Identification of plant proteins in adulterated skimmed milk powder by high-performance liquid chromatography—mass spectrometry

Journal of Chromatography A, 1164 (2007) 189–197

Identification of plant proteins in adulterated skimmed milk powder by high-performance liquid chromatography—mass spectrometry Dion M.A.M. Luykx a,∗ , Jan H.G. Cordewener b , Pasquale Ferranti c , Rob Frankhuizen a , Maria G.E.G. Bremer a , Hendricus Hooijerink a , Antoine H.P. America b a b

RIKILT – Institute of Food Safety, P.O. Box 230, 6700 AE Wageningen, The Netherlands PRI – Plant Research International, P.O. Box 16, 6700 AA Wageningen, The Netherlands c University of Napels, Dipartimento di Scienza degli Alimenti, I-80055 Portici, Italy Received 3 July 2007; received in revised form 6 July 2007; accepted 9 July 2007 Available online 13 July 2007

Abstract The EU subsidises the use of skimmed-milk powder (SMP) in compound feeding stuffs. There are indications of falsified SMP content due to the addition of plant proteins. These proteins are not allowed in SMP and cannot be identified by the official reference method. Since soy and pea proteins are most likely to be added to SMP, manufactured SMP containing 1 and 5% of these plant proteins was used to develop a sensitive protein identification method based on mass spectrometry (MS). The method included a pre-fractionation step to enrich for plant proteins by using a borate buffer. A very fast perfusion liquid chromatography method including sensitive and selective intrinsic fluorescence detection was developed for monitoring and quantifying the efficiency of the pre-fractionation and screening for plant proteins. After tryptic digestion of the enriched fraction from manufactured adulterated SMP, numerous peptides originating from the major seed proteins of soy (glycinin, ␤-conglycin) and pea (legumin, vicilin) could be identified by MS/MS analysis on a quadrupole time-of-flight MS instrument. © 2007 Elsevier B.V. All rights reserved. Keywords: Protein identification; Milk powder; Plant proteins; Adulteration; Mass spectrometry; HPLC; Enrichment; Borate buffer

1. Introduction The EU subsidises the use of skimmed-milk powder (SMP) in compound feeding stuffs (EC regulation 2799/1999). In this way, European dairy surpluses can be reduced. A precondition for receiving any subsidy is that the processed milk powder meets certain specifications. The Institute of Food Safety (RIKILT) in The Netherlands checks these specifications on assignment from the General Inspection Service of the Dutch Ministry of Agriculture, Nature and Food Quality. There are indications now of falsified SMP content due to the addition of plant proteins. The low prices of these proteins make them attractive as potential adulterants in SMP for feed. Soybean is a very important and cheap source for plant proteins that are added as protein extender in the manufacture of various food and feed products all over the world [1–4]. Another attractive source for non-milk

∗

Corresponding author. Tel.: +31 317 484437; fax: +31 317 417717. E-mail address: [email protected] (D.M.A.M. Luykx).

0021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.chroma.2007.07.017

proteins is pea, especially in Europe [4,5]. Levels of adulteration below 1% of these two types of plant proteins in SMP are not of commercial interest [6]. Plant proteins in SMP for feed are not allowed. So far several methods and techniques, mostly based on electrophoretic, chromatographic and immunological principles, have been developed to screen for plant proteins in milk products [7–12]. However, each of them has difficulties and limitations and is not applicable for identifying the fraudulently added proteins. This holds also for the official reference method. This method is based on capillary zone electrophoresis (CZE) and is used for the detection of soy proteins [13]. Although aberrant proteins can be detected via the CZE profiles, the method gives no clear evidence about the origin of the added proteins. Therefore, an analytical method is needed not only for screening adulterations in SMP, but also to identify the origin of the aberrant proteins. Nowadays, mass spectrometry (MS) is often applied for the identification of proteins in food and pharmaceutical products [14–18]. In general, the MS procedure includes the preparation of tryptic digests from the protein samples first. Next,

