN, a-LA, and b-LG were most
affected (5–6 modification sites).
Most proteins identified here were already reported as carrying
other non-enzymatic modifications. For example, a significant
number of lysine residues in caseins and whey proteins a-LA and
b-LG are modified by lactose, hexoses, and advanced glycation
end-products (AGEs) in the course of the Maillard reaction
(Milkovska-Stamenova & Hoffmann, 2016a, 2016b, 2017;
Renzone, Arena, & Scaloni, 2015). The Maillard reaction seems to
have a significant effect on protein oxidation by generating dicar-
bonyl compounds, such as GO, that can react with lysine, cysteine,
and arginine residues. However, the same dicarbonyls could also
result from lipid peroxidation products, which appears also likely
owing to the high lipid contents of milk. Thus, two third of all mod-
ifications sites were protein-bound carbonyls formed from dicar-
bonyls (mostly GO-derived) and the numbers increased
significantly in processed milk, especially in infant formula. This
was further supported by eight GO-modified peptides of a-LA, b-
LG, BSA, and lactotransferrin, which contents were considerably
higher in IFs than the other milk samples. This indicates that the
carbonylation sites identified here can be used for quantitative
studies to better understand the impact of milk processing on pro-
tein oxidation and carbonylation. Most carbonyl modifications
were identified on lysine residues (57%), which corresponds to ear-
lier reports in human plasma and rat cardiac mitochondria
(Bollineni et al., 2014; Chavez, Bisson, & Maier, 2010), while mod-
ifications at cysteine, arginine, and proline residues were less com-
mon. We could not detect carbonylated histidine and threonine
residues, which is in partial contrast to a report that His146 is
modified in b-LG (Fenaille et al., 2005). However, this modification
was identified by MALDI-TOF MS after DNPH derivatization, which
might indicate that ARP-derivatization or biotin-avidin affinity
chromatography is not the best approach to identify carbonylation
of histidine.
Interestingly, milk proteins appeared to be affected by different
carbonylation reactions. Although reactive carbonyls derived from
dicarbonyls (mainly GO) were most abundant in the studied sam-
ples and affected the majority of proteins (12/15), j-CN was the
main target of MCO which converted five of its 20 proline residues
into glutamic semialdehyde. The higher susceptibility to direct oxi-
dation attack compared to other caseins might be related to its
423
exposure to the surface of casein micelles. Additionally, one proline
and two lysine residues of aS1-CN appeared also to be oxidized by
metal ions. Both caseins have a strong tendency to bind metal ions
that might explain the observed oxidation reactions. Interestingly,
glutamic and aminoadipic semialdehydes as the main oxidation
products triggered by metal ions were also the dominating modifi-
cations in proteins isolated from HeLa cells, human plasma, and rat
liver (Bollineni, Hoffmann, & Fedorova, 2014; Bollineni et al., 2014;
Requena, Chao, Levine, & Stadtman, 2001).
The identified carbonylation sites and the presumed underlying
mechanisms producing them will allow testing strategies for
reducing carbonylation degrees in bovine milk, especially in infant
formula by altering processing conditions, such as fast cooling after
heat treatment and reduced oxygen or metal ion contents. How-
ever, this will also require quantitative approaches, ideally based
on isotope-labelled peptide standards, which have to be developed.
Generally, ARP-labeling and avidin affinity chromatography should
allow quantitative analyses, but it has to be optimized for milk
samples first. Such techniques would also allow evaluating the
impact of carbonyls on the quality of milk products and human
health.
5. Conclusions
In total, 53 unique carbonylated peptides (37 carbonylation
sites, 15 proteins) were identified in raw milk and differently pro-
cessed milk products (different brands of pasteurized, UHT milk,
and IFs). Protein-bound carbonyls in milk proteins were derived
from dicarbonyls (GO, MGO, and 3-DG), a,b-unsaturated aldehydes
(acrolein, pentenal, and OHE), and MCO (glutamic and aminoadipic
semialdehydes). Protein carbonyls derived from dicarbonyls
(mainly GO) were most common. The number of carbonylation
sites increased from raw milk to pasteurized milk and UHT milk
and was even higher in infant formula indicating that the extent
of carbonylation correlates to severity of industrial processing.
Conflict of interest
None.
Acknowledgements
Financial support from the Deutsche Forschungsgemeinschaft
(HO2222/7-1) and the European Fund for Regional Structure
Development (EFRE, European Union and Free State Saxony;
100055720, 100092961 and 100146238) is gratefully acknowl-
edged. We thank Dr. Ravi Chand Bollineni and Dr. Maria Fedorova
for their support and Dr. Alessandra Altomare for her help on opti-
mizing sample preparations.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2017.
02.102.
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