Article pubs.acs.org/jpr

Analysis of the Endogenous Peptide Profile of Milk: Identification of 248 Mainly Casein-Derived Peptides Florian Baum,† Maria Fedorova,‡ Jennifer Ebner,† Ralf Hoffmann,‡ and Monika Pischetsrieder*,† †

Department of Chemistry and Pharmacy, Food Chemistry, Emil Fischer Center, University of Erlangen-Nuremberg, Schuhstrasse 19, 91052 Erlangen, Germany ‡ Center for Biotechnology and Biomedicine, Universität Leipzig, Deutscher Platz 5, 04103 Leipzig, Germany ABSTRACT: Milk is an excellent source of bioactive peptides. However, the composition of the native milk peptidome has only been partially elucidated. The present study applied matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) directly or after prefractionation of the milk peptides by reverse-phase high-performance liquid chromatography (RP-HPLC) or OFFGEL fractionation for the comprehensive analysis of the peptide profile of raw milk. The peptide sequences were determined by MALDITOF/TOF or nano-ultra-performance liquid chromatography−nanoelectrospray ionization-LTQ-Orbitrap-MS. Direct MALDI-TOF-MS analysis led to the assignment of 57 peptides. Prefractionation by both complementary methods led to the assignment of another 191 peptides. Most peptides originate from αS1-casein, followed by β-casein, and αS2-casein. κCasein and whey proteins seem to play only a minor role as peptide precursors. The formation of many, but not all, peptides could be explained by the activity of the endogenous peptidases, plasmin or cathepsin D, B, and G. Database searches revealed the presence of 22 peptides with established physiological function, including those with angiotensin-converting-enzyme (ACE) inhibitory, immunomodulating, or antimicrobial activity. KEYWORDS: peptide profiling, bioactive peptides: milk, MALDI-TOF-MS, ESI-MS: caseins, OFFGEL fractionation, HPLC prefractionation, plasmin, cathepsin

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digested milk samples.7−10 However, less is known about the composition and biological activity of peptides originally present in raw milk. They may exert activity in the upper gastrointestinal tract independent from digestive processes. Additionally, endogenous physiologically active milk peptides may render whey, which is an abundant byproduct in cheese production, a valuable source of bioactive peptides for a possible use as nutraceuticals. Recent studies suggest that the peptide fraction of raw milk contains a great variety of structures.11 While peptides that are products of high proteolytic activity in mastitic milk12,13 or heat exposure14 have already been investigated, only a few amino acid sequences have been identified in fresh and unprocessed milk.11,15−17 Mass spectrometric proteomic techniques have been successfully applied to characterize the protein composition of milk or milk compartments.18−21 The aim of the present project was a comprehensive sequence mapping of the native cow’s milk peptidome, which comprises peptide structures smaller than 5000 Da, by mass spectrometry techniques. Recording of the peptide profile of cow’s milk was used to identify bioactive sequences. Additionally, the

INTRODUCTION Dietary proteins and peptides are not only valuable nutrients but also display various additional physiological functions. Bioactive peptides have been defined as “specific protein fragments that have a positive impact on body functions or conditions and may ultimately influence health”.1 Milk is an excellent source of bioactive peptides.2,3 Milk peptides may affect the cardiovascular, digestive, immune, and nervous system after oral administration.4 Exorphins, opioid peptides with morphine-like effects and similarity to endogenous ligands (endorphins and enkephalins), were identified as one of the first bioactive peptides derived from milk.5 Since then, a variety of bioactive milk peptides have been detected that exert antimicrobial, antioxidative, antithrombotic, antihypertensive, mineral binding, immunomodulatory, and other activities. Crucial for the bioactivity is the primary amino acid sequence of the peptide, which can vary from 2 to about 20 amino acid residues. Multifunctional peptides can modulate two or more physiological processes.6 An important source of bioactive milk peptides are the major milk proteins, which release the peptides by enzymatic digestion, microbial fermentation, gastrointestinal digestion, or a combination of these processes. Thus, most of the bioactive milk peptides were isolated from fermented or © 2013 American Chemical Society

Received: April 10, 2013 Published: November 18, 2013 5447

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

Journal of Proteome Research

Article

detector (JASCO Deutschland, Groß-Umstadt, Germany) at an excitation wavelength of 280 nm and an emission wavelength of 340 nm. Continuous fractionation started after 5.8 min and stopped at 59.6 min. On the basis of peak separation, 12 fractions were collected continuously in 4 mL vials. The fractions were frozen at −80 °C, lyophilized overnight, and redissolved in 10 μL of MALDI-TOF matrix (see below).

composition of the peptide fraction revealed information on the mechanisms of peptide formation in milk.

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EXPERIMENTAL SECTION

Milk Samples and Peptide Extraction

Raw cow’s milk was obtained from a local farm directly after morning milking and immediately cooled on ice. A pooled sample from 30 animals was used [Fleckvieh cattle, with an average age of 4.5 years; about one-third of the animals were primiparous and 10% were in the fifth lactation period, the average somatic cell count of the herd was 168 ± 71 × 103 cells/mL (mean ± SD, n = 10 analyses/year) indicating a good health status]. After centrifugation of the sample at 1100g for 60 min at 4 °C, the upper milk fat layer was removed, the liquid milk was aliquoted, and the defatted milk aliquots were frozen at −20 °C. In summary, the following measures were applied to minimize microbiological acitivity in the samples: immediate cooling on ice after milking; very rapid transport and processing  during this time the samples were always kept ≤4 °C; and storage at −20 °C. For matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) analysis peptides were extracted directly from the defatted milk with C18 ZipTip (Millipore, Billerica, USA) pipet tips. The column bed was wetted by aspirating and dispensing acetonitrile for five times with a piston pipet adjusted to 10 μL. In the same way, the column was preconditioned with 0.1% trifluoroacetic acid (TFA). The sample was loaded by pipetting 10 μL of sample 10 times. After the column was washed with 0.1% TFA five times as described, the peptides were eluted by aspirating and dispensing 5 μL of 60% acetonitrile, containing 0.1% TFA. Peptide fractionation by high-performance liquid chromatography (HPLC) and OFFGEL fractionator, as well as nano-ultraperformance liquid chromatography (UPLC)-nano electrospray ionization mass spectrometry (ESI-MS) and MALDI-TOF/ TOF required further sample preparation steps: Prior to fractionation, the samples were acidified with 1% (v/v) acetic acid solution. The pH of samples for LC−MS/MS analysis was adjusted by 2 M sodium acetate buffer (pH 4.0) to 4.6. Precipitated casein was removed from both kinds of samples by centrifugation (1100g, 15 min, 4 °C) and filtration through a 0.22 μm membrane filter (Carl Roth, Karlsruhe, Germany). Prior to OFFGEL fractionation, MALDI-TOF/TOF or nanoUPLC-nano-ESI-LTQ-Orbitrap-MS, 2 mL of the filtrate was applied to a C18 SPE column (200 mg C18-E, 3 mL, Phenomenex, Aschaffenburg, Germany) and freeze-dried after elution with 4 mL of 60% acetonitrile/0.1% TFA (v/v). The lyophilizate was dissolved in 2 mL of H2O. This pretreatment was omitted before fractionation by HPLC. The samples were then filtered in a stirred cell through an Ultracel ultrafiltration membrane (Millipore, Billerica, USA) with a nominal molecular weight limit of 10 kDa.

