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Quantification of lactosylation of whey proteins in stored milk powder using multiple reaction monitoring
Food Chemistry 141 (2013) 1203–1210
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Analytical Methods
Quantification of lactosylation of whey proteins in stored milk powder using multiple reaction monitoring Thao T. Le a,b, Hilton C. Deeth a,⇑, Bhesh Bhandari a, Paul F. Alewood b, John W. Holland b a b
School of Agriculture and Food Sciences, The University of Queensland, Brisbane 4072, Australia Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072, Australia
a r t i c l e
i n f o
Article history: Received 3 September 2012 Received in revised form 21 January 2013 Accepted 20 March 2013 Available online 29 March 2013 Keywords: Lactosylation Multiple reaction monitoring MRM Quantification Milk powder
a b s t r a c t Lactosylation in stored milk powder was quantified by multiple reaction monitoring (MRM), a mass spectrometry-based quantification method. The MRM method was developed from a knowledge of peptide fragmentation. The neutral losses of 162 Da (cleavage of galactose) and 216 Da (the formation of furylium ion) which were representative of lactosylated peptides were specifically selected as MRM transitions. Quantification of lactosylated protein was based on the peak areas of these fragmentation ions. The MRM results showed an increase in peak areas of the two transition fragments from tryptic digests of whey proteins in stored milk protein concentrate powder. A good correlation between the MRM and furosine results indicated that MRM based on tryptic digests of whole products was a feasible method for quantification of modified milk proteins. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The covalent attachment of lactose to protein forming lactulosyllysine (Amadori product) readily occurs in dairy products, particularly milk powder, during processing and storage (Thomas, Scher, Desobry-Banon, & Desobry, 2004). The lactulosyllysine content has mostly been quantified by indirect methods, such as by measuring furosine which is a product formed from lactulosyllysine by acid hydrolysis (Erbersdobler & Somoza, 2007). MALDITOF-MS has been used successfully for the quantification of lactosylated whey proteins in heat-treated milk (Meltretter, Birlouez Aragon, Becker, & Pischetsrieder, 2009). LC–ESI-MS, based on the deconvolution of ESI spectra or chromatographic traces arising from multiple ion current extraction, was used for quantification of lactosylated species of whey proteins in pasteurised and UHT milk by Losito, Carbonara, Monaci, and Palmisano (2007). However, those methods have some drawbacks. For example, the Amadori product which is hydrolysed to form furosine, also produces pyridosine and lysine; the yield of furosine depends on the conversion rate which varies according to the hydrolysis conditions and sugar moiety of the Amadori product (Krause, Knoll, & Henle, 2003). Furthermore, the Amadori product can be formed by different sugar sources in a complex sample. Therefore, the lactulosyllysine cannot be absolutely quantified by the furosine method. For the mass spectrometry methods, MALDI-TOF-MS gives a ⇑ Corresponding author. Tel.: +61 7 3870 8251. E-mail address: [email protected] (H.C. Deeth). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.073
poor signal in some severely thermally processed milk due to the denaturation of b-lactoglobulin (Meltretter et al., 2009). The deconvolution method failed to quantify low concentrations of proteins such as the bi-lactosylated forms of a-La and b-Lg in UHT and pasteurised milk (Losito et al., 2007). Although quantification based on multiple ion current extraction proved to be more sensitive than the deconvolution method, it has only been applied on pasteurised and UHT milk. These products contain a maximum of two lactose adducts. The more severe heat treatment processes (e.g., milk powder processing) or storage conditions could lead to more protein modifications (e.g., whey protein denaturation, lactosylation, etc.), which could interfere in LC chromatograms of native and/or lactosylated forms. This study used multiple reaction monitoring (MRM) to quantify lactosylation of whey proteins in milk powder after storage. MRM using a triple quadrupole (QqQ) instrument has been used in quantification of post-translational modifications of proteins (Cox et al., 2005). The first quadrupole (Q1) acts as a filter where only ions of a specific m/z are allowed to pass through the second quadrupole. Here, these ions collide with gas molecules and fragment. The third quadrupole (Q3) passes one of the fragments with specific m/z to the detector. The combination of triple quadrupole with chromatography makes MRM ideal for quantifying compounds in a complex mixture. There is an unlikely chance of isobaric compounds co-eluting with the target compounds and having the same fragments.
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2. Materials and methods 2.1. Materials Whey protein concentrate (80% protein, 6% lactose and 7% fat, hereafter termed WPC80) and milk protein concentrate (81.1% protein, 4.2% lactose and 1.4% fat, hereafter termed MPC80) were obtained from Murray Goulburn Co-op Ltd. (Victoria, Australia). WPC80 was stored at 30 °C and 84% relative humidity. MPC80 powder was stored at a range of temperatures (25–40 °C) and relative humidities (44–84%) for up to 12 weeks. Control samples of both WPC80 and MPC80 were stored at 20 °C. Modified porcine trypsin (proteomics grade), iodoacetamide, dithiothreitol (DTT), and triethylammonium bicarbonate (TEAB) buffer (1 M) were obtained from Sigma–Aldrich (Sydney, Australia). Hydrochloric acid, acetonitrile and formic acid were analytical grade. 2.2. Methods 2.2.1. In-solution tryptic digestion For WPC80: approximately 10 mg of WPC80 powder was dissolved in 1 mL of Milli-Q water. A 100 lL aliquot of reconstituted WPC80 was diluted with 700 lL of 40 mM TEAB, pH 8, to obtain a 1 mg/mL protein solution. One microliter of a reducing agent (10 mg/mL DTT in 40 mM TEAB, pH 8) was added to 20 lL of the protein solution, followed by incubation for 10 min at 60 °C. Protein thiol groups from the reduced solution were then alkylated with iodoacetamide; 1 lL of 50 mg/mL iodoacetamide in 40 mM TEAB was added to the reduced solution, followed by incubation for 30 min at 37 °C in the dark. The solution was digested with 20 lL of trypsin (10 lg/mL in 40 mM TEAB, pH 8) and incubated at 37 °C overnight. The solution digests were acidified by 6.25% formic acid to obtain a final concentration of 1% before LC/MS/MS analysis. For MPC80: 10 mg of MPC80 powder was dissolved in 8 mL of 40 mM TEAB, pH 8. The reduction, alkylation and digestion steps were the same as described for WPC80. 