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the peptides are usually separated via liquid chromatography (LC) before electrospray ionisation (ESI) and entering the MS instrument. For protein identification the ionised peptides have to be fragmented (MS/MS) revealing the amino acid sequence. Preliminary LC–ESI–MS/MS measurements at our laboratory with a quadrupole time-of-flight (Q-TOF) MS instrument on manufactured SMP containing 5% of soy protein isolate or 5% of pea protein isolate showed that hardly any soy or pea peptides, respectively, could be identified. Nearly, all identified peptides were originating from the abundant milk proteins (e.g., caseins and whey proteins). This indicated that an enrichment step of the plant proteins was needed before analysing manufactured SMP containing low amounts of plant protein. Earlier experimental data showed that a borate buffer treatment can be used for that purpose when it concerns soy and pea proteins [7,19]. Therefore, our main goal was to investigate the suitability of the LC–MS/MS method in combination with the borate enrichment step for the identification of soy or pea proteins in manufactured SMP containing between 1 and 5% of these plant proteins. At first, a fast, sensitive and straightforward method was needed to monitor and quantify the efficiency of the enrichment step with borate buffer. Perfusion reversed-phase HPLC constitutes a great advance in the separation and detection of proteins and peptides, permitting their separation in very short analysis times (few minutes) without impairing resolution [20,21]. This technique has already been described for the detection of soybean proteins in cheese and milk [22,23]. From earlier studies it appeared also that HPLC with intrinsic fluorescence detection compared favourably to UV detection with respect to sensitivity and selectivity [24,25]. Therefore, an adjusted perfusion reversed-phase HPLC method in combination with a fluorescence detector was applied in our study to monitor and quantify the enrichment procedure. Finally, the protein fractions enriched with plant proteins were digested with trypsin, and the resulting peptide mixtures were investigated by nano-LC–MS/MS. 2. Experimental 2.1. Samples and chemicals Skimmed-milk powder samples were purchased from Friesland Foods (Leeuwarden, The Netherlands). Commercially available soy protein isolate (SPI) (Supro 500 E from Protein Technologies, Ieper, Belgium) and pea protein isolate (PPI) (Pisane HD from Cosucra, Fontenoy, Belgium) were used for preparing adulterated SMP. Adulterated SMP was manufactured at NIZO Food Research (Ede, The Netherlands); each of the plant protein isolates at levels of 1% (SMP-S1 and SMP-P1 containing 1% SPI and 1% PPI, respectively) or 5% (SMPS5 and SMP-P5 containing 5% SPI and 5% PPI, respectively) were added to skimmed milk after which the mixtures were pasteurised and powdered [6]. Acetonitrile (ACN) (HPLC Supra-gradient, Biosolve, Valkenswaard, The Netherlands), trifluoroacetic acid (TFA) (Pierce, Rockford, IL, USA), formic acid (FA) (Merck, Darm-

stadt, Germany) and HPLC-grade water (Milli-Q system; Millipore, Bedford, MA, USA) were used for the preparation of the mobile phases. Urea (>99.5%; Fluka) and dithiothreitol (DTT) (>99%; Sigma) were used as denaturing and reducing agent, respectively. Disodium tetraborate decahydrate (Merck) and ethylenedinitrilotetraacetic acid disodium salt dehydrate (EDTA) (Merck) were employed for the preparation of the borate buffer. Ammonium hydrogencarbonate (Fluka Biochemica) and iodoacetamide (Amersham Biosciences) were applied before MS analysis. 2.2. Sample preparation 2.2.1. HPLC SMP, SPI and PPI (3–5 mg) were dissolved in 500 ␮l 6 M urea. After adding 500 ␮l 20 mM DTT and mixing, these samples were directly applied to the HPLC column for their individual HPLC profiles. For the borate treatment 5–10 mg SMP, SPI, PPI and manufactured SMP containing 1 or 5% of SPI or PPI were separately mixed with 1 ml of borate buffer, pH 8.3 (30 mM disodium tetraborate decahydrate containing 40 mM EDTA). After vortex-mixing (1 min), incubation for 15 min and vortex-mixing again (1 min), the samples were centrifuged at 10,000 × g for 30 min. The supernatant was carefully removed with a pipette and the pellet was washed with 1 ml of borate buffer. The final pellet was dissolved in 250–500 ␮l 6 M urea before 250–500 ␮l 20 mM DTT was added and mixed. The obtained supernatants and dissolved pellets were directly applied to the reversed-phase perfusion column. 2.2.2. Mass spectrometry For MS analysis, samples of SMP and manufactured SMP containing SPI or PPI (SMP-S1, SMP-S5, SMP-P1 and SMPP5) were pre-treated with borate buffer as described in Section 2.2.1, except that 10 ml of borate buffer was added to 50.0 mg of sample. The final borate pellet was suspended in 50 ␮l of 50 mM ammonium hydrogen carbonate (pH 7.8) with addition of 0.1% RapiGest SF (Waters, Milford, MA, USA). Protein was reduced in the presence of 5 mM DTT at 60 ◦ C for 30 min. Half of the sample (25 ␮l) was used for gel electrophoretic analysis. The remaining 25 ␮l of sample was alkylated in the presence of 15 mM iodoacetamide at room temperature for 30 min in the dark. Proteolytic digestion was initiated by adding 5 ␮l of modified porcine trypsin (0.5 ␮g/␮l, Promega). The sample was incubated for 4 h at 37 ◦ C, after which the tryptic digestion was terminated by freezing immediately at −20 ◦ C. After thawing, TFA was added to a final concentration of 0.5% and the peptide mixture was incubated at 37 ◦ C for 30 min for detergent degradation. After centrifugation at 15,000 × g for 10 min, the supernatant was applied to a Supelclean LC-18 1 ml SPE column (Supelco, Bellefonte, PA, USA) equilibrated with 0.1% TFA for sample clean-up. After washing with 0.1% TFA, the peptides were eluted with 84% ACN containing 0.1% FA and then dried down in a vacuum centrifuge. The samples were dissolved in 50 ␮l 0.1% FA prior to LC–MS analysis.