OFFGEL Peptide Fractionation

OFFGEL peptide fractionation was carried out by a 3100 OFFGEL fractionator (Agilent Technologies, Boeblingen, Germany) according to the manufacturer’s instructions using a commercially available kit (3100 OFFGEL starter kit, Agilent Technologies, Boeblingen, Germany). Focusing of 12 fractions was performed for 20 kVh with a current of 50 μA and a power of 200 mW, followed by ZipTip extraction of each peptide fraction as described above prior to MALDI-TOF-MS. MALDI-TOF Analysis

The eluates were mixed 1:1 with matrix (4-chloro-αcyanocinnamic acid, 5 mg/mL in 60% acetonitrile, containing 0.1% TFA (v/v)), spotted onto a ground steel target (Bruker Daltonics, Bremen, Germany), and air-dried before analysis in a Bruker Autoflex MALDI-TOF mass spectrometer in positive reflector mode (acceleration voltage 20 kV, 140 ns delay) with a mass range from 700 to 5000 m/z. For each spectrum, at least 300 single subspectra were averaged. External calibration was performed with Bruker peptide calibration standard II (Bruker Daltonics, Bremen, Germany). 4-Chloro-α-cyanocinnamic acid was used as matrix, because its superiority had been reported in terms of the number of identified peptides, obtained sequence coverage, and peptide detection compared to α-cyano-4hydroxycinnamic acid.22 MALDI-TOF/TOF

After ZipTip extraction, samples were mixed with an equal volume of matrix (α-cyano-4-hydroxycinnamic acid, 4 mg/mL in 60% acetonitrile containing 0.5% formic acid (v/v)). An aliquot of 1 μL of the mixture was spotted onto a polished steel target and air-dried. MALDI-TOF/TOF tandem mass spectrometry was carried out with a 4700 proteomic analyzer (Applied Biosystems, Darmstadt, Germany). For external calibration, 4700 analyzer mix (Applied Biosystems, Darmstadt, Germany) was used. Fragment ion spectra of selected precursor ions were acquired in postsource-decay mode without collision gas in the reaction cell (acceleration voltage 8 kV, 70% grid voltage, 1.277 ns delay, detector voltage 2.1 kV, collision energy 1 kV, and laser intensity 5500). Every single spectrum consisted of 60 subspectra with 100 laser shots each. Mass spectrum analysis was done by 4000 Series Data Explorer software (version 4.8, Applied Biosystems, Darmstadt, Germany). Since α-cyano-4-hydroxycinnamic acid leads to more intensive fragments in postsource-decay mode compared to 4-chloro-α-cyanocinnamic acid, the former matrix was used for MALDI-TOF/TOF analyses.23

Peptide Fractionation by HPLC

Analysis by nano-UPLC-nano-ESI-MS

Peptide fractionation was carried out on a JASCO HPLC (JASCO Deutschland, Groß-Umstadt, Germany) with a Zorbax Eclipse XDB C8 column (4.6 × 150 mm, 5 μm particle diameter, 80 Å pore size, Agilent, Boeblingen, Germany) using a gradient from 5−70% aqueous methanol, containing 0.1% formic acid (v/v), in 50 min after initial 5 min with 5% aqueous methanol/0.1% formic acid (flow rate 0.5 mL/min, injection volume 50 μL, room temperature). Peptides were detected by a MD-1510 multiwavelength detector and FP-920 fluorescence

Nano-UPLC was performed on a Waters nanoacquity UPLC (Waters, Eschborn, Germany) coupled to an LTQ-Orbitrap XL mass spectrometer equipped with a nanospray source (Thermo Fisher Scientific, Bremen, Germany). One microliter of the peptide extract diluted 1:50 with eluent A (0.1% formic acid in water) was injected into a Waters 5 μm symmetry C18 (180 μm × 20 mm) trap column with a flow rate of 5 μL/min eluent consisting of 99% eluent A (0.1% aqueous formic acid) and 1% 5448

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

Journal of Proteome Research

Article

and for MALDI-TOF/TOF. Search parameters for Sequest were as follows: peptide mass tolerance−15 ppm; peptide mass units−ppm; mass type parent−monoisotopic masses; fragment ion tolerance−250 ppm; mass type fragment−monoisotopic masses; ion series−b, y, b(neutral loss), y(neutral loss); enzyme number−none; diff search options−oxidation (+16.0, methionine), phosphorylation (+80.0, serine, threonine) for nano-ESIMS/MS. Peptide assignment of the 100 peptides with the lowest scores was verified by manual checking of product ion spectra. Additionally, peptide assignment could be confirmed by their detection with two different ionization methods (MALDITOF/TOF and nano-ESI-MS, see Table 1) and/or by the coverage of amino acid sequences by more than one peptide (see Figures 1−3). A literature search was performed for all peptides that had been detected either by direct measurement or after HPLC and OFFGEL fractionation. The search was conducted in the literature databases ISI Web of Knowledge, PubMed, Scopus, and Google Scholar with the peptide sequence in single letter code as search term. Direct MALDI-TOF analysis, HPLC- and OFFGELfractionation were performed in duplicates, and nano-Orbitrap-MS/MS and MALDI-TOF/TOF MS in quadruplicates using pooled milk samples from different days. Only peptides that were present in both investigated samples (MALDI-TOF analysis, HPLC- and OFFGEL-fractionation), or in three out of four samples, respectively (nano-Orbitrap-MS/MS and MALDI-TOF/TOF-MS), were considered.

eluent B (0.1% formic acid in acetonitrile) and separated on a 1.7 μm BEH 130 C18 column (100 μm × 100 mm, Waters, Eschborn, Germany) with a flow rate of 400 nL/min. After 1 min, the gradient increased from 1% eluent B to 50% eluent B in 30 min and to 85% eluent B in 2 min. The temperature of the transfer capillary was set to 200 °C and the tube lens voltage to 110 V. A source voltage of 1.5 kV was applied, using a nano-ESI emitter with silica coating and a diameter of 15 μm (New Objective, Berlin, Germany). Spectra in the mass range from 400 to 2000 m/z were acquired in positive mode with a resolution of 60 000 in the Orbitrap. Tandem mass spectra of the nine most abundant peaks with a charge state of two or higher were recorded in collision-induced dissociation (CID) mode (isolation width 2.0, collision energy 35%, collision gas helium, activation Q 0.25, activation time 30 ms) at the same time in the linear ion trap. Spectra were analyzed by Xcalibur software (version 2.0.7). Plasmin Activity

Raw milk was collected on three different days, skimmed as described above, and immediately used for further analysis. Sample preparation was performed according to the protocols of Korycka-Dahl et al.,24 later modified by Politis et al..25 Each milk sample was analyzed in duplicate. One milliliter of milk was first centrifuged for 1 h at 100000g at 4 °C to separate the milk serum from the casein pellet. After removal of the serum, the casein micelles were resuspended in 1 mL of 50 mM TrisHCl buffer (pH 8.0) containing 110 mM NaCl and 50 mM εaminocaproic acid (Acros Organics, Geel, Belgium). During incubation at room temperature for 2 h, plasmin dissociated from the casein micelles and was released into the buffer. After another hour of centrifugation at 100000g at 4 °C, the plasmincontaining supernatant was collected and directly used for measurement. Plasmin activity was determined using the substrate D-Ala-Leu-Lys-7-amido-4-methylcoumarin (SigmaAldrich, Taufkirchen, Germany) as described by Kato et al.26 with minor modifications. The substrate was dissolved in ethanol and diluted to a final concentration of 0.4 mM with Tris-HCl buffer (pH 7.4) containing 150 mM NaCl. Fifty microliters of the plasmin-containing supernatant was mixed with 950 μL of substrate buffer in a cuvette thermostatted at 37 °C. Emission was measured in intervals of 15 min in a JASCO FP-6200 spectrofluorometer (JASCO Deutschland, GroßUmstadt, Germany), and 7-amino-4-methylcoumarin (AMC)release was calculated by a standard curve of AMC (SigmaAldrich, Taufkirchen, Germany). The enzyme activity was calculated from the linear part of the resulting curves depicting liberated AMC versus time. One unit (U) was defined as the quantity of plasmin liberating 1 nM of AMC per minute under these specific conditions. Possible substrate degradation under the assay conditions was monitored using buffer as control and could be considered negligible.