2.2.2. LC/MS After resuspension in 1% formic acid, the tryptic digests of WPC80 stored at 30 °C and 84% RH for 4 weeks were analysed on a Triple TOF LC/MS/MS 5600 (ABSciex). Aliquots (2 lL) of the digests were injected into an C18 Agilent ZORBAX nano column (150 mm 75 lm, with a particle size of 3.5 lm) of a Shimadzu LC-20AD coupled to the triple TOF mass spectrometer. The solvents used were (A) 0.1% formic acid in Milli-Q water and (B) 90% acetonitrile in Milli-Q water with 0.1% formic acid. The gradient was run as follows: 0–40% B over 95 min and increasing 80% B over 5 min at a flow rate of 0.008 mL/min. The triple TOF mass spectrometer was equipped with a nano-spray ionization source and was operated in positive ion mode with a capillary voltage of 2.6 kV. Spectra were acquired for 120 min in data-dependent MS/MS mode. The MS survey scans were conducted on peptides from a m/z of 350 to 1,800. Twenty precursors in each MS survey scan were subjected to MS/ MS each second. 2.2.3. Multiple reaction monitoring (MRM) The digests were analysed on an Ultimate 3000 LC system (Dionex) coupled to a 4000 QTRAP mass spectrometer (ABSciex). Peptides were separated on a C18 column (2.1 150 mm, 5 lm, Grace Davison) at 45 °C. The mobile phases contained (A) 0.1% formic acid in Milli-Q water and (B) 90% acetonitrile in Milli-Q water with 0.1% formic acid at a flow rate of 250 lL/min. The gradient
was as follows: 0–30% B over 35 min and increasing 80% B over 5 min. The injection volume was 20 lL. The 4000 QTRAP was operated in MRM mode with unit (1 Da) resolution in Q1 and low resolution (5 Da) in Q3, dwell time of 50 ms and declustering potential of 45 V. Analyst 1.5 software was used to control both the LC system and QTRAP 4000. A list of Q1/Q3 masses of peptides of interest were saved and submitted as a batch for data acquisition. Based on preliminary MRM experiments on 10 (2 transitions each) and 12 (2 transitions each) lactosylated peptides of a-La and b-Lg, respectively, the best three peptides of each protein were chosen for quantification. The method was tested and optimised for collision energy (CE) based on whey proteins obtained from WPC80. Experiment 1: WPC80 powder was used as a standard to optimise the MRM conditions. WPC80 was reconstituted, in-solution digested with trypsin and directly loaded on a LC/MS system for analysis. The MRM transitions of a-La and b-Lg were developed based on the knowledge of fragmentation of lactosylated peptides obtained from a full LC/MSMS scan. Due to lactose attachment on investigated peptides, it is very challenging to choose the right CE for each transition. Thus, three different CEs were selected for each transition of these a-La and b-Lg lactosylated peptides and labelled ‘‘normal’’, ‘‘low’’ and ‘‘very low’’ CE (see Supplementary data 1 and 2). Normal and low CE values were calculated as a linear function of parent ion m/z ratios of lactosylated and unmodified peptides, respectively. Very low CE values were 25% lower than ‘‘low’’ CE values. The optimal CE and six best candidates of lactosylated peptides were selected. Each of best candidates were examined by a full scan MSMS to confirm the identity. Control peptides which were consistently observed in the spectra were used as internal standards for relative quantification. In order to test the linearity and reproducibility of the MRM assay, a Q1/Q3 list containing the six best candidates of lactosylated peptides and four control peptides of a-La and b-Lg was set up in an MRM mode. The WPC80 (stored at 30 °C and 84% RH for 4 weeks) was reconstituted with water to produce a solution containing 1 mg protein/mL and digested with trypsin. The digests were diluted with 1% formic acid in the ratios 1:3, 1:1, and 3:1. Thus, the relative concentration (based on initial protein content) was 0.25, 0.5, 0.75 and 1 mg/ml. The peak area of each transition was calculated using Analyst 1.5 (ABSciex) and plotted against the relative concentration of the initial protein. Experiment 2: The optimised MRM assay on WPC80 (mainly whey proteins) was applied to MPC80 (approximately 80% casein and 20% whey proteins) for lactosylation quantification. The MPC80 samples had been stored in a range of temperature (25–40 °C) and humidity (44–84% RH) conditions for 12 weeks. A few changes were made to improve the signal to noise ratio for MRM transitions of lactosylated peptides of the MPC80 samples. Lactosylation was normalised by multiplying peak areas of the chromatograms of selected MRM transitions by 1000 and dividing them by the corresponding peak areas of control peptides. The normalised lactosylation levels were correlated with furosine levels (Le, Bhandari, & Deeth, 2011) to test the applicability of the MRM assay.
3. Results and discussion 3.1. Experiment 1 3.1.1. MRM transition development Protein quantification by MRM involves many steps, but the selection of specific peptides and their fragments (MRM transitions) that uniquely identify lactosylated peptides is a key part. The neutral loss of 162 Da followed by the loss of three water
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molecules (total loss: 216 Da) that accounted for the formation of a furylium ion were specifically observed in the fragmentation of lactosylated peptides (Molle, Morgan, Bouhallab, & Leonil, 1998; Morgan, Leonil, Molle, & Bouhallab, 1997). Thus, the loss of 162 and 216 Da could be monitored for MRM transitions. To examine the fragmentation patterns of lactosylated peptides, WPC80 tryptic digests were subjected to a full MS scan by LC–MSMS. Lactosylated peptides were detected by the increase in mass of 324 Da compared to the unmodified peptides. Several lactosylated peptides were identified by LC–MSMS that matched with our previous results (Le, Deeth, Bhandari, Alewood, & Holland, 2011) and the study of Holland, Gupta, Deeth, and Alewood (2011) on lactosylated whey proteins detected by MALDI-TOF-MS. Figure 1A shows the MS/ MS result of the a-La peptide 134LDQWLCEKL142 with a lactose adduct on K141 and a mass of 1528.71. The peptide was detected as a doubly charged ion with m/z 764.86. Similarly, the b-Lg peptide 57 VYVEELKPTPEGDLEILLQK76 with a lactose adduct on K63 and a mass of 2637.36 detected as a triply charged ion with m/z 879.7 is shown in Fig. 1B. The patterns of the neutral losses of 162 and 216 Da were readily observed in both a-La and b-Lg lactosylated peptides. For example, it can be seen very clearly that the precursor ion with m/z 764.86 lost one hexose to form an ion with m/z 683.84. The appearance of ions with m/z 674.84, 665.83 and 656.35 was the consequence of three dehydrations to form furylium ion. A similar fragmentation pattern was observed for the precursor ion with m/ z 879.79 of lactosylated b-Lg. However, the signal intensity of [M 216]3+ ion was very low as the collision energy was not optimised. Overall, Q1/Q3 pairs were selected based on the knowledge of fragmentation of lactosylated peptides. Candidate peptides (i.e., Q1) were all theoretical peptides with an internal missed cleavage at a lysine residue and a mass 324 Da higher than the calculated mass from the peptide alone. Q1 was calculated as [M + z H]/z (M = mass of lactosylated peptides and z = number of charges). The neutral losses [M 162] and [M 216] were set as Q3; they