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2.3. HPLC measurements 2.3.1. Instrumentation HPLC experiments were performed on an Agilent 1100 system including a G1379A micro vacuum degasser, G1312A binary pump, G1329A auto-sampler, G1330B autosampler thermostat, G1316A thermostatted column compartment, G1315B diode array detector and G1321A fluorescence scanning detector. This Agilent HPLC system was operated by ChemStation software. UV detection was performed at 214 nm, fluorescence emission detection at 340 nm with an excitation wavelength of 280 nm.

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B (1 pmol/␮l) (Sigma) was delivered from a syringe pump (Harvard Apparatus, USA) to the reference sprayer of the NanoLockSpray source at a flow rate of 1 ␮l/min. The lock mass channel was sampled every 10 s. LC–MS was performed with the Q-TOF-2 operating in either (continuum) MS mode or in MS/MS mode for data dependent acquisition (DDA) of MS/MS peptide fragmentation spectra. Processing and searching of the MS/MS data sets was performed using Protein Lynx Global Server V2.2.5 (Waters, Manchester, UK) and the NCBI nonredundant protein database, taking fixed (carbamidomethyl) and variable (oxidation of methionine) modifications into account. 3. Results

2.3.2. Reversed-phase perfusion chromatography Samples (20 ␮l) of SMP, SPI, PPI and manufactured SMP containing SPI or PPI were applied either directly or after borate treatment (see Section 2.2.1) to a POROS R2/10 (Applied Biosystems, Foster City, USA) reversed-phase perfusion column (50 mm × 4.6 mm, I.D.). The column had a volume of 0.8 ml and was packed with cross-linked polystyrene–divinylbenzene beads (10 ␮m particle size) equilibrated with 0.1% TFA (solvent A). The organic mobile phase contained ACN with 0.1% TFA (solvent B). The column was eluted at 60 ◦ C with a flow rate of 2 ml/min. The gradient consisted of isocratic conditions at 5% B for 1 min, a linear gradient to 35% B over 1 min, a linear gradient to 45% B over 2 min, isocratic conditions at 45% B for 1 min, and then a linear gradient to 80% B over 1 min. After isocratic conditions at 80% B for 1 min a linear gradient back to 5% B over 1 min was applied. 2.4. Gel electrophoresis Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed on 12.5% Midget gels (Pharmacia) according to Laemmli [26]. The gel was calibrated with broad range molecular mass markers (Biorad). All samples were heated for 5 min at 95 ◦ C in presence of SDS sample buffer before loading the gel. Gels were stained with Coomassie Brilliant Blue R-250 (CBB). 2.5. Nano-liquid chromatography–mass spectrometry The tryptic digests were analysed by one-dimensional LC–MS using an EttanTM MDLC system (GE Healthcare) in high-throughput configuration directly connected to a Q-TOF-2 mass spectrometer (Waters, Manchester, UK). Samples (5 ␮l) were loaded on 5 mm × 300 ␮m I.D. Zorbax 300 SB C18 trap columns (Agilent Technologies), and the peptides were separated on 15 cm × 100 ␮m I.D. Chromolith CapRod monolithic C18 capillary columns at a flow rate of approx. 1 ␮l/min. Solvent A contained an aqueous 0.1% FA solution and solvent B contained 84% ACN in 0.1% FA. The gradient consisted of isocratic conditions at 5% B for 10 min, a linear gradient to 30% B over 40 min, a linear gradient to 100% B over 10 min, and then a linear gradient back to 5% B over 5 min. MS analyses were performed in positive mode using ESI with a NanoLockSpray source. As lock mass, [Glu1 ]fibrinopeptide