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RESULTS

Peptide Analysis by Direct MALDI-TOF-MS

The aim of the study was the comprehensive analysis of the native bovine milk peptidome to gain information on the presence and formation mechanisms of bioactive peptides in milk. For this purpose, defatted milk was directly subjected to MALDI-TOF analysis after C18 ZipTip treatment. MALDITOF-MS gave access to 57 signals (Table 1). For the structural assignment of these signals, product ion spectra were generated by MALDI-TOF/TOF and an ESI-LTQ-Orbitrap mass spectrometer coupled online to nano-UPLC. The tandem mass spectra were used to retrieve the sequences from SwissProt-Database with the search engines Mascot and Sequest. The determination of the primary sequence showed that peptides are almost exclusively fragments of the main milk proteins αS1-casein (accession number P02662 in UniProtKB Protein-Database), αS2-casein (accession number P02663), and β-casein (accession number P02666). Most peptides originate from αS1-casein (30), followed by β-casein (17), and αS2-casein (10) (Table 1). The regions releasing peptides are not evenly distributed along the total sequence of the caseins. On the one side, there are specific “hot spots” in the amino acid sequence that are particularly prone to proteolysis and release a large number of peptides. On the other hand, there are insusceptible sequence motifs without measurable hydrolysis events. While peptides from αS1-casein mainly originate from the region between the N-terminus and K36 and from H80 to L98, the majority of peptides from β-casein and αS2-casein originate from the region between Y193 and the C-terminus (V209) or between T198 and the C-terminus (L207) (Figures 1−3). Heat maps of peptide distribution demonstrate the locations of highest proteolysis activity, which are located at the edges of

Database and Literature Search

Spectra from MALDI-TOF/TOF and nano-ESI-MS were searched against Swissprot Database (September 11, 2009; 495 880 sequences in the database, 64 755 sequences in taxonomy mammals) by Mascot (Version 2.4, Matrix Science, London, UK) or Sequest (Proteome Discoverer, Thermo Fisher Scientific). Search parameters for Mascot were as follows: taxonomy−mammals; enzyme−none; variable modifications−oxidation (methionine); phosphorylation−serine, threonine; mass tolerance−15 ppm (150 ppm) for precursor ions and 0.8 u (0.5 u) for product ions for nano-ESI-MS/MS 5449

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

found [M + H]+

806.41 863.43 869.38 871.40 884.55 905.50 905.45 905.47 915.41 934.53 943.42 962.50 970.47 991.55 991.54 1,012.58 1,018.46 1,035.53 1,044.40 1,047.56 1,052.48 1,052.56 1,052.44 1,055.41 1,080.56 1,090.57 1,092.48 1,099.45 1,099.57 1,104.70 1,109.58 1,117.69 1,120.66 1,122.36 1,143.42 1,168.69 1,175.62 1,179.60 1,181.60 1,200.52 1,217.80 1,219.65

no.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42

806.41 863.43 869.47 871.50 884.56 905.48 905.48 905.48 915.48 934.54 943.51 962.50 970.57 991.52 991.55 1,012.65 1,018.57 1,035.51 1,044.52 1,047.58 1,052.55 1,052.55 1,052.53 1,055.54 1,080.57 1,090.59 1,092.54 1,099.56 1,099.61 1,104.64 1,109.57 1,117.64 1,120.57 1,122.54 1,143.56 1,168.62 1,175.71 1,179.64 1,181.58 1,200.62 1,217.73 1,219.64

calculated [M + H]+ +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2

charge state of the precursor ion

Table 1. Profile of Native Milk Peptides

αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein

parent proteina 26−32 26−33 10−17 16−22 95−101 17−23 24−31 25−32 81−88 94−100 14−21 25−33 15−22 26−34 93−100 95−102 16−23 85−93 81−89 94−101 17−24 24−32 80−89 11−19 28−36 25−34 191−199 146−153 14−22 92−100 24−33 15−23 26−35 83−91 110−119 11−20 94−102 28−37 80−89 81−90 92−101 25−35

AAb H, D, H H, H, H, H, H, H H, H H, H, H, H, H, O H, H, H, H, H, H, H O H, H H, H, H, H, H, H, H, H H H, H, H, H, H H,

5450

O

O O O O

O O O O O O O

O

O O O O O O

O O O O O

O

O O O O O

O H, O

fractionationc MALDI TOF/TOF

score/ xcorrd 42/1.30 47/1.81 44/2.04 49/2.07 60/2.00 46/2.25 30 38 33 45/2.38 22/1.81 58/1.45 31/1.93 67/2.10 52/2.24 42/2.47 61 27/1.95 44/2.49 56/2.21 42/1.85 48/1.41 31/2.51 50/1.88 37/2.18 55/1.37 46/2.16 30/1.62 38/2.05 72/2.64 44/1.69 51/2.14 55/3.13 62/2.28 51/1.67 44/2.36 39/2.30 49/2.79 39/2.51 61/2.46 62 45/1.60

nano- ESI-MS/MS ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● APFPEVF APFPEVFG GLPQEVLN LNENLLR LEQLLRL NENLLRF FVAPFPEV VAPFPEVF IQKEDVPS YLEQLLR EVLNENLL VAPFPEVFG VLNENLLR APFPEVFGK GYLEQLLR LEQLLRLK LNENLLRF DVPSERYLG IQKEDVPSE YLEQLLRL NENLLRFF FVAPFPEVF HIQKEDVPS LPQEVLNEN FPEVFGKEK VAPFPEVFGK SEKTTMPLW YPELFRQF EVLNENLLR LGYLEQLLR FVAPFPEVFG VLNENLLRF APFPEVFGKE KEDVPSERY EIVPNSAEER LPQEVLNENL YLEQLLRLK FPEVFGKEKV HIQKEDVPSE IQKEDVPSER LGYLEQLLRL VAPFPEVFGKE

sequence

Journal of Proteome Research Article

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

found [M + H]+

1,227.66 1,237.66 1,246.75 1,248.61 1,250.67 1,256.63 1,256.58 1,281.74 1,292.62 1,303.76 1,336.61 1,337.68 1,338.76 1,347.73 1,377.41 1,384.52 1,384.73 1,390.35 1,420.48 1,437.41 1,437.84 1,446.91 1,453.62 1,476.84 1,494.80 1,494.58 1,500.74 1,504.87 1,533.55 1,566.58 1,580.83 1,584.95 1,590.65 1,593.58 1,603.59 1,613.70 1,641.87 1,660.93 1,670.79 1,679.98 1,689.95 1,708.71

no.

43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84

1,227.67 1,237.66 1,246.68 1,248.66 1,250.60 1,256.64 1,256.65 1,281.70 1,292.65 1,303.81 1,336.61 1,337.68 1,338.73 1,347.73 1,377.68 1,384.70 1,384.73 1,390.65 1,420.71 1,437.71 1,437.81 1,446.80 1,453.73 1,476.77 1,494.80 1,494.83 1,500.70 1,504.69 1,533.79 1,566.82 1,580.83 1,584.87 1,590.82 1,593.87 1,603.84 1,613.83 1,641.87 1,660.79 1,670.85 1,679.90 1,689.82 1,708.92

calculated [M + H]+

Table 1. continued

+2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +3

charge state of the precursor ion αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein

parent proteina 13−22 24−34 14−23 26−36 82−91 109−119 23−33 11−21 83−93 95−104 109−119 80−90 10−21 26−37 8−19 108−119 23−34 185−197 82−93 125−137 11−22 25−37 86−97 81−92 24−36 10−22 80−91 184−197 81−93 86−98 106−119 11−23 26−39 24−37 8−21 80−92 23−36 106−119 80−93 86−99 185−199 105−119

AAb H, D, H H, H, H, H, H H H H D, H D, O O D, O O O H, H, H, H, D, O D, H O O H, H, O O O H, D, H H, H, H, O

5451

O O O

O H, O

O O

H, O

O O O O H, O

H, O

H, O

H, O

O O O O

O H, O

●

fractionationc MALDI TOF/TOF

score/ xcorrd 49/2.53 61/1.84 38 51/3.01 43/1.21 52 67/2.10 71/1.99 55/2.66 23/1.96 40 49/3.89 70/2.11 54/3.22 42/2.01 51/1.92 58/2.22 76/2.76 23/2.01 28/1.50 85/3.78 66/2.82 35/2.33 32/2.43 65/3.00 75/3.64 52/2.62 43/2.23 34/2.15 51/2.26 78/3.62 85/3.58 61/2.62 74/3.31 60/1.96 70/3.28 58/2.61 33 65/4.06 55 88/3.35 30/2.39

nano- ESI-MS/MS ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

sequence QEVLNENLLR FVAPFPEVFGK EVLNENLLRF APFPEVFGKEK QKEDVPSERY FFVAPFPEVFG LEIVPNSAEER LPQEVLNENLL KEDVPSERYLG LEQLLRLKKY LEIVPNSAEER (1 × phosphoserine) HIQKEDVPSER GLPQEVLNENLL APFPEVFGKEKV HQGLPQEVLNEN QLEIVPNSAEER FFVAPFPEVFGK PIGSENSEKTTMP QKEDVPSERYLG EGIHAQQKEPMIG LPQEVLNENLLR VAPFPEVFGKEKV VPSERYLGYLEQ IQKEDVPSERYL FVAPFPEVFGKEK GLPQEVLNENLLR HIQKEDVPSERY NPIGSENSEKTTMP IQKEDVPSERYLG VPSERYLGYLEQL VPQLEIVPNSAEER LPQEVLNENLLRF APFPEVFGKEKVNE FVAPFPEVFGKEKV HQGLPQEVLNENLL HIQKEDVPSERYL FFVAPFPEVFGKEK VPQLEIVPNSAEER (1 x phosphoserine) HIQKEDVPSERYLG VPSERYLGYLEQLL PIGSENSEKTTMPLW KVPQLEIVPNSAEER

Journal of Proteome Research Article

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

found [M + H]+

1,714.79 1,730.77 1,731.64 1,759.73 1,764.00 1,790.57 1,828.08 1,829.81 1,833.75 1,836.73 1,845.78 1,852.80 1,864.13 1,871.96 1,907.87 1,916.85 1,917.80 1,932.84 1,983.80 1,991.11 1,999.81 2,013.99 2,030.00 2,079.13 2,129.02 2,204.10 2,216.06 2,232.07 2,234.21 2,235.00 2,317.17 2,340.08 2,347.29 2,460.38 2,586.36 2,616.48 2,618.24 2,763.56 2,827.55 2,910.62 2,955.64 3,246.79

no.