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were calculated as [M + z H 162 (or 216)]/z. The list of Q1/Q3 pairs included 20 transitions for a-La and 24 transitions for b-Lg (see Supplementary data 1 and 2). Those lactosylated peptides were previously detected by MALDI-TOF-MS by Holland et al. (2011) and Arena et al. (2010). 3.1.2. Optimisation of collision energy Ionisation and fragmentation are the two main factors affecting signal intensity of MRM transitions. In this study, we focused more on fragmentation conditions, particularly optimisation of collision energy (CE) for all MRM transitions listed in Supplementary data 1 and 2. Fig. 2 illustrates the effect of CE on the two MRM transitions (neutral losses) of the doubly charged ion at m/z 764.86. It can be clearly seen that there is considerable change in the total ion current (TIC) (shown in the left hand column) and the extracted ion chromatogram (XIC) (shown in the right hand column) of MRMIDA experiments operated at three different CE values. At a ‘‘normal’’ CE (42.77 eV), the loss of 216 Da (formation of furylium ion) which is represented as a red peak Fig. 2B was more prominent than the loss of 162 (cleavage of one hexose) (blue peak) (Fig. 2A). Reducing the CE to 34.35 eV (‘‘low’’ value) increased the loss of 162 and decreased the loss of 216 (Fig. 2B). A further decrease in CE to 25.76 eV (‘‘very low’’ CE) leads to an increase in signal intensity of [M162]2+ ion (Fig. 2C). From the study of Gadgil, Bondarenko, Treuheit, and Ren (2007) on glycated peptides, the parent ions were dissociated differently at different collision energies. The maximum neutral loss of the sugar moiety (162 Da) of glycated peptides has been reported at a collision energy of 20 eV (Gadgil et al., 2007). Morgan et al. (1997) confirmed that the sufficient CE for fragmentation of lactosylated peptides to form furylium ions was 30 eV. These values were variable for different peptides and their charge states. Hence, the CE that gave reliable and reproducible signals for both transitions could be selected as the optimal CE. For this doubly charged ion, ‘‘very low’’ CE gave a better signal intensity for [M 162]2+ than [M 216]2+ ion; this means the collision energy
Fig. 1. Product-ion tandem MS of the doubly and triply charged precursor ions at m/z (A) 764.86 (full scan); (B) 879.79 (full scan); (C) 764.86 (zoomed in); (D) 879.79 (zoomed in) of a-La and b-Lg, respectively. Two major losses of the ions: the loss of 162 Da and subsequent loss of 3H2O to form furylium ions were labelled.
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Fig. 2. Effects of CE on signal intensity. WPC80 stored at 30 °C and 84% RH for 4 weeks, digested with trypsin and analysed by MRM. TIC (left hand column) and XIC (right hand column) of precursor ion with m/z at 764.86 using (A and B) normal; (C and D) low; (E and F) very low CE.
was sufficient for the cleavage of hexose and the formation of furylium ion; but not too strong to form other unnecessarily smaller fragments. Different optimal CEs were obtained for 9 and 12 other MRM transitions of a-Lac and b-Lg lactosylated peptides, respectively.
3.1.3. Selection of best candidates for Q1/Q3 list The best MRM candidates were selected based on signal intensities of both transitions from XIC. The ions with m/z 764.86 (shown in Fig. 2C), 535.28 and 665.35 (see Supplementary data 3B and 3E) produced a single peak with reproducible signal intensities for both transitions (losses of 162 and 216 Da). These ions were eluted at different times, approximately 6.67, 21.89 and 20.21 min, respectively. However, a number of ions appeared with multiple peaks (see Supplementary data 3); consequently, they were not considered for quantitative purposes. That does not mean lactosylated peptides were not present in the samples. The specific signals from these peptides could not be resolved reliably from the other MS signals. Thus, m/z 535.28, 665.35 and 764.86 were chosen as the best candidates for MRM quantification of a-Lac. Similarly, only three out of 12 transitions were selected as the best MRM candidates for b-Lg; the remaining ions were either too weak or appeared as multiple peaks. The three ions were those with m/z 491.94, 638.01 and 879.79 corresponding to elution times at
14.39, 19.77 and 26.34 min, respectively. As a result, the Q1/Q3 list was shortened to six lactosylated peptides including 12 transitions in total for a-Lac and b-Lg (see highlights in grey in Supplementary 1 and 2).
3.1.4. Validation of transitions The best MRM candidates in this study were acquired by MS/MS in order to confirm that the detected signals derive from the targeted peptide. The MS/MS spectra of two out of three best candidate ions from each lactosylated a-La and b-Lg was shown in Supplementary data 4. They included precursor ions with m/z 665.35, 764.86 (from a-La), 491.94 and 638.01 (from b-Lg). The lactosylation sites of these peptides have been confirmed by MALDI-TOF-MS (Holland et al., 2011). From the MS/MS spectra of the precursor ion with m/z 764.86 (see Supplementary data 4B), a loss of 162 Da was observed at m/z 684.3 and the appearance of m/z 657.5 indicated the subsequent loss of three H2O from the [M-162]2+ ion to form a furylium ion. Including the fact that b3 and b4 fragmentation ions were detected at m/z 357.2 and 543.5, this confirmed the identity of this candidate peptide 134LDQWLCEKL142. Although the loss of galactose ion was not clearly shown in the MS/MS spectra of the ion with m/z 665.35 (see Supplementary data 4A), a series of y ions (i.e., y3, y4, y5 and y6) and the formation of furylium ion were enough to confirm the identity of the peptide 114ILDKVGINYWLAHK127. Similar results
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were obtained for precursors ions with m/z 491.94 and 638.01 from b-Lg (see Supplementary data 4C and 4D). Again, the loss of galactose, formation of furylium and a series of y ions (i.e., y1–y5) identified the candidate ion with m/z 491.94 from the lactosylated peptide of b-Lg (see Supplementary data 4C). The presence of ions [M 162], [y12-216], b2 and y12 sufficiently confirmed the identity of the ion at m/z 638.01. Thus, the combination of backbone fragments (e.g., mixture of b and y ions) and sugar loss enabled the identity of the candidate lactosylated peptides to be confirmed. 