3.1. Efficiency of plant protein enrichment by borate treatment First, SMP and the plant protein isolates of soy (SPI) and pea (PPI) were applied separately to the RP perfusion column to optimise the HPLC conditions (gradient, flow, temperature) and to select the best detection system for monitoring milk and plant proteins. While 70–80% of the soy and pea proteins eluted from the perfusion column in the same time range as the milk proteins, i.e. between 2.5 and 4.5 min, the remaining 20–30% of plant proteins eluted at higher acetonitrile concentration, i.e. between 5.8 and 7.0 min (data not shown). From the elution patterns it was also clear that fluorescence detection compared favourably to UV detection with respect to selectivity (no fluctuating baseline due to the effect of matrix/eluents) and sensitivity (data not shown). Therefore, the efficiency of borate buffer to separate plant proteins from milk proteins by their solubility in the buffer was monitored and quantified using a fast gradient for the RP perfusion column in combination with intrinsic fluorescence detection. The solubility behaviour of milk proteins (SMP) and plant proteins (SPI and PPI) in borate buffer were first separately analysed. The fluorescence HPLC profiles of the soluble (supernatant) and insoluble proteins (dissolved pellet) of SMP, SPI and PPI after borate treatment are shown in Fig. 1. On basis of the peak area 98% of the milk proteins were present in the supernatant and 2% in the pellet (Fig. 1A). In case of the plant isolates, 94% of the soy proteins and 87% of the pea proteins were retrieved in the pellet (Fig. 1B and C). So the solubility of plant proteins in borate buffer contrasted strongly to that of milk proteins. Furthermore, the dissolved pellets of borate treated SPI and PPI revealed distinct fluorescence HPLC profiles. Notably, the plant proteins present in the borate supernatants of SPI and PPI eluted at rather low ACN concentration. Next step was to test the suitability of borate buffer to separate plant proteins from milk proteins when present in a mixture as is the case for manufactured SMP containing plant proteins. Therefore, SMP containing 1 or 5% of SPI (SMP-S1 and SMPS5, respectively) or PPI (SMP-P1 and SMP-P5, respectively) were subjected to borate treatment and the pellets were analysed by fast RP perfusion chromatography as described above. As shown in Fig. 2, the intrinsic fluorescence profiles of the dissolved pellets of borate treated SMP-S5 and SMP-P5 were similar to the profiles of the dissolved pellets of borate treated

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Fig. 1. HPLC intrinsic fluorescence profiles of supernatant (grey line) and dissolved pellet (black line) of SMP (A) SPI (B) and PPI (C) after borate treatment (5 mg sample/ml borate buffer).

SPI and PPI, respectively (Fig. 1B and C). This indicated that the solubility behaviour of the plant proteins in borate buffer was not considerably changed by the presence of milk proteins and/or by the process used to produce the milk powder. The differences in solubility between plant and milk proteins in borate buffer was also analysed by SDS-PAGE and subsequent CBB-staining. The SDS-PAGE protein patterns of the various borate pellets are shown in Fig. 3A. Whereas the lanes containing the borate pellets of two control samples of SMP showed only a few weak bands (lanes C1 and C2), the lanes containing the dissolved pellets of SMP-S1 and SMP-S5 (lanes S1 and S5)

Fig. 2. HPLC intrinsic fluorescence profiles of dissolved pellet of SMP-S5 (grey line) and SMP-P5 (black line) after borate treatment (10 mg sample/ml borate buffer).

and SMP-P1 and SMP-P5 (lanes P1 and P5) showed numerous bands. SMP containing SPI revealed a different protein band pattern compared to SMP containing PPI, indicating that mainly plant proteins were not dissolving in borate buffer. As expected, the presence of 5% plant protein isolate in SMP resulted in higher intensity bands for the corresponding borate pellets than when 1% of plant protein isolate was present. The protein pattern of the supernatants from borate treated SMP and manufactured SMP containing low amounts of plant protein isolate were very similar

Fig. 3. SDS-PAGE patterns of dissolved pellet (A) and supernatant (B) of SMP and adulterated SMP samples subjected to borate treatment. For each of the samples, 50 mg of powder was suspended in 10 ml of borate buffer (see also Section 2.2.2). Five microliters dissolved pellet (total volume 50 ␮l) and 7.5 ␮l supernatant (total volume 10 ml) were loaded on gel. Lane M, molecular mass standards; lane S1, SMP-S1; lane S5, SMP-S5; lane P1, SMP-P1; lane P5, SMPP5; lane C1, SMP 1; lane C2, SMP 2. Milk proteins: ␣-casein (␣-cas), ␤-casein (␤-cas), ␬-casein (␬-cas), ␤-lactoglobulin (␤-LG) and ␣-lactalbumin (␣-LA).

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Fig. 4. Base peak intensity (BPI) chromatograms of the borate pellet from SMP (A), SMP-S1 (B) and SMP-P1 (C). 5 ␮l of tryptic digest, originating from 2.5 mg of (adulterated) milk powder, was injected for each LC–MS run. Arrows indicate peptide peaks with corresponding m/z-value and specific retention time present in all three samples (␤-lactoglobulin (␤-LG), ␣-casein (␣-Cas)). The intensity of the largest peak (Fig. B, m/z = 713.4 and BPI = 504) was set to 100% for all three chromatograms.