85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126

1,714.83 1,730.83 1,731.94 1,759.95 1,764.00 1,790.93 1,827.92 1,829.86 1,833.91 1,836.95 1,845.85 1,852.97 1,864.01 1,871.99 1,907.93 1,916.89 1,918.00 1,932.89 1,984.02 1,991.13 2,000.08 2,014.00 2,029.99 2,079.13 2,129.02 2,204.10 2,216.06 2,232.06 2,234.21 2,235.24 2,317.18 2,340.20 2,347.30 2,460.39 2,586.37 2,616.49 2,618.25 2,763.56 2,827.55 2,910.62 2,955.64 3,246.80

calculated [M + H]+

Table 1. continued

+2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +3 +2 +3 +2 +3 +2 +2 +2 +3 +2 +2 +2 +2 +3 +2 +2 +3 +3 +3 +3 +3 +2 +4 +3 +2 +3 +3 +3 +3 +3

charge state of the precursor ion αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein αS1-casein

parent proteina 182−197 182−197 11−24 8−22 1−15 26−41 182−198 181−197 80−94 24−39 181−197 4−19 174−188 104−119 125−141 180−197 106−122 180−197 23−39 1−17 103−119 182−199 182−199 4−21 181−199 80−97 180−199 180−199 1−19 4−22 80−98 104−123 1−20 1−21 80−100 1−22 176−199 1−23 80−102 1−24 80−103 80−105

AAb H, H H, O D O H H O O H O H H, H, H, O H, O D, O D, H, D, D, D, D, H, D, O D, O D, D, D, D D, D D, D D D

5452

O

O

O O O

O

H, O O O H, O O H, O O O

H

O

O O O

O

O

● ● ● ● ●

●

fractionationc MALDI TOF/TOF

score/ xcorrd 57/3.22 82/4.49 91 95/3.32 46 44/1.87 63/2.90 57/2.60 48/3.04 63 79/2.89 64/2.34 38/2.35 78/2.35 27/1.82 43/1.44 59/3.32 65/2.98 61 35 61/3.90 68/3.64 61/3.59 56/2.90 78/2.16 40/3.27 85/3.71 69 66 58/2.30 48 52 52 77/3.17 42 65 67 71 48/3.10 75 32 44

nano- ESI-MS/MS ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

sequence IPNPIGSENSEKTTMP IPNPIGSENSEKTTMP (Ox) LPQEVLNENLLRFF HQGLPQEVLNENLLR RPKHPIKHQGLPQEV APFPEVFGKEKVNELS IPNPIGSENSEKTTMPL DIPNPIGSENSEKTTMP HIQKEDVPSERYLGY FVAPFPEVFGKEKVNE DIPNPIGSENSEKTTMP (Ox) HPIKHQGLPQEVLNEN FALPQYLKTVYQHQK YKVPQLEIVPNSAEER EGIHAQQKEPMIGVNQE SDIPNPIGSENSEKTTMP VPQLEIVPNSAEERLHS SDIPNPIGSENSEKTTMP (Ox) FFVAPFPEVFGKEKVNE RPKHPIKHQGLPQEVLN KYKVPQLEIVPNSAEER IPNPIGSENSEKTTMPLW IPNPIGSENSEKTTMPLW (Ox) HPIKHQGLPQEVLNENLL DIPNPIGSENSEKTTMPLW HIQKEDVPSERYLGYLEQ SDIPNPIGSENSEKTTMPLW SDIPNPIGSENSEKTTMPLW (Ox) RPKHPIKHQGLPQEVLNEN HPIKHQGLPQEVLNENLLR HIQKEDVPSERYLGYLEQL YKVPQLEIVPNSAEERLHSM RPKHPIKHQGLPQEVLNENL RPKHPIKHQGLPQEVLNENLL HIQKEDVPSERYLGYLEQLLR RPKHPIKHQGLPQEVLNENLLR APSFSDIPNPIGSENSEKTTMPLW RPKHPIKHQGLPQEVLNENLLRF HIQKEDVPSERYLGYLEQLLRLK RPKHPIKHQGLPQEVLNENLLRFF HIQKEDVPSERYLGYLEQLLRLKK HIQKEDVPSERYLGYLEQLLRLKKYK

Journal of Proteome Research Article

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

found [M + H]+

3,982.19 4,239.34 4,338.40 819.41 923.53 961.52 970.52 975.53 979.53 986.45 1,018.56 1,022.60 1,080.50 1,098.54 1,107.61 1,118.50 1,131.42 1,138.66 1,150.64 1,178.53 1,199.58 1,211.79 1,215.56 1,251.75 1,270.67 1,279.60 1,291.64 1,318.83 1,323.70 1,386.62 1,402.63 1,434.78 1,466.69 1,474.64 1,505.79 1,514.61 1,514.73 1,517.71 1,520.75 1,594.46 1,594.73 1,610.51

no.

127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 167 168

3,982.20 4,239.34 4,338.41 819.50 923.54 961.57 970.52 975.60 979.56 986.52 1,018.52 1,022.61 1,080.61 1,098.59 1,107.62 1,118.56 1,131.60 1,138.66 1,150.70 1,178.58 1,199.66 1,211.68 1,215.66 1,251.78 1,270.68 1,279.63 1,291.66 1,318.59 1,323.73 1,386.65 1,402.64 1,434.88 1,466.61 1,474.79 1,505.80 1,514.74 1,514.74 1,517.81 1,520.81 1,594.71 1,594.71 1,610.71

calculated [M + H]+

Table 1. continued

+3 +4 +5 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +3 +2 +2 +2

charge state of the precursor ion αS1-casein αS1-casein αS1-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein

parent proteina 1−34 1−36 1−37 198−204 201−207 176−183 189−196 198−205 174−181 189−196 154−161 200−207 174−182 105−113 172−180 71−79 153−161 198−206 199−207 155−163 189−198 104−113 189−198 198−207 171−180 154−163 155−164 14−23 175−185 138−149 138−149 191−202 138−149 151−162 153−164 137−149 138−150 176−187 152−163 137−149 138−150 138−150

AAb D D D H, D, H, D, H H, H, H, D, O H H, H H, D, H H, H H H D, H, H, H, H H, H, H H H H, H, H, H, O H, O O O

5453

O

O O O O

O O

H, O O O O

O

O H

O

O O O H, O

O H, O O O

●

●

●

●

fractionationc MALDI TOF/TOF ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

nano- ESI-MS/MS 63 44 31 51/2.15 46/1.60 51/2.37 41/2.27 47/2.03 46/1.90 40 28/2.15 44/1.78 49/2.08 41/1.98 41/2.27 58/2.43 43/2.51 40/2.17 34/2.38 33/1.77 42/2.74 60/2.16 21/2.01 58/2.95 42/2.03 39/2.81 46/2.11 37 39/1.72 96/4.11 83/3.29 59/2.11 61 50/2.94 46/1.95 61/3.30 79/3.02 47/2.96 36/2.73 51 41 48

score/ xcorrd sequence

RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGK RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEK RPKHPIKHQGLPQEVLNENLLRFFVAPFPEVFGKEKV TKVIPYV IPYVRYL LPQYLKTV AMKPWIQP TKVIPYVR FALPQYLK AMKPWIQP (Ox) TEEEKNRL VIPYVRYL FALPQYLKT VLNPWDQVK QKFALPQYL ITVDDKHYQ LTEEEKNRL TKVIPYVRY KVIPYVRYL EEEKNRLNF AMKPWIQPKT IVLNPWDQVK AMKPWIQPKT (Ox) TKVIPYVRYL YQKFALPQYL TEEEKNRLNF EEEKNRLNFL IISQETYKQE (1 x phosphoserine) ALPQYLKTVYQ TVDMESTEVFTK TVDMESTEVFTK (Ox) KPWIQPKTKVIP TVDMESTEVFTK (1 x phosphoserine) TKLTEEEKNRLN LTEEEKNRLNFL KTVDMESTEVFTK TVDMESTEVFTKK LPQYLKTVYQHQ KLTEEEKNRLNF KTVDMESTEVFTK (1 × phosphoserine) TVDMESTEVFTKK (1 × phosphoserine) TVDMESTEVFTKK (oxidation (M), 1 × phosphoserine)