3.1.5. Selection of control peptides One of the challenges of the MRM method is variation in signal intensities from LC/MS analyses. This could be due to differences in efficiency of the digestion, peptide recovery and/or injection volume. As MRM is used to quantify the amount of specific fragments produced from tryptic digests of lactosylated proteins, it is very important to have control peptides. These peptides could also be considered as internal standards; therefore, they can be used to normalise signal intensities of lactosylated peptides for relative quantification. The control peptides included two a-La peptides 134 LDQWLCEK141 and 82DDQNPHSSNICNISCDK98 with m/z 546.27 and 668.63, respectively; and 2 b-Lg peptides 108VLVLDTDYKK117 and 165LSFNPTQLEEQCHI178 with m/z 597.36 and 858.78 (see Supplementary data 5). These peptides were chosen because they were observed consistently in the spectra of both control and stored samples. The control peptides were subjected to MS/MS in order to select the best fragments for MRM transitions. Hence, the two prominent y-ions of each peptide were chosen for Q3. For example, the y5 and y7 fragments were selected from the fragmentation of peptide sequence 134LDQWLCEK141 from a-La (see Supplementary data 5B), so the Q1/Q3 pairs were 546.3/735.4 and 546.3/978.5. Similarly, the y4 and y6 ions were selected for 82DDQNPHSSNICNISCDK98 from a-La (see Supplementary data 5D), so the Q1/Q3 pairs were 668.6/509.2 and 668.6/736.3. For 108VLVLDTDYKK117 from b-Lg (see Supplementary data 5C) the y6 and y8 fragments were selected so the Q1/Q3 pairs were 597.4/769.4 and 597.4/ 981.5. For 165LSFNPTQLEEQCHI178 from b-Lg (see Supplementary data 5E) the y7 and y10 ions were selected so the Q1/Q3 pairs were 858.8/928.4 and 858.8/1254.6. 3.1.6. Linearity and reproducibility of the selected transitions Figs. 3 and 4 demonstrate responses of 20 MRM transitions from candidate peptides in addition to control peptides. The a-La peptide 114ILDKVGINYWLAHK127 could be detected as a triply or quadruply charged ion. The signal-to-noise ratio of the XIC of the triply charged ion (m/z 665.35) was relatively low. Hence, the ion with m/z 449.27 (a quadruply charged ion of the peptide) was included in the MRM list. Generally, only 16 out of 20 MRM transitions showed a reasonably linear response with r2 ranging between 0.89 and 0.97. The results of four other MRM transitions on the Q1/Q3 list 499.27/458.75, 499.27/445.25 (Fig. 3B), 879.79/ 825.78 and 879.79/807.76 (Fig. 4D) varied considerably between samples. MRM transitions of control peptides (Figs. 3A and 4A) had a good reproducibility due to their high signal intensity from the mass spectra. Although the precursor ion with m/z 665.35 (from a-La) (Fig. 3C) and 491.94 (from b-Lg) (Fig. 4B) showed variation between samples, the linearity and reproducibility of precursor ions with m/z 764.86 (from a-Lac) (Fig. 3D) and 638.01 (from b-Lg) (Fig. 4C) were better. The considerable variability of the lactosylated peptides with m/z 665.35 and 491.94 could be due to the lower abundance of these peptides resulting in relatively low signal-to-noise ratio of the extracted ions. From the demonstration of linearity of lactosylation measurement on WPC80, the MRM list was shortened to two control peptides (four transitions) and two lactosylated peptides (four transitions) for
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each whey protein. The peptides with m/z 499.27 and 879.79 were excluded from the final list. 3.2. Experiment 2 3.2.1. MRM quantification of stored MPC80 The lower abundance of whey proteins in MPC80 compared to that in WPC80 could lead to the low signal-to-noise ratio of the transitions in MPC80 samples. Moreover, the presence of abundant non-target peptides obtained from casein could result in the generation of a broad range of fragment ions that could interfere with those from the targeted peptides. Thus, dropping an EPI scan and shortening the MRM list (one control and two lactosylated peptides for each protein) allowed the dwell time to be increased from 50 to 80 ms/transition, while one data point was still acquired every second across the elution profile. In addition, by scheduling MRM transitions, the full cycle time is deployed to detect the peptides expected to elute in a given time window. In this study, 12 transitions were split into two periods. The first period was set up from 1 to 16.5 min. The second period started from 16.5 to 42 min. Therefore, the first period includes precursor ions with m/z 491.94 (from lactosylated b-Lg), 546.38 and 597.34 (control peptides of a-La and b-Lg, respectively). The second period involved precursor ions with m/z 638.01 (from lactosylated b-Lg), 665.35 and 764.86 (from lactosylated a-La). Six transitions were scanned in the full cycle time (little over 1 s). This allowed the dwell time to be increase to 160 ms/transition, resulting in an increase in signal-to-noise ratio. These modifications were incorporated into the assay and it was re-applied to MPC80 samples. Fig. 5 shows the normalised lactosylation levels of precursor ions with m/z 665.35 and 764.86 from a-La in MPC80 stored at 30 °C, 44% and 84% RH. There was a considerable increase in normalised lactosylation levels of these transitions with increasing RH values. The time effect on lactosylation was clearly seen in all transitions; however, the loss of 162 of precursor ion with m/z 665.35 slightly increased (Figs. 5A and B). This could be due to low signal-to-noise ratio of the transition which leads to insignificant changes between MPC80 samples stored at different times. Similarly, the effects of humidity and time on the normalised lactosylation level obtained by MRM were observed on both precursor ions with m/z at 491.94 and 638.01 from b-Lg (see Supplementary data 6); however, the loss of 162 of m/z 491.94 was not considerably changed with time. This could be explained by a low signalto-noise ratio of the transition. The normalised lactosylation levels in both a-La and b-Lg seem to reach a plateau at 12 weeks of storage for samples stored at 84% RH. 3.2.2. Correlation with furosine content Normalised lactosylation levels were correlated with furosine levels to test the applicability of the MRM assay. Fig. 6 shows a good linear response (r2 = 0.98–0.99) of precursor ion with m/z 764.86 at two transitions (the loss of 162 and 216 Da). The precursor ion with m/z 764.86 also gave higher signal-to-noise ratio compared to the ion with m/z 665.35 which only showed a reasonable linear response (r2 = 0.89–0.92). For b-Lg, the precursor ions with m/z 638.01 and m/z 491.94 showed good correlations (r2 = 0.950.99) with furosine content, at both transitions (see Supplementary data 7). The variation in slope of the lines reflects the differences in ionisation and fragmentation efficiency of the peptides. These differences would be overcome by using matched isotopically-labelled internal standards for normalisation as they should have the same ionisation and fragmentation behaviour as the peptides of interest. As mentioned in the Introduction, the Amadori products have mostly been quantified by indirect ways (e.g., furosine). This hydrolysed compound cannot be considered as the absolute
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Fig. 3. Demonstration of linearity of lactosylation measurement. Peak areas of extracted ions from 10 MRM transitions of a-La were calculated by using Analyst 1.5 quantification module software. (A) d, m/z 546.38/735.35; s, 546.38/978.43; ., 668.61/509.20; D, 668.61/736.33; (B) d, 499.27/458.75; s, 499.27/445.25; (C) d, 665.35/ 611.34; s, 665.35/593.33; (D) d, 764.86/683.83; s 764.86/656.82.