(Fig. 3B). The major proteins in the supernatants correspond to typical milk proteins; ␣-, ␤- and ␬-casein, ␤-lactoglobulin and ␣-lactalbumin. 3.2. Identification of plant proteins by LC–MS/MS Nano-LC–MS/MS was applied to identify the proteins present in the pellets of borate treated SMP, SMP-S1, SMP-S5, SMP-P1 and SMP-P5. After digestion of the proteins in the various pellets with trypsin the peptides were separated by nanoflow reversed-phase LC. First, MS data were acquired on-line with the Q-TOF operating in MS mode. As presented in Fig. 4, the base peak chromatograms of the pellet fraction of borate treated SMP (A), SMP-S1 (B) and SMP-P1 (C) differ both in signal intensity and retention time of eluting peptides. The adulterated SMPs feature a variety of peaks (with overall higher intensity) compared to SMP, and only a limited number of peaks having the same m/z value were present in two or all three chromatograms (arrows in Fig. 4). Next step was to obtain sequence information of the tryptic peptides present in the various samples. Aliquots were run once more using nano-LC, but now MS/MS data were acquired using real time data dependent switching of the Q-TOF between MS and MS/MS mode. The MS/MS spectra generated in this way were then processed and database searched using ProteinLynx GlobalServer. For most of the peptide peaks observed in the chromatograms of Fig. 4, MS/MS data were obtained resulting in sequences that could be assigned to proteins in the NCBI NR database. In case of the pellet from borate treated SMP in total 12 sequence hits were obtained corresponding to the milk proteins ␤-lactoglobulin (8), ␣-s1-casein (2) and ␬-casein

(2). Table 1 summarises the proteins with a sequence coverage of 5% and higher that were identified in the pellet of borate treated SMP-S1 and SMP-S5. For SMP-S1 in total 43 peptides could be assigned to the soy proteins glycinin (types G1–G5) and ␤-conglycinin (␣- and ␤-subunit). In case of SMP-S5 in total 64 peptides were assigned to these soy proteins. Whereas for SMP-S1 and SMP-S5 both peptides of soy lipoxygenase-1 and -3 were detected, for SMP-S5 also peptides of lipoxygenase2, alcohol dehydrogenase and glucose binding protein from soy were revealed. Furthermore, in the borate pellets from SMP-S1 and SMP-S5 nine peptides were identified originating from the milk proteins bovine ␤-lactoglobulin (7) and ␣-s1-casein (2), and in SMP-S1 four extra peptides were detected corresponding to milk butyrophilin (Table 1). Querying the NR database with the peptide sequences found in the pellets from borate treated SMP-P1 and SMP-P5 resulted in hits for pea legumin (A, B, J and K precursor), provicilin (A and B precursor), vicilin, convicilin and albumin (Table 2). For SMP-P1 and SMP-P5 in total 39 and 68 peptides, respectively, could be assigned to these pea seed proteins. As was also found for the pellets of borate treated SMP containing soy protein, a limited number of peptides (13 and 11 peptides for SMP-P1 and SMP-P5, respectively) originated from milk proteins (␤-lactoglobulin, ␣-s1-casein and butyrophilin). In Fig. 4, the most abundant tryptic peptides of ␤lactoglobulin and ␣-s1-casein detected in the pellets of borate treated SMP, SMP-S1 and SMP-P1 are indicated, showing that the absolute amounts of these milk peptides differ between the samples. However, the majority of peptides observed in the borate pellets of adulterated SMP originated from plant proteins. This is clearly demonstrated in Fig. 5, showing the base

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Table 1 Identified proteins (accession number, description and molecular mass (Mw)) in the borate pellets of SMP-S5 and SMP-S1 (“Matched Peptides” gives the number of peptide sequences identified by LC–MS/MS for SMP-S5 and SMP-S1 (between brackets) that matched to the corresponding protein. The percent protein coverage is given only for the matched peptides of SMP-S5) Accession

Description

Matched peptides

Coverage (%)

Mw (Da)

CAA33215 CAA33216.1 CAA33217.1 AAB23212.1 BAA19058.1 AAA33986.1 AAA33987.1 AAB41272.1 AAO45103.1 BAA23361.2 AAN03476.1 AAB03894.1 AAA30411.1 AAA30428.1 CAB07533.1

Glycinin G1—A1ab1b subunit [Glycine max] Glycinin G2—A2B1a subunit [G. max] Glycinin G3—A1bB2 subunit [G. max] Glycinin G4—A5A4B3 subunit [G. max] Glycinin G5—A3B4 subunit [G. max] Lipoxygenase-1 [G. max] Lipoxygenase-2 [G. max] Lipoxygenase-3 [G. max] Beta-conglycinin—alpha subunit [G. max] Beta-conglycinin—beta chain [G. max] Alcohol dehydrogenase 1 [G. max] Glucose binding protein [G. max] Beta-lactoglobulin [Bos taurus] Alpha-s1-casein [B. taurus] Butyrophilin [B. taurus]

17 (10) 12 (5) 3 (4) 10 (7) 9 (4) 5 (1) 7 (0) 6 (3) 6 (7) 7 (6) 2 (0) 3 (0) 7 (7) 2 (2) 0 (4)