Journal of Proteome Research Article

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

found [M + H]+

1,621.79 1,633.84 1,722.57 1,734.94 1,735.82 1,749.83 1,862.98 1,863.04 2,300.08 2,331.34 2,347.33 2,766.17 2,812.17 3,215.79 3,855.17 826.38 877.26 879.32 898.42 941.25 994.46 994.52 997.47 1,038.56 1,052.54 1,094.67 1,102.53 1,136.58 1,151.69 1,157.51 1,204.55 1,220.53 1,224.65 1,233.46 1,264.71 1,284.64 1,312.74 1,323.67 1,328.74 1,343.65 1,359.61 1,361.61

no.

169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210

1,621.85 1,633.89 1,722.80 1,734.93 1,735.92 1,749.95 1,863.03 1,863.03 2,300.14 2,331.34 2,347.34 2,766.53 2,812.33 3,215.79 3,855.17 826.46 877.41 879.44 898.55 941.48 994.57 994.51 997.62 1,038.61 1,052.63 1,094.67 1,102.54 1,136.58 1,151.70 1,157.63 1,204.61 1,220.60 1,224.63 1,233.56 1,264.71 1,284.65 1,312.70 1,323.80 1,328.69 1,343.68 1,359.67 1,361.62

calculated [M + H]+

Table 1. continued

+2 +2 +2 +2 +3 +3 +3 +2 +2 +2 +3 +3 +3 +4 +4 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2

charge state of the precursor ion αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein αS2-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein

parent proteina 151−163 153−165 137−150 151−164 174−187 150−163 150−164 151−165 115−135 189−207 189−207 151−172 1−24 182−207 151−181 199−206 72−78 109−115 202−209 49−56 194−202 109−116 201−209 197−206 199−208 200−209 178−186 107−115 199−209 193−202 109−118 109−118 111−120 39−48 195−206 1−11 84−95 165−176 84−95 178−188 178−188 38−48

AAb H, O H, O O D, H, O O H, O D, H, H, O D, H O O O D D H, O O O O O H, O O H H, O O D H, O H, O D, H, H, O H,O H O H, O D, H, D, H, H, O H, O H H H H, O

5454

●

● ● O O

●

●

●

●

O

O

O

fractionationc MALDI TOF/TOF

score/ xcorrd 43/3.26 47/3.09 60 59/3.63 53/1.96 48/2.30 39/2.48 67/3.09 74 54/2.71 46/2.59 53 78/4.66 32 40 34 35/1.40 32/1.63 25 29/1.80 46 /1.93 35/1.50 33/1.56 40/1.29 37 44/1.45 32/1.95 30/1.26 39/2.23 49/1.87 31/1.32 36/2.05 44/1.32 45/2.50 72 66/1.50 37/1.83 68/1.88 37/2.69 42/2.67 62/1.72

nano- ESI-MS/MS ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

sequence TKLTEEEKNRLNF LTEEEKNRLNFLK KTVDMESTEVFTKK (1 × phosphoserine) TKLTEEEKNRLNFL FALPQYLKTVYQHQ KTKLTEEEKNRLNF KTKLTEEEKNRLNFL TKLTEEEKNRLNFLK NAVPITPTLNREQLSTSEENS AMKPWIQPKTKVIPYVRYL AMKPWIQPKTKVIPYVRYL (Ox) TKLTEEEKNRLNFLKKISQRYQ KNTMEHVSSSEESIISQETYKQEK TVYQHQKAMKPWIQPKTKVIPYVRYL TKLTEEEKNRLNFLKKISQRYQKFALPQYLK GPVRGPFP TVDDKHY MPFPKYP RGPFPIIV IHPFAQTQ QEPVLGPVR MPFPKYPV (Ox) VRGPFPIIV VLGPVRGPFP GPVRGPFPII PVRGPFPIIV VPYPQRDMP KEMPFPKYP GPVRGPFPIIV YQEPVLGPVR MPFPKYPVEP MPFPKYPVEP (Ox) FPKYPVEPFT QQTEDELQDK EPVLGPVRGPFP RELEELNVPGE VPPFLQPEVMGV LSQSKVLPVPQK VPPFLQPEVMGV (Ox) VPYPQRDMPIQ VPYPQRDMPIQ (Ox) QQQTEDELQDK

Journal of Proteome Research Article

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

5455

1,377.78 1,392.76 1,396.69 1,410.71 1,414.69 1,430.68 1,460.89 1,490.87 1,505.78 1,555.82 1,581.66 1,589.94 1,618.99 1,673.54 1,713.03 1,717.99 1,768.59 1,782.00 1,881.07 1,883.60 1,981.71 1,994.15 1,997.01 2,013.00 2,107.23 2,109.71 2,141.98 2,254.30 2,278.22 2,390.82 2,432.28 2,479.99 2,560.15 2,696.19 2,802.21 3,029.26 933.41

1,132.52

211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247

248

1,132.62

1,377.79 1,392.76 1,396.66 1,410.83 1,414.71 1,430.71 1,460.90 1,490.88 1,505.85 1,555.83 1,581.77 1,589.94 1,618.93 1,673.86 1,712.87 1,718.00 1,768.88 1,782.00 1,881.07 1,883.89 1,981.86 1,994.15 1,996.97 2,012.93 2,107.23 2,109.96 2,142.01 2,254.30 2,278.08 2,391.17 2,432.17 2,480.18 2,560.15 2,696.35 2,802.40 3,029.53 933.54

calculated [M + H]+

+2

+2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +2 +3 +2 +2 +2 +2 +2 +2 +2 +3 +3 +2 +3 +3 +3 +3 +3 +2

charge state of the precursor ion β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein β-casein βlactoglobulin κ-casein

parent proteina

159−169

195−207 194−206 114−125 164−176 178−189 178−189 196−209 195−208 194−207 193−206 109−121 195−209 194−208 178−191 1−15 194−209 111−125 193−208 193−209 109−124 32−47 192−209 109−125 108−124 191−209 32−48 108−125 190−209 106−124 106−125 1−22 29−48 29−48 139−161 1−25 1−27 1−8

AAb

H

O D, H H H, H D D, H, D, H D, H O H D, O D, D, O H D, H H, D, O H D H, O H H D, D O O O ● ●

●

●

H, O H, O

H, O O O

O ●

●

●

H, O

O

●

● ●

H, O

H, O O H, O

O

H, O

fractionationc MALDI TOF/TOF

62/1.70

●

● ● ●

50/1.34 44/1.74 55/2.32 48/2.66 45/2.83 38/2.34 63/2.01 56/1.92 60 55/3.34 29/2.24 48/2.00 47 35/2.68 82/3.05 75/2.16 53/2.73 77/3.59 60/4.00 64/3.81 28/3.36 72/2.50 80/3.36 56/3.79 90/3.44 105/4.44 70 76 53/2.74 67 124 98/5.28 55 32 72 94 35

score/ xcorrd

● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ● ●

nano- ESI-MS/MS

sequence

INTVQVTSTAV

EPVLGPVRGPFPI QEPVLGPVRGPFP YPVEPFTESQSL SLSQSKVLPVPQK VPYPQRDMPIQA VPYPQRDMPIQA (Ox) PVLGPVRGPFPIIV EPVLGPVRGPFPII QEPVLGPVRGPFPI YQEPVLGPVRGPFP MPFPKYPVEPFTE EPVLGPVRGPFPIIV QEPVLGPVRGPFPII VPYPQRDMPIQAFL RELEELNVPGEIVES QEPVLGPVRGPFPIIV FPKYPVEPFTESQSL YQEPVLGPVRGPFPII YQEPVLGPVRGPFPIIV MPFPKYPVEPFTESQS KFQSEEQQQTEDELQD LYQEPVLGPVRGPFPIIV MPFPKYPVEPFTESQSL EMPFPKYPVEPFTESQS LLYQEPVLGPVRGPFPIIV KFQSEEQQQTEDELQDK EMPFPKYPVEPFTESQSL (Ox) FLLYQEPVLGPVRGPFPIIV HKEMPFPKYPVEPFTESQS HKEMPFPKYPVEPFTESQSL RELEELNVPGEIVESLSSSEES KIEKFQSEEQQQTEDELQDK KIEKFQSEEQQQTEDELQDK (1 × phoshoserine) LLQSWMHQPHQPLPPTVMFPPQS RELEELNVPGEIVESLSSSEESITR RELEELNVPGEIVESLSSSEESITRIN LIVTQTMK