Fig. 4. Demonstration of linearity of lactosylation measurement. Peak areas of extracted ions from 10 MRM transitions of b-Lg were calculated by using Analyst 1.5 quantification module software. (A) d, m/z 597.34/769.37; s, 597.34/981.53; ., 858.41/928.42; D, 858.41/1254.58; (B) d, 491.94/437.92; s, 491.94/419.91; (C) d, 638.01/ 583.99; s, 638.01/565.98; (D) d, 879.79/825.78; s 879.79/807.76.
concentration of the Amadori products. The MS approaches such as LC–ESI-MS and MALDI-TOF-MS allows direct detection of lactulosyllysine (Losito et al., 2007; Meltretter et al., 2009). The advantage of this MS analysis of lactosylation is minimal sample workup, without protein hydrolysis resulting in minimal artifact formation and fast analysis. In fact, MALDI-TOF-MS is even faster and easier than LC–ESI-MS as the milk sample can be directly loaded onto the MALDI target for MS analysis without chromatography and other sample preparation steps (e.g., protein extraction or tryptic digestion). It was first optimized for relative quantifica-
tion of lactosylation in milk samples by Meltretter et al. (2009). That study failed to quantify lactosylated whey proteins in severely heated samples (e.g., condensed milk) as whey protein are largely aggregated. However, LC–ESI-MS was shown to be a sensitive method for detecting and quantifying lactosylated whey proteins in pasteurised milk with a low degree of lactosylation. The method has not been applied on severely heated samples. Since, under high temperature of processing and storage, milk proteins can undergo many modifications such as denaturation and lactosylation, these changes could affect multiple extracted ion chromatograms of both
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Fig. 5. MRM quantification of lactosylated peptides from a-La in stored MPC80. Normalised lactosylation levels were calculated from the peak areas of two selected precursor ions including loss of 162 (d) and 216 Da (s). (A and B) m/z 665.35 from a-La of MPC80 stored at 30 °C, (A) 44% and (B) 84% RH; (C and D) 764.86 from a-La of MPC80 stored at 30 °C, (C) 44% and (D) 84% RH.
Fig. 6. Correlation of normalised lactosylation level (from MRM results) with furosine content (Le, Bhandari, et al., 2011; Le, Deeth, et al., 2001). MPC80 samples were stored at different temperatures and humidities before tryptic digestion for MRM analysis: (A) 25 °C, 44% RH; (B) 25 °C, 84% RH; (C) 30 °C, 44% RH; (D) 30 °C, 84% RH. Two selected precursor ions from a-La with m/z 665.35 with the loss of 162 Da (d) and 216 Da (s); and m/z 764.86 with the loss of 162 (.) and 216 Da (D).
native or lactosylated forms of proteins. Thus, the MRM assay used in this study could become a useful method for a direct quantification of lactosylation in low-, medium- and high-heat-treated milk samples. In addition to the advantages of MS-based quantification methods (as listed above), there is less interference of structural modifications of proteins as only specific fragment ions of targeted lactosylated peptides are detected and quantified. However, the method requires further optimisation to become an applicable method for assessing the quality of dairy products. To assess the quantitative performance of the MRM assay, the
limits of detection and quantification (LOD and LOQ, respectively) should be determined using an internal standard. Aliquots of isotopically labelled lactosylated-peptides can be spiked into the tryptic digests of stored MPC80 samples at a range of concentrations, allowing determination of LOD and LOQ. 4. Conclusion Lactosylation in stored MPC80 can be quantified by the MRM assay. A good correlation between normalised lactosylation levels
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(obtained by MRM) and furosine content proves the feasibility of the MRM assay for quantification of protein changes during storage of milk powders. The approach of MRM in quantification of lactosylation could lead to a new method of detecting and quantifying other modifications of milk protein such as phosphorylation, deamidation, lactosylated casein and protein cross-linking. However, in order to replace traditional methods of quantifying lactosylation, an internal standard needs to be used to obtain absolute quantification of lactose adducts by MRM. Acknowledgements We thank Alun Jones from IMB Mass Spectrometry Facility for expert technical assistance and Dairy Innovation Australia Ltd. and the Australian Research Council for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 03.073. References Arena, S., Renzone, G., Novi, G., Paffetti, A., Bernardini, G., Santucci, A., et al. (2010). Modern proteomic methodologies for the characterization of lactosylation protein targets in milk. Proteomics, 10, 3414–3434. Cox, D. M., Zhong, F., Du, M., Duchoslav, E., Sakuma, T., & McDermott, J. C. (2005). Multiple reaction monitoring as a method for identifying protein posttranslational modifications. Journal of Biomolecular Techniques, 16, 83–90.
Erbersdobler, H. F., & Somoza, V. (2007). Forty years of furosine-forty years of using Maillard reaction products as indicators of the nutritional quality of foods. Molecular Nutrition and Food Research, 51, 423–430. Gadgil, H. S., Bondarenko, P. V., Treuheit, M. J., & Ren, D. (2007). Screening and sequencing of glycated proteins by neutral loss scan LC/MS/MS method. Analytical Chemistry, 79, 5991–5999. Holland, J. W., Gupta, R., Deeth, H. C., & Alewood, P. F. (2011). Proteomic analysis of temperature-dependent changes in stored UHT milk. Journal of Agricultural and Food Chemistry, 59, 1837–1846. Krause, R., Knoll, K., & Henle, T. (2003). Studies on the formation of furosine and pyridosine during acid hydrolysis of different Amadori products of lysine. European Food Research and Technology, 216, 277–283. Le, T. T., Bhandari, B. R., & Deeth, H. C. (2011). Chemical and physical changes of milk protein concentrate (MPC80) powder during storage. Journal of Agricultural and Food Chemistry, 59, 5465–5473. Le, T. T., Deeth, H. C., Bhandari, B., Alewood, P. F., & Holland, J. W. (2011). A proteomic approach to detect lactosylation and other chemical changes in milk protein concentrate. Food Chemistry, 132, 655–662. Losito, I., Carbonara, T., Monaci, L., & Palmisano, F. (2007). Evaluation of the thermal history of bovine milk from the lactosylation of whey proteins: An investigation by liquid chromatography–electrospray ionization mass spectrometry. Analytical and Bioanalytical Chemistry, 389, 2065–2074. Meltretter, J., Birlouez Aragon, I., Becker, C., & Pischetsrieder, M. (2009). Assessment of heat treatment of dairy products by MALDI TOF MS. Molecular Nutrition & Food Research, 53, 1487–1495. Molle, D., Morgan, F., Bouhallab, S., & Leonil, J. (1998). Selective detection of lactolated peptides in hydrolysates by liquid chromatography/electrospray tandem mass spectrometry. Analytical Biochemistry, 259, 152–161. Morgan, F., Leonil, J., Molle, D., & Bouhallab, S. (1997). Nonenzymatic lactosylation of bovine beta-lactoglobulin under mild heat treatment leads to structural heterogeneity of the glycoforms. Biochemical and Biophysical Research Communications, 236, 413–417. Thomas, M. E. C., Scher, J., Desobry-Banon, S., & Desobry, S. (2004). Milk powders ageing: Effect on physical and functional properties. Critical Reviews in Food Science and Nutrition, 44, 297–322.