40 41 6 22 27 10 12 12 15 17 8 10 44 10 8

55,671 54,356 54,207 63,640 58,141 94,310 97,085 96,737 44,991 47,946 39,981 60,484 17,155 24,513 59,207

Table 2 Identified proteins (accession number, description and molecular mass (Mw)) in the borate pellets of SMP-P5 and SMP-P1 (“Matched Peptides” gives the number of peptide sequences identified by LC–MS/MS for SMP-P5 and SMP-P1 (between brackets) that matched to the corresponding protein. The percent protein coverage is given only for the matched peptides of SMP-P5) Accession

Description

Matched peptides

Coverage (%)

Mw (Da)

CAA10722.1 CAA47809.1 CAA30067.1 CAA30068.1 P02855 P02854 P13918 CAB82855.1 AAA33641.1 CAA72090.1 CAA68497.1 AAA30411.1 AAA30428.1 CAB07533.1

Legumin A precursor [Pisum sativum] Legumin B precursor [P. sativum] Legumin J precursor [P. sativum] Legumin K precursor [P. sativum] Provicilin type A precursor [P. sativum] Provicilin type B precursor [P. sativum] Vicilin precursor [P. sativum] Convicilin [P. sativum] Major seed albumin PA2 [P. sativum] P54 protein (sucrose-binding protein) [P. sativum] Lectin precursor [P. sativum] Beta-lactoglobulin [Bos taurus] Alpha-s1-casein [B. taurus] Butyrophilin [B. taurus]

15 (8) 5 (3) 3 (2) 3 (1) 4 (2) 9 (5) 8 (4) 10 (7) 11 (7) 4 (2) 2 (1) 8 (7) 3 (2) 0 (4)

35 10 5 11 22 24 28 24 58 11 11 52 18 8

58,753 64,833 56,860 39,776 31,521 46,356 52,199 72,019 26,221 54,627 30,251 17,155 24,513 59,207

peak chromatograms of the borate pellets from adulterated SMP containing 5% soy (Fig. 5A) and 5% pea (Fig. 5B). Glycinin G1 is the most prominent protein in the SMP-S5 borate pellet, followed by glycinin G2 and G4. In case of the pellet from borate treated SMP-P5, the majority of the LC–MS high intensity peaks were assigned to pea legumin A (5), legumin B (3), albumin (4) and provicilin (3) peptides (Fig. 5B). 4. Discussion A new method is described to screen and identify low amounts of plant proteins in manufactured adulterated SMP. The addition of plant proteins to SMP is illegal practise and the aberrant proteins cannot be identified by the current official reference CZE method [13]. From a financial point of view, soy and pea are attractive sources for plant proteins to adulterate SMP (from 1%), so the new method should be reliable in screening and identifying these types of proteins down to this level of

contamination [1–6]. Direct LC–MS/MS analysis of the tryptic peptides obtained from SMP adulterated with 5% soy protein isolate or 5% pea protein isolate resulted in the detection of only a few peptides originating from soy or pea proteins, respectively (data not shown). The main reason for this is that with the used MS instrument (Q-TOF-2) primarily the more abundant (milk) peptides in a tryptic digest will be fragmented and sequenced. Therefore, an additional enrichment step based on a borate treatment was a prerequisite for a reliable detection of low levels of plant proteins in adulterated SMP. A fast HPLC method, using an RP perfusion column combined with fluorescence detection, was developed and tested to monitor and quantify the efficiency of the enrichment step. This HPLC method may be used for fast screening of adulterations in SMP samples. The applied HPLC method resulted in distinct fluorescence profiles for SMP, SPI and PPI (data not shown). Whereas the milk proteins and majority of plant proteins eluted from the perfusion column between 35 and 45% of ACN (2.5–4.5 min),

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Fig. 5. Base peak intensity (BPI) chromatograms of the borate pellet from SMPS5 (A) and SMP-P5 (B). Five microliters of tryptic digest, originating from 2.5 mg of adulterated milk powder, was injected for each LC–MS run. Assigned peaks correspond to peptides (with sequence numbering between brackets) from ␤-lactoglobulin (␤-LG), ␣-casein (␣-Cas), glycinin (G1, G2, G4), ␤-conglycinin (Con␣ and Con␤), lipoxygenase (Lip1-3), legumin (LegA, LegB, LegK), albumin (Alb), provicilin (ProA and ProB), P54 protein (P54), vicilin (Vic) and convicilin (Con). The accession numbers of these proteins in the NCBI protein database are presented in Tables 1 and 2. The intensity of the largest peak was set to 100% for (A) BPI = 1270, and (B) BPI = 806.