The amino acid sequence of the parent proteins is based on UniProtKB entries with the accession numbers below: αS1-casein: P02662, αS2-casein: P02663, β-casein: P02666; κ-casein: P02668; βlactoglobulin: P02754. bAA = amino acid. cD = direct measurement, H = HPLC fractionation, O = OFFGEL fractionation. dIon score (Mascot) and/or xcorr-value (Sequest) obtained for peptide sequence by database search. Only top ranking peptides were included. Ion scores >46 indicate identification (p < 0.05).

a

found [M + H]+

no.

Table 1. continued

Journal of Proteome Research Article

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

Journal of Proteome Research

Article

Figure 1. Sequence of αS1-casein in single letter code. Peptides detected by direct MALDI-TOF-MS analysis are annotated in horizontal black double arrows. Cleavage sites of endogenous milk proteases are marked with colored vertical arrows28,31−33,46 and possible phosphorylation sites by S.

Figure 2. Sequence of αS2-casein in single letter code. Peptides detected by direct MALDI-TOF-MS analysis are annotated in horizontal black double arrows. Cleavage sites of endogenous milk proteases are marked with colored vertical arrows29,30,32 and possible phosphorylation sites by S.

In order to check if the peptide release could indeed be caused by plasmin, the plasmin activity was determined. Thus, a plasmin activity of 26.6 ± 13.4 U/L milk (n = 3) was measured, which is in very good accordance to previous studies applying a similar assay system.34

the highlighted regions (Figure 4A−C). Thus, rapid changes in color scale indicate many cleavage sites or intensive use of single cleavage sites. In vitro digestion with proteases, which are considered to be endogenous in bovine milk such as plasmin and cathepsin D, B, and G, revealed several specific cleavage sites in αS1-, αS2-, and β-casein that were recognized by these enzymes (Figures 1−3).27−33 In the present study, the cleavage sites R22−F23, K34−E35, K79−H80, which can be explained by plasmin activity, and F23−F24, possibly cleaved by cathepsin B, D or G, were revealed as locations of high proteolysis activity in αS1-casein. In αS2-casein, K150−K151 is the predominant cleavage site. For this site, plasmin activity has also been reported. β-Casein shows the most cleavages at the positions L192−Y193, possibly mediated by cathepsin D, Q194−E195, and P206−I207.

MALDI-TOF-MS Peptide Profiling after Prefractionation by Reverse-Phase HPLC

Because of the complexity of the sample, peptides may be suppressed or discriminated during the ionization process of MALDI-TOF-MS leading to an underestimation of the peptide spectrum. Therefore, prefractionation of the peptides was performed by two complementary methods. For this purpose, reverse-phase high-performance liquid chromatography (RPHPLC) and OFFGEL fractionation were applied. 5456

dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462

Journal of Proteome Research

Article

Figure 3. Sequence of β-casein in single letter code. Peptides detected by direct MALDI-TOF-MS analysis are annotated in horizontal black double arrows. Cleavage sites of endogenous milk proteases are marked with colored vertical arrows27,32,33 and possible phosphorylation sites by S.

(Table 1). Ninety-eight peptides originated from αS1-casein, 43 peptides from β-casein and 39 peptides from αS2-casein. Additionally, one fragment of β-lactoglobulin (accession number P02754) was detected. The heat map of αS1-casein identifies the regions between H4 and V37 and the region between H80 and L101 as important sources of peptides. Other peptides originated from positions V106 to R119 and between S180 and the C-terminus (Figure 4F). Additionally to the sites detected after HPLC fractionation, intensive cleavage was attributed to K36−V37, R100−L101 and K102−K103, which had been reported as plasmin cleavage sites in the literature.31 V37−N38 was not described in the literature to be cleaved by any of these proteases, but led to intensive peptide release. G10−L11 and D181−I182 were also detected as cleavage sites, but of minor relevance. Peptides derived from αS2-casein appeared especially from the regions T138−L164, F174−T182, and I201−V204 (Figure 4G). Identical cleavage sites with high activity were detected similar to the findings after HPLC fractionation, with the exception of K188−A189 and K197−T198, which have been detected more rarely here. β-Casein released peptides mainly at the C-terminus from Y193−V209 and, in minor degree, from M109−S124 (Figure 4H). OFFGEL fractionation revealed cleavage sites that were similar to those detected after direct measurement or after HPLC fractionation and resulted in two regions where peptides were released in high amounts. The first section was from M109 to S124 and the second section from Y193 to the C-terminus. Considering all cleavage sites detected in MALDI-MS by direct measurement, HPLC fractionation, or OFFGEL fractionation, a clear preference for hydrolysis after lysine can be observed. For identification of bioactive peptides in the native milk peptidome, the sequences of the assigned peptides from all experiments were submitted in single letter code to four literature databases to determine if biological activity has been reported for any of the peptides (see Experimental Section). Overall 22 peptides with established physiological or biological function were detected (Figure 5).

MALDI-TOF measurement after HPLC prefractionation gave 366 signals; 174 peptides thereof could be assigned to a structure (Table 1). Again, αS1-casein contributed most peptides (89). Similar to direct measurement, HPLC prefractionation revealed more peptides from β-casein (43) than from αS2-casein (41) and additionally one fragment of κcasein (accession number P02668). The peptides from αS1casein mainly derive from three parts of the protein, most of them from L11 to K36, H80 to L101, and S180 to W199. Additionally to the cleavage sites R22−F23, K34−E35, K79−H80, and F23−F24, which had already been observed by direct measurement, 11 more intensively used cleavage sites could be detected. R119−L120 has been previously described as a cleavage site for plasmin. F24−V25 is known as cleavage site for cathepsin B and D, V25−A26 for cathepsin B, G33−K34 for cathepsins B and G, and F179−S180 for cathepsin G. The cleavage sites G10− L11, H80−I81, Y91−L92, G93−Y94, D181−I182, and P197−L198 have not been reported for one of these proteases but contribute in relevant amounts to the formation of peptides. Most of the peptides cleaved from αS2-casein are from the segments T138 to L164, L176 to L180, and A189 to L207. Besides the plasmin cleavage site K150−K151, as described above, K149−K150, K188−A189, and K197−T198 have also been reported for plasmin in αS2-casein. Plasmin cleavage at position K137−T138 is not described in the literature, but could be possible, since plasmin cuts specifically after the amino acids lysine and arginine. F163− L164 and L164−K165 also show intensive cleavage but have not been assigned to one of the milk proteases mentioned above. The sections E39 to D47, M109 to S124, V178 to P186, and Y193 to V209 are the origin of most peptides cleaved from β-casein (Figure 4D−F). In contrast to direct measurement, position Q194−E195 was not found to be one of the dominant cleavage sites, but the cleavage between E108−M109, which can be attributed to cleavage by cathepsin B and/or G, K48−I49, A177− V178, and Y193−Q194 was observed. MALDI-TOF-MS Peptide Profiling after OFFGEL Prefractionation

OFFGEL fractionation of the milk peptides was performed, which resulted in 466 signals in subsequent MALDI-TOF-MS analysis, of which 181 peptides could be assigned to structures 5457

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Figure 4. Heat maps of αS1-, αS2-, and β-casein by direct measurement and after fractionation with RP-HPLC and OFFGEL fractionation. Each amino acid was colored according to the number of peptides in which this amino acid has been found.