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Analytical Methods
Quantification of lactosylation of whey proteins in stored milk powder using multiple reaction monitoring Thao T. Le a,b, Hilton C. Deeth a,⇑, Bhesh Bhandari a, Paul F. Alewood b, John W. Holland b a b
School of Agriculture and Food Sciences, The University of Queensland, Brisbane 4072, Australia Institute for Molecular Bioscience, The University of Queensland, Brisbane 4072, Australia
a r t i c l e
i n f o
Article history: Received 3 September 2012 Received in revised form 21 January 2013 Accepted 20 March 2013 Available online 29 March 2013 Keywords: Lactosylation Multiple reaction monitoring MRM Quantification Milk powder
a b s t r a c t Lactosylation in stored milk powder was quantified by multiple reaction monitoring (MRM), a mass spectrometry-based quantification method. The MRM method was developed from a knowledge of peptide fragmentation. The neutral losses of 162 Da (cleavage of galactose) and 216 Da (the formation of furylium ion) which were representative of lactosylated peptides were specifically selected as MRM transitions. Quantification of lactosylated protein was based on the peak areas of these fragmentation ions. The MRM results showed an increase in peak areas of the two transition fragments from tryptic digests of whey proteins in stored milk protein concentrate powder. A good correlation between the MRM and furosine results indicated that MRM based on tryptic digests of whole products was a feasible method for quantification of modified milk proteins. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction The covalent attachment of lactose to protein forming lactulosyllysine (Amadori product) readily occurs in dairy products, particularly milk powder, during processing and storage (Thomas, Scher, Desobry-Banon, & Desobry, 2004). The lactulosyllysine content has mostly been quantified by indirect methods, such as by measuring furosine which is a product formed from lactulosyllysine by acid hydrolysis (Erbersdobler & Somoza, 2007). MALDITOF-MS has been used successfully for the quantification of lactosylated whey proteins in heat-treated milk (Meltretter, Birlouez Aragon, Becker, & Pischetsrieder, 2009). LC–ESI-MS, based on the deconvolution of ESI spectra or chromatographic traces arising from multiple ion current extraction, was used for quantification of lactosylated species of whey proteins in pasteurised and UHT milk by Losito, Carbonara, Monaci, and Palmisano (2007). However, those methods have some drawbacks. For example, the Amadori product which is hydrolysed to form furosine, also produces pyridosine and lysine; the yield of furosine depends on the conversion rate which varies according to the hydrolysis conditions and sugar moiety of the Amadori product (Krause, Knoll, & Henle, 2003). Furthermore, the Amadori product can be formed by different sugar sources in a complex sample. Therefore, the lactulosyllysine cannot be absolutely quantified by the furosine method. For the mass spectrometry methods, MALDI-TOF-MS gives a ⇑ Corresponding author. Tel.: +61 7 3870 8251. E-mail address: [email protected] (H.C. Deeth). 0308-8146/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.foodchem.2013.03.073
poor signal in some severely thermally processed milk due to the denaturation of b-lactoglobulin (Meltretter et al., 2009). The deconvolution method failed to quantify low concentrations of proteins such as the bi-lactosylated forms of a-La and b-Lg in UHT and pasteurised milk (Losito et al., 2007). Although quantification based on multiple ion current extraction proved to be more sensitive than the deconvolution method, it has only been applied on pasteurised and UHT milk. These products contain a maximum of two lactose adducts. The more severe heat treatment processes (e.g., milk powder processing) or storage conditions could lead to more protein modifications (e.g., whey protein denaturation, lactosylation, etc.), which could interfere in LC chromatograms of native and/or lactosylated forms. This study used multiple reaction monitoring (MRM) to quantify lactosylation of whey proteins in milk powder after storage. MRM using a triple quadrupole (QqQ) instrument has been used in quantification of post-translational modifications of proteins (Cox et al., 2005). The first quadrupole (Q1) acts as a filter where only ions of a specific m/z are allowed to pass through the second quadrupole. Here, these ions collide with gas molecules and fragment. The third quadrupole (Q3) passes one of the fragments with specific m/z to the detector. The combination of triple quadrupole with chromatography makes MRM ideal for quantifying compounds in a complex mixture. There is an unlikely chance of isobaric compounds co-eluting with the target compounds and having the same fragments.