a considerable amount of plant proteins eluted at higher ACN concentration (5.8–7.0 min) indicating the presence of highly hydrophobic proteins. Reversed-phase perfusion chromatography has been described in earlier studies for the detection of soybean proteins in cheese and milk [22,23]. These studies applied an HPLC method in which the eluting proteins were monitored via UV detection at 254 nm. This resulted in complex UV profiles with low peak intensities and fluctuating baselines due to interference of matrix compounds and eluents. Furthermore, the used gradient did not exceed ACN concentrations above 45%, meaning that the more hydrophobic soy proteins most probably were not eluting from the column and thus not detected. As was clearly observed in the intrinsic fluorescence profiles of our study (Figs. 1 and 2), a considerable amount of soy proteins do elute at high ACN concentration. The distinct intrinsic fluorescence HPLC profiles of SMP, SPI and PPI were used for analysing the efficiency of borate buffer

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in separating plant proteins from milk proteins. In general, milk proteins should dissolve in borate buffer while most plant proteins are insoluble [7,19]. This is in agreement with the HPLC profiles of the supernatant and dissolved pellet of borate treated SMP, SPI and PPI recorded in our study (Fig. 1). Whereas 98% of the milk proteins in SMP were soluble in borate (supernatant), the majority of plant proteins in the plant protein isolates (94% for SPI and 87% for PPI) were insoluble in borate (pellet). Most of the borate soluble plant proteins eluted at rather low ACN concentration indicating the presence of more hydrophilic proteins or peptides (its presence in the plant protein isolates is known). In case of peptides, these cannot be detected by SDS-PAGE. Although the exact mechanism that determines the solubilisation of proteins in borate buffer is still unknown (Olieman, C., personal communication), the observed HPLC profiles indicate that the hydrophobicity of a protein affects to a great extent its solubility characteristics. The HPLC profiles of the borate pellets of manufactured SMP samples adulterated with soy or pea protein isolate (Fig. 2) resembled the profiles of the borate pellets of SPI and PPI (Fig. 1B and C), respectively. This means that the solubility behaviour of the plant proteins in borate is not severely changed after mixing the plant protein isolates with the skimmed milk and the subsequent process of pasteurisation and powdering. Notably, the ratio of proteins eluting below and above 45% ACN differed somewhat for the borate pellets from adulterated SMP and the plant protein isolates. Whereas the majority of proteins in the pellet from the borate treated plant protein isolate eluted below 45% ACN, this is not the case for the proteins present in the borate pellet from adulterated SMP. It seems that in the latter case a relatively higher amount of proteins eluting above 45% ACN (i.e., more hydrophobic proteins) are retrieved in the borate pellet. Nevertheless, the borate buffer treatment appeared to be a very effective way to obtain a fraction enriched in plant proteins from adulterated SMP. Although the HPLC profiles of the borate pellets from adulterated SMP strongly indicate the presence of mainly plant proteins, SDS-PAGE (Fig. 3) was used as a second independent method to determine the efficiency of borate to enrich for plant proteins. Whereas the supernatants of borate treated SMP and adulterated SMP all show the same typical band pattern of milk proteins [27], the corresponding borate pellets show different band patterns. The rather broad bands observed for the milk proteins in the borate supernatants of non-adulterated and adulterated SMP can be explained by lactosylation of the caseins and the whey proteins ␤-lactoglobulin and ␣-lactalbumin. During the production of SMP (at high pressure, high temperature) several lactose molecules can attach to the milk proteins [28,29] resulting in broader bands on gel. As expected, the SDS-PAGE band patterns of SMP-S1 or SMP-S5 clearly differ from those obtained of SMP-P1 and SMP-P5. Furthermore, the intensity differences between the lanes reflect the differences in percentage of plant material originally present in the adulterated SMPs. On the basis of these results and the HPLC findings, the borate treatment can be used as a (semi)quantitative method to estimate the percentage of plant proteins present in adulterated SMP. The chromatographic and electrophoretic techniques discussed so far are well suited for routine screening of the presence