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DISCUSSION

Figure 6. MALDI-TOF-MS can be applied directly to defatted milk after peptide extraction with ZipTip. Thus, this method can provide a rapid and easy overview of the peptide profile of cow’s milk. However, the disadvantage of this approach is the high number of peptides and proteins competing in the ionization process. This may lead to the negative discrimination of peptides with lower ionization efficiency or of peptides that require higher laser energy for ionization. Both properties are influenced by the side chains of the amino acids in the peptide. For example, peptides containing arginine, histidine, and lysine residues in their sequence yield higher ionization efficiency.35

Peptide Profiling of Milk Using Three Complementary Workup Methods

In the present study, a comprehensive analysis of endogenous cow’s milk peptides was performed leading to the assignment of 248 peptides. For this purpose, three complementary analytical workup methods were applied including direct analysis by MALDI-TOF-MS and MALDI-TOF-MS analysis after RPHPLC or after OFFGEL fractionation. The number of peptides found by each method is illustrated in the Venn diagram in 5458

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Figure 5. Endogenous milk peptides with established bioactivities.

were removed prior to analysis by casein precipitation at pH 4.6 and subsequent ultrafiltration of whey proteins. To compensate for the dilution effect caused by HPLC fractionation, samples must be lyophilized and redissolved before MALDI-TOF-MS. Protein precipitation may lead to the loss or depletion of certain peptides, which could explain the fact that 74 of the peptides detected by direct analysis or after OFFGEL fractionation were not revealed after HPLC analysis. Instead, lower abundant peptides may appear which are not detected by the other methods. OFFGEL fractionation, in contrast to HPLC, follows the principle of pI-based separation in an immobilized pH gradient. Isoelectric focusing is performed in precast polyacrylamide gel strips, while sample loading and recovery are carried out in a subdivided reservoir attached to the strip. Peptides were distributed over all 12 fractions of the gel strip indicating that OFFGEL fractionation is a suitable method for the separation of milk peptides. To achieve satisfying peptide separation, proteins and salts must be removed beforehand by casein precipitation, ultrafiltration of whey proteins as well as solid phase extraction. Since buffer salts, which are used for OFFGEL fractionation, interfere with MALDI-TOF measurement, subsequent cleanup of the peptide fractions with ZipTips is required. OFFGEL fractionation works automatically and can be performed overnight. The time need compared to direct measurement is considerably higher when a prefractionation technique is used but is comparable between both methods. Although peptide fractionation by RP-HPLC takes only 50 min compared to the approximately 24 h of OFFGEL fractionation, the necessary lyophilization after RP-HPLC separation is done

Figure 6. The Venn diagram shows the number of peptides detected by direct MALDI-TOF-MS measurement or after fractionation by RPHPLC or OFFGEL fractionation. Intersections of two or three circles give the number of peptides which were found by both or all three methods.

Prefractionation of the peptides reduces highly competitive processes during MALDI-TOF-MS, thus minimizing discriminating effects in the mixtures resulting from differences in the primary structure.36,37 RP-HPLC is a well-established technology that leads to peptide separation according to the peptide polarity. Since milk proteins, which are abundant in the samples, could overlay peptide signals in the RP-HPLC chromatograms, milk proteins 5459

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αS1-Casein contains nine serine residues that can be phosphorylated. Eight of them are located in the section between amino acids L40 and H80. αS2-Casein comprises 12 possible phosphoserines, 9 of them at the N-terminus. β-Casein contains five possible phosphorylation sites, which are located in the area from R1 to Q40. Phosphoserines are often found in clusters.38 The electronegativity of the phosphoryl groups results in low ionization efficiency, which makes the detection of multiphosphorylated peptides especially challenging.40 Thus, the phosphopeptides can be suppressed by nonphosphorylated peptides in complex peptide mixtures.41 In the present study, seven monophosphorylated peptides could be identified but none of the multiphosphorylated ones. Therefore, specific enrichment techniques for phosphopeptides are required to fully elucidate the endogenous phosphopeptidome of milk.42 Heat map analysis of the detected peptides clearly demonstrates that protein cleavage is not evenly distributed throughout the protein sequences. Instead, several hot spots of cleavage sites were identified. Therefore, it can be concluded that peptides are not released by spontaneous chemical hydrolysis processes but rather by enzymatic hydrolysis. However, it cannot be excluded that other peptides/cleavage sites exist, which were not detected due to poor ionization properties of these peptides. Plasmin is the major milk protease and responsible for the formation of the γ- and λ-caseins and heat-stable, acid-soluble proteolytic fragments of αS1-, αS2-, and β-casein, which are assigned to the proteose peptone fraction. This mixture of glycoproteins, phosphoproteins, and peptides contains probably at least 38 components. Major proteose peptone components such as component 5, component 8 slow and component 8 fast have later been identified as β-casein fragments (f 1−105), (f 29−105), and (f 1−28),43 which result from plasmin cleavage, while κ-casein is resistant to plasmin proteolysis. In milk, plasmin is associated to the casein micelles. It cleaves Lys−X and Arg−X bonds but prefers Lys− X.44 In the present experiments, we found also a clear preference for Lys−X cleavage sites, which is consistent with the measured plasmin activity. The association of plasmin within the casein micelles and the relatively flexible tertiary structure of the caseins may be the reason for almost exclusive hydrolysis of αS1-, αS2-, and β-casein as precursors for milk peptides, while κ-casein is resistant to hydrolysis,44 and the whey proteins α-lactalbumin and β-lactoglobulin are not or only a little affected.45 Additionally, cathepsin B, D, and G have been described as endoproteases endogenous to milk, having a rather broad specificity toward the caseins.32,33,46 Besides abrupt changes in the color scale of the heat maps, which indicate a highly specific cleavage by endoproteases, also soft color transitions were observed. These effects could be explained by initial formation of polypeptides like γ-caseins and proteose peptones through the activity of selective endogenous milk proteases like plasmin or cathepsin B, D, and G, and subsequent further degradation by endo- and exopeptidases. The occurrence of homologous peptide series missing one amino acid at the N-terminus, for example 190−, 191−, 192−, 193−, 194−, 195−, and 196−209 of β-casein, or at the C-terminus, for example, peptides 1−19, −20, −21, −22, −23, −24 of αS1-casein, indicates the activity of aminopeptidases and carboxypeptidases. Most likely, similar processes occur as in human milk, where formation of small peptides through the hydrolysis of intermediate polypeptides has been described.47 Although the presence of endogenous proteases in

overnight. In addition, the fact that the OFFGEL fractionator can fractionate 16 samples in parallel compensates for the shorter fractionation time by RP-HPLC. A disadvantage of OFFGEL fractionation is the relatively high price of IPG-strips. The yield of assigned peptides was very similar with both prefractionation methods, leading to 174 (RP-HPLC) and 181 (OFFGEL fractionation) peptides, respectively. Interestingly, also the yield of unique peptides, which were exclusively assigned by one method, was almost the same (50/RPHPLC; 49/OFFGEL). Using the fractionation techniques, more than four times as many peptides could be detected than by direct analysis. Although HPLC and OFFGEL fractionation led to a large overlap of peptides detected by both methods, the different prefractionation steps also revealed peptides which could be detected exclusively with one method. Thus, the application of both methods proved to be complementary and increased the peptide coverage considerably. Direct MALDI-TOF-MS analysis also revealed 15 peptides that were not detectable after prefractionation, having probably been lost during the fractionation processes. Although the application of prefractionation techniques considerably increases the number of assigned peptides, it cannot be excluded that further higher abundant peptides are not covered by the method. In particular, ionization efficiency of peptides with low pI, such as monophosphorylated peptides, may be too low for efficient detection. Additionally, negative discrimination may occur during peptide assignment. Peptide structures were identified by their mass and fragmentation spectra using two different search engines. Therefore, only peptides could be identified which were encrypted in bovine proteins and posttranscriptionally released by endoproteases. Peptides formed by other pathways, such as small peptide synthesis, were not covered by this method and would rather require de novo peptide sequencing for identification. Peptide assignment of signals in the MALDI-TOF mass spectra was achieved by product ion spectra using MALDITOF/TOF or nano-UPLC-nano-ESI-MS. Since the mass accuracy of MALDI-TOF is lower compared to ESI-LTQOrbitrap-MS, mismatches could occur at this step. However, since all detected peptides originated from only five different milk proteins, chances of wrong peptide assignment were relatively low. Formation Mechanism of Endogenous Milk Peptides