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2. Materials and methods 2.1. Materials Whey protein concentrate (80% protein, 6% lactose and 7% fat, hereafter termed WPC80) and milk protein concentrate (81.1% protein, 4.2% lactose and 1.4% fat, hereafter termed MPC80) were obtained from Murray Goulburn Co-op Ltd. (Victoria, Australia). WPC80 was stored at 30 °C and 84% relative humidity. MPC80 powder was stored at a range of temperatures (25–40 °C) and relative humidities (44–84%) for up to 12 weeks. Control samples of both WPC80 and MPC80 were stored at 20 °C. Modified porcine trypsin (proteomics grade), iodoacetamide, dithiothreitol (DTT), and triethylammonium bicarbonate (TEAB) buffer (1 M) were obtained from Sigma–Aldrich (Sydney, Australia). Hydrochloric acid, acetonitrile and formic acid were analytical grade. 2.2. Methods 2.2.1. In-solution tryptic digestion For WPC80: approximately 10 mg of WPC80 powder was dissolved in 1 mL of Milli-Q water. A 100 lL aliquot of reconstituted WPC80 was diluted with 700 lL of 40 mM TEAB, pH 8, to obtain a 1 mg/mL protein solution. One microliter of a reducing agent (10 mg/mL DTT in 40 mM TEAB, pH 8) was added to 20 lL of the protein solution, followed by incubation for 10 min at 60 °C. Protein thiol groups from the reduced solution were then alkylated with iodoacetamide; 1 lL of 50 mg/mL iodoacetamide in 40 mM TEAB was added to the reduced solution, followed by incubation for 30 min at 37 °C in the dark. The solution was digested with 20 lL of trypsin (10 lg/mL in 40 mM TEAB, pH 8) and incubated at 37 °C overnight. The solution digests were acidified by 6.25% formic acid to obtain a final concentration of 1% before LC/MS/MS analysis. For MPC80: 10 mg of MPC80 powder was dissolved in 8 mL of 40 mM TEAB, pH 8. The reduction, alkylation and digestion steps were the same as described for WPC80. 2.2.2. LC/MS After resuspension in 1% formic acid, the tryptic digests of WPC80 stored at 30 °C and 84% RH for 4 weeks were analysed on a Triple TOF LC/MS/MS 5600 (ABSciex). Aliquots (2 lL) of the digests were injected into an C18 Agilent ZORBAX nano column (150 mm 75 lm, with a particle size of 3.5 lm) of a Shimadzu LC-20AD coupled to the triple TOF mass spectrometer. The solvents used were (A) 0.1% formic acid in Milli-Q water and (B) 90% acetonitrile in Milli-Q water with 0.1% formic acid. The gradient was run as follows: 0–40% B over 95 min and increasing 80% B over 5 min at a flow rate of 0.008 mL/min. The triple TOF mass spectrometer was equipped with a nano-spray ionization source and was operated in positive ion mode with a capillary voltage of 2.6 kV. Spectra were acquired for 120 min in data-dependent MS/MS mode. The MS survey scans were conducted on peptides from a m/z of 350 to 1,800. Twenty precursors in each MS survey scan were subjected to MS/ MS each second. 2.2.3. Multiple reaction monitoring (MRM) The digests were analysed on an Ultimate 3000 LC system (Dionex) coupled to a 4000 QTRAP mass spectrometer (ABSciex). Peptides were separated on a C18 column (2.1 150 mm, 5 lm, Grace Davison) at 45 °C. The mobile phases contained (A) 0.1% formic acid in Milli-Q water and (B) 90% acetonitrile in Milli-Q water with 0.1% formic acid at a flow rate of 250 lL/min. The gradient
was as follows: 0–30% B over 35 min and increasing 80% B over 5 min. The injection volume was 20 lL. The 4000 QTRAP was operated in MRM mode with unit (1 Da) resolution in Q1 and low resolution (5 Da) in Q3, dwell time of 50 ms and declustering potential of 45 V. Analyst 1.5 software was used to control both the LC system and QTRAP 4000. A list of Q1/Q3 masses of peptides of interest were saved and submitted as a batch for data acquisition. Based on preliminary MRM experiments on 10 (2 transitions each) and 12 (2 transitions each) lactosylated peptides of a-La and b-Lg, respectively, the best three peptides of each protein were chosen for quantification. The method was tested and optimised for collision energy (CE) based on whey proteins obtained from WPC80. Experiment 1: WPC80 powder was used as a standard to optimise the MRM conditions. WPC80 was reconstituted, in-solution digested with trypsin and directly loaded on a LC/MS system for analysis. The MRM transitions of a-La and b-Lg were developed based on the knowledge of fragmentation of lactosylated peptides obtained from a full LC/MSMS scan. Due to lactose attachment on investigated peptides, it is very challenging to choose the right CE for each transition. Thus, three different CEs were selected for each transition of these a-La and b-Lg lactosylated peptides and labelled ‘‘normal’’, ‘‘low’’ and ‘‘very low’’ CE (see Supplementary data 1 and 2). Normal and low CE values were calculated as a linear function of parent ion m/z ratios of lactosylated and unmodified peptides, respectively. Very low CE values were 25% lower than ‘‘low’’ CE values. The optimal CE and six best candidates of lactosylated peptides were selected. Each of best candidates were examined by a full scan MSMS to confirm the identity. Control peptides which were consistently observed in the spectra were used as internal standards for relative quantification. In order to test the linearity and reproducibility of the MRM assay, a Q1/Q3 list containing the six best candidates of lactosylated peptides and four control peptides of a-La and b-Lg was set up in an MRM mode. The WPC80 (stored at 30 °C and 84% RH for 4 weeks) was reconstituted with water to produce a solution containing 1 mg protein/mL and digested with trypsin. The digests were diluted with 1% formic acid in the ratios 1:3, 1:1, and 3:1. Thus, the relative concentration (based on initial protein content) was 0.25, 0.5, 0.75 and 1 mg/ml. The peak area of each transition was calculated using Analyst 1.5 (ABSciex) and plotted against the relative concentration of the initial protein. Experiment 2: The optimised MRM assay on WPC80 (mainly whey proteins) was applied to MPC80 (approximately 80% casein and 20% whey proteins) for lactosylation quantification. The MPC80 samples had been stored in a range of temperature (25–40 °C) and humidity (44–84% RH) conditions for 12 weeks. A few changes were made to improve the signal to noise ratio for MRM transitions of lactosylated peptides of the MPC80 samples. Lactosylation was normalised by multiplying peak areas of the chromatograms of selected MRM transitions by 1000 and dividing them by the corresponding peak areas of control peptides. The normalised lactosylation levels were correlated with furosine levels (Le, Bhandari, & Deeth, 2011) to test the applicability of the MRM assay.
3. Results and discussion 3.1. Experiment 1 3.1.1. MRM transition development Protein quantification by MRM involves many steps, but the selection of specific peptides and their fragments (MRM transitions) that uniquely identify lactosylated peptides is a key part. The neutral loss of 162 Da followed by the loss of three water
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molecules (total loss: 216 Da) that accounted for the formation of a furylium ion were specifically observed in the fragmentation of lactosylated peptides (Molle, Morgan, Bouhallab, & Leonil, 1998; Morgan, Leonil, Molle, & Bouhallab, 1997). Thus, the loss of 162 and 216 Da could be monitored for MRM transitions. To examine the fragmentation patterns of lactosylated peptides, WPC80 tryptic digests were subjected to a full MS scan by LC–MSMS. Lactosylated peptides were detected by the increase in mass of 324 Da compared to the unmodified peptides. Several lactosylated peptides were identified by LC–MSMS that matched with our previous results (Le, Deeth, Bhandari, Alewood, & Holland, 2011) and the study of Holland, Gupta, Deeth, and Alewood (2011) on lactosylated whey proteins detected by MALDI-TOF-MS. Figure 1A shows the MS/ MS result of the a-La peptide 134LDQWLCEKL142 with a lactose adduct on K141 and a mass of 1528.71. The peptide was detected as a doubly charged ion with m/z 764.86. Similarly, the b-Lg peptide 57 VYVEELKPTPEGDLEILLQK76 with a lactose adduct on K63 and a mass of 2637.36 detected as a triply charged ion with m/z 879.7 is shown in Fig. 1B. The patterns of the neutral losses of 162 and 216 Da were readily observed in both a-La and b-Lg lactosylated peptides. For example, it can be seen very clearly that the precursor ion with m/z 764.86 lost one hexose to form an ion with m/z 683.84. The appearance of ions with m/z 674.84, 665.83 and 656.35 was the consequence of three dehydrations to form furylium ion. A similar fragmentation pattern was observed for the precursor ion with m/ z 879.79 of lactosylated b-Lg. However, the signal intensity of [M 216]3+ ion was very low as the collision energy was not optimised. Overall, Q1/Q3 pairs were selected based on the knowledge of fragmentation of lactosylated peptides. Candidate peptides (i.e., Q1) were all theoretical peptides with an internal missed cleavage at a lysine residue and a mass 324 Da higher than the calculated mass from the peptide alone. Q1 was calculated as [M + z H]/z (M = mass of lactosylated peptides and z = number of charges). The neutral losses [M 162] and [M 216] were set as Q3; they
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were calculated as [M + z H 162 (or 216)]/z. The list of Q1/Q3 pairs included 20 transitions for a-La and 24 transitions for b-Lg (see Supplementary data 1 and 2). Those lactosylated peptides were previously detected by MALDI-TOF-MS by Holland et al. (2011) and Arena et al. (2010). 3.1.2. Optimisation of collision energy Ionisation and fragmentation are the two main factors affecting signal intensity of MRM transitions. In this study, we focused more on fragmentation conditions, particularly optimisation of collision energy (CE) for all MRM transitions listed in Supplementary data 1 and 2. Fig. 2 illustrates the effect of CE on the two MRM transitions (neutral losses) of the doubly charged ion at m/z 764.86. It can be clearly seen that there is considerable change in the total ion current (TIC) (shown in the left hand column) and the extracted ion chromatogram (XIC) (shown in the right hand column) of MRMIDA experiments operated at three different CE values. At a ‘‘normal’’ CE (42.77 eV), the loss of 216 Da (formation of furylium ion) which is represented as a red peak Fig. 2B was more prominent than the loss of 162 (cleavage of one hexose) (blue peak) (Fig. 2A). Reducing the CE to 34.35 eV (‘‘low’’ value) increased the loss of 162 and decreased the loss of 216 (Fig. 2B). A further decrease in CE to 25.76 eV (‘‘very low’’ CE) leads to an increase in signal intensity of [M162]2+ ion (Fig. 2C). From the study of Gadgil, Bondarenko, Treuheit, and Ren (2007) on glycated peptides, the parent ions were dissociated differently at different collision energies. The maximum neutral loss of the sugar moiety (162 Da) of glycated peptides has been reported at a collision energy of 20 eV (Gadgil et al., 2007). Morgan et al. (1997) confirmed that the sufficient CE for fragmentation of lactosylated peptides to form furylium ions was 30 eV. These values were variable for different peptides and their charge states. Hence, the CE that gave reliable and reproducible signals for both transitions could be selected as the optimal CE. For this doubly charged ion, ‘‘very low’’ CE gave a better signal intensity for [M 162]2+ than [M 216]2+ ion; this means the collision energy
Fig. 1. Product-ion tandem MS of the doubly and triply charged precursor ions at m/z (A) 764.86 (full scan); (B) 879.79 (full scan); (C) 764.86 (zoomed in); (D) 879.79 (zoomed in) of a-La and b-Lg, respectively. Two major losses of the ions: the loss of 162 Da and subsequent loss of 3H2O to form furylium ions were labelled.
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Fig. 2. Effects of CE on signal intensity. WPC80 stored at 30 °C and 84% RH for 4 weeks, digested with trypsin and analysed by MRM. TIC (left hand column) and XIC (right hand column) of precursor ion with m/z at 764.86 using (A and B) normal; (C and D) low; (E and F) very low CE.
was sufficient for the cleavage of hexose and the formation of furylium ion; but not too strong to form other unnecessarily smaller fragments. Different optimal CEs were obtained for 9 and 12 other MRM transitions of a-Lac and b-Lg lactosylated peptides, respectively.
3.1.3. Selection of best candidates for Q1/Q3 list The best MRM candidates were selected based on signal intensities of both transitions from XIC. The ions with m/z 764.86 (shown in Fig. 2C), 535.28 and 665.35 (see Supplementary data 3B and 3E) produced a single peak with reproducible signal intensities for both transitions (losses of 162 and 216 Da). These ions were eluted at different times, approximately 6.67, 21.89 and 20.21 min, respectively. However, a number of ions appeared with multiple peaks (see Supplementary data 3); consequently, they were not considered for quantitative purposes. That does not mean lactosylated peptides were not present in the samples. The specific signals from these peptides could not be resolved reliably from the other MS signals. Thus, m/z 535.28, 665.35 and 764.86 were chosen as the best candidates for MRM quantification of a-Lac. Similarly, only three out of 12 transitions were selected as the best MRM candidates for b-Lg; the remaining ions were either too weak or appeared as multiple peaks. The three ions were those with m/z 491.94, 638.01 and 879.79 corresponding to elution times at
14.39, 19.77 and 26.34 min, respectively. As a result, the Q1/Q3 list was shortened to six lactosylated peptides including 12 transitions in total for a-Lac and b-Lg (see highlights in grey in Supplementary 1 and 2).
3.1.4. Validation of transitions The best MRM candidates in this study were acquired by MS/MS in order to confirm that the detected signals derive from the targeted peptide. The MS/MS spectra of two out of three best candidate ions from each lactosylated a-La and b-Lg was shown in Supplementary data 4. They included precursor ions with m/z 665.35, 764.86 (from a-La), 491.94 and 638.01 (from b-Lg). The lactosylation sites of these peptides have been confirmed by MALDI-TOF-MS (Holland et al., 2011). From the MS/MS spectra of the precursor ion with m/z 764.86 (see Supplementary data 4B), a loss of 162 Da was observed at m/z 684.3 and the appearance of m/z 657.5 indicated the subsequent loss of three H2O from the [M-162]2+ ion to form a furylium ion. Including the fact that b3 and b4 fragmentation ions were detected at m/z 357.2 and 543.5, this confirmed the identity of this candidate peptide 134LDQWLCEKL142. Although the loss of galactose ion was not clearly shown in the MS/MS spectra of the ion with m/z 665.35 (see Supplementary data 