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of soy or pea proteins in SMP. However, as these methods are based only on deviations in protein profiles compared to control SMP, they cannot be used as an official reference method to prove fraudulent addition of plant proteins to SMP. Therefore, an analytical tool was required leading to the identification of the added plant material. Recently, new methods for the detection of adulterations in milk products like cheese have been described based on mass spectrometric instrumentation [30–32]. Matrix-assisted laser desorption ionisation (MALDI) MS has been shown to be suitable for monitoring changes in milk protein profiles caused by adulterations [30,31]. Nowadays, the most obvious approach for protein identification is based on peptide sequencing of (tryptic) digests of either pure proteins or mixtures of proteins by LC–MS/MS. Here, the (complex) mixture of peptides resulting from tryptic digestion of the proteins present in the borate pellets of the various (adulterated) SMP samples, were separated by nanoflow reversed-phase LC in combination with on-line automatic data-dependent acquisition of MS/MS peptide fragmentation spectra. This resulted in the identification of numerous peptides of soy and pea proteins in manufactured adulterated SMP (Tables 1 and 2). In the borate pellets of SMP-S1 or SMP-S5 mainly glycinin, and to a lesser extent ␤-conglycinin and lipoxygenases, were identified (Table 1). Glycinin and ␤-conglycinin represent the predominant storage proteins in soybean. Because of their abundance in soy and because they are easily extractable, these proteins are rather cheap and used as soy protein isolates on the market [1–4]. The predominant glycinin subunits found in soy are encoded by five genes, designated as Gy1–Gy5 [33]. Peptides of all five glycinin subunits were detected in the borate pellets of SMP adulterated with SPI, indicating that oligomeric protein complexes formed by all five subunits were insoluble in borate buffer. Peptides of both the ␣- and ␤-subunit of the trimeric protein ␤-conglycinin (ca. 210 kDa) were also detected in SPI adulterated SMP. In the borate pellets of SMP-P1 and SMP-P5 mainly legumin, vicilin, convicilin and albumin were identified by LC–MS/MS (Table 2). The seeds of pea consist of two major classes of proteins, i.e. water-soluble albumins and salt-soluble globulins. Approximately, 30% of the total protein content of pea consists of albumins and the remaining 70% is mainly represented by globulins [5]. Albumin PA2 is among the most hydrophobic albumins and was detected with high protein coverage (58%) in the pellet of SMP-P5. [34]. Leguminous globulins can be separated into two major fractions, legumins and vicilins/convicilins. For pea, 11 legumin genes (legumin A–K) are known, showing sequence identities between 50 and 95% [35]. We detected in the pellet of borate treated SMP adulterated with PPI several peptides belonging to the legumin gene products designated as A, G, J and K. Vicilin corresponds to up to 35% of the total protein content of pea seeds and consists of three major subunits assembled into a less well-defined oligomer of about 150 kDa [5]. Peptides of all three subunits were detected in the borate pellets of SMP containing PPI (Table 2). Finally, peptides of a third globulin protein, convicilin, that is highly homologous to vicilin, but possesses an extended Nterminus, were observed in the borate pellets obtained from PPI adulterated SMP.

In the borate pellets from non-adulterated and adulterated SMP several peptides were identified originating from ␤lactoglobulin and ␣-s1-casein (Tables 1 and 2). It is known that heat treatment of milk induces the formation of covalent crosslinks (e.g., lysinoalanine) in milk proteins [36]. Therefore, it is conceivable that during the production of SMP ␤-lactoglobulin and ␣-s1-casein formed a protein complex that causes insolubility in borate buffer. In the presence of plant proteins it appears that more of these complexes are formed as higher peak intensities for the peptides corresponding to the milk proteins were observed in the LC–MS chromatograms of adulterated SMP than in the LC–MS chromatogram of SMP (Fig. 4). This suggests that complexes are formed of the specific milk proteins with plant proteins. The fact that these protein complexes are normally highly hydrophobic explains the HPLC profiles of the borate pellets of adulterated SMP (Fig. 2) where a relatively high amount of proteins eluted above 45% ACN. Eventually, the presence of several milk peptides in the borate pellets from adulterated SMP did not complicate the identification of plant proteins as primarily soy or pea peptides were retrieved in the pellet (Tables 1 and 2; Fig. 5). 5. Conclusions We demonstrated an LC–MS/MS method which, in combination with a borate enrichment step, allowed the identification of numerous soy or pea storage proteins in manufactured SMP containing low amounts of soy protein isolate or pea protein isolate, respectively. Although the method was developed to detect 1–5% plant material in adulterated SMP, the method is easily applicable to detect plant proteins at concentrations well below 1%. Since mainly plant proteins are retrieved in the borate pellet, higher amounts of adulterated SMP as starting material for the borate treatment will proportionally increase the amount of protein available for LC–MS/MS analysis. Although soy and pea are the most attractive protein sources for potential adulterations in SMP, the presented method can be extended to the detection of other plant proteins in SMP as well. The fast HPLC method for screening plant proteins and peptides in SMP after the borate enrichment step, in combination with the LC–MS/MS identification method, provides means to assess the quality and authenticity of SMP. Acknowledgement This work was partially funded by the Netherlands Proteomics Centre (NPC-Hotel). References [1] M.C. Garc´ıa, M. Torre, M.L. Marina, F. Laborda, CRC Crit. Rev. Food Sci. Nutr. 37 (1997) 361. [2] Soy Protein Council, Soy Protein Products—Characteristics, Nutritional Aspects and Utilization, Washington, 1987. [3] E.W. Lusas, M.N. Riaz, J. Nutr. 125 (1995) 573S. [4] A. Maraboli, T.M.P. Cattaneo, R. Giangiacomo, J. Near Infrared Spectrosc. 10 (2002) 63.

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