Sequence analysis of the detected peptides by product ion spectra and high-resolution spectra revealed that the vast majority of the peptides originate from caseins. This observation may be explained by the higher prevalence of casein in the milk proteome. In average, 34% of skim milk protein is αS1-casein, followed by β-casein with 25% and αS2casein with 8%,38 which have been identified as the major peptide sources in this study. Additionally, caseins may also be more prone to enzymatic degradation. Partially due to their high proline content, caseins have a more open and flexible structure than typical globular whey proteins. Probably as a result of this flexibility, they show a higher proteolysis rate than globular proteins when incubated with proteases.27 Comparison with results of three-dimensional molecular modeling revealed that residues which are hydrolyzed more rapidly are located in the flexible regions like turns or between hydrophobic and polar sections.39 5460

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(7) Lopez Exposito, I.; Quiros, A.; Amigo, L.; Recio, I. Casein hydrolysates as a source of antimicrobial, antioxidant and antihypertensive peptides. Dairy Sci. Technol. 2007, 87, 241−249. (8) Clare, D. A.; Swaisgood, H. E. Bioactive milk peptides: a prospectus. J. Dairy Sci. 2000, 83, 1187−1195. (9) Phelan, M.; Aherne, A.; FitzGerald, R. J.; O’Brien, N. M. Caseinderived bioactive peptides: Biological effects, industrial uses, safety aspects and regulatory status. Int. Dairy J. 2009, 19, 643−654. (10) FitzGerald, R. J.; Murray, B. A.; Walsh, D. J. Hypotensive peptides from milk proteins. J. Nutr. 2004, 134, 980S−988S. (11) Meltretter, J.; Schmidt, A.; Humeny, A.; Becker, C. M.; Pischetsrieder, M. Analysis of the peptide profile of milk and its changes during thermal treatment and storage. J. Agric. Food Chem. 2008, 56, 2899−2906. (12) Larsen, L. B.; Hinz, K.; Jorgensen, A. L.; Moller, H. S.; Wellnitz, O.; Bruckmaier, R. M.; Kelly, A. L. Proteomic and peptidomic study of proteolysis in quarter milk after infusion with lipoteichoic acid from Staphylococcus aureus. J. Dairy Sci. 2010, 93, 5613−5626. (13) Napoli, A.; Aiello, D.; Di Donna, L.; Prendushi, H.; Sindona, G. Exploitation of endogenous protease activity in raw mastitic milk by MALDI-TOF/TOF. Anal. Chem. 2007, 79, 5941−5948. (14) Gaucheron, F.; Molle, D.; Briard, V.; Leonil, J. Identification of low molar mass peptides released during sterilization of milk. Int. Dairy J. 1999, 9, 515−521. (15) Wedholm, A.; Moller, H. S.; Lindmakr-Mansson, H.; Rasmussen, M. D.; Adren, A.; Larsen, L. B. Identification of peptides in milk as a result of proteolysis at different levels of somatic cell counts using LC MALDI MS/MS detection. J. Dairy Res. 2008, 75, 76−83. (16) Farrell, H. M.; Jimenez-Flores, R.; Bleck, G. T.; Brown, E. M.; Butler, J. E.; Creamer, L. K.; Hicks, C. L.; Hollar, C. M.; Ng-KwaiHang, K. F.; Swaisgood, H. E. Nomenclature of the proteins of cows’ milk - sixth revision. J. Dairy Sci. 2004, 87, 1641−1674. (17) Pinto, G.; Caira, S.; Cuollo, M.; Fierro, O.; Nicolai, M. A.; Chianese, L.; Addeo, F. Lactosylated casein phosphopeptides as specific indicators of heated milks. Anal. Bioanal. Chem. 2012, 402, 1961−1972. (18) Le, A.; Barton, L. D.; Sanders, J. T.; Zhang, Q. Exploration of bovine milk proteome in colostral and mature whey using an ionexchange approach. J. Proteome Res. 2011, 10, 692−704. (19) Kussmann, M.; Panchaud, A.; Affolter, M. Proteomics in nutrition: status quo and outlook for biomarkers and bioactives. J. Proteome Res. 2010, 9, 4876−4887. (20) Pischetsrieder, M.; Baeuerlein, R. Proteome research in food science. Chem. Soc. Rev. 2009, 38, 2600−2608. (21) Bislev, S. L.; Deutsch, E. W.; Sun, Z.; Farrah, T.; Aebersold, R.; Moritz, R. L.; Bendixen, E.; Codrea, M. C. A Bovine Peptide Atlas of milk and mammary gland proteomes. Proteomics 2012, 12, 2895− 2899. (22) Jaskolla, T. W.; Papasotiriou, D. G.; Karas, M. Comparison between the matrices alpha-cyano-4-hydroxycinnamic acid and 4chloro-alpha-cyanocinnamic acid for trypsin, chymotrypsin, and pepsin digestions by MALDI-TOF mass spectrometry. J. Proteome Res. 2009, 8, 3588−3597. (23) Leszyk, J. D. Evaluation of the new MALDI matrix 4-chloroalpha-cyanocinnamic acid. J. Biomol. Tech. 2010, 21, 81−91. (24) Korycka-Dahl, M.; Ribadeau Dumas, B.; Chene, N.; Martal, J. Plasmin activity in milk. J. Dairy Sci. 1983, 66, 704−711. (25) Politis, I.; Zavizion, B.; Barbano, D. M.; Gorewit, R. C. Enzymatic assay for the combined determination of plasmin plus plasminogen in milk: revisited. J. Dairy Sci. 1993, 76, 1260−1267. (26) Kato, H.; Adachi, N.; Ohno, Y.; Iwanaga, S.; Takada, K.; Sakakibara, S. New fluorogenic peptide substrates for plasmin. J. Biochem. 1980, 88, 183−190. (27) Swaisgood, H. E. Chemistry of milk proteins. In Developments in Dairy Chemistry; Fox, P. F., Ed.; Applied Science Publishers: London, 1982; pp 1−59. (28) Le Bars, D.; Gripon, J.-C. Hydrolysis of αS1-casein by bovine plasmin. Lait 1993, 73, 337−344.

milk is well established, it cannot be excluded that enzymes of microorganisms, which can infect milk during the milking process, also participate in peptide generation. In order to minimize microbial processes, milk was collected directly after milking, immediately cooled on ice, and rapidly processed. Bioactive Peptides in Raw Milk

The milk peptide database generated in the present study was applied to identify bioactive peptide sequences already present in milk. Out of the 22 peptides, for which bioactivity has been reported before, eight peptides have an inhibitory effect on the angiotensin-converting enzyme or have an antihypertensive effect.48−53 Eight peptides cause a bitter taste.54−61 An immunomodulating effect has been reported for three peptides,62,63 antimicrobial properties for three peptides,63−65 and an antioxidative effect for two peptides.7,66 Furthermore, a peptide which inhibits prolyl endopeptidases linked to amnesia67 and one peptide interacting with calmodulin were found.68 Casecidin 17 (β-casein193−209) shows plurifunctional properties including immunomodulation, bitter taste, ACEinhibition, and antimicrobial activity.51,58−62,65 Isracidin (αS1casein1−23) displays antimicrobial and immunomodulating properties,63 and the fragment FALPQYLK (αS2-casein174−181) shows antioxidant and ACE-inhibitory effects.7,64 αS1-Casein23−34 is described as bitter and ACE-inhibitory.49,54,66,69 However, it can be assumed that the physiological activity of the majority of peptides has not been tested so far, so that the description of the endogenous milk peptidome may help to identify novel bioactive peptides, for example, by the generation of peptide libraries, which can be subjected to screening assays.

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AUTHOR INFORMATION

Corresponding Author

*Telephone: +49-9131-8524102, fax: +49-9131-8522587, email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Funding support by the Bavarian Research and Innovation Network “BayFood” − Cluster Nutrition to M.P. and from the European Regional Development Fund (ERFD, European Union and Free State Saxony) and the Federal Ministry of Education and Research (BMBF) to R.H. is gratefully acknowledged.

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REFERENCES

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dx.doi.org/10.1021/pr4003273 | J. Proteome Res. 2013, 12, 5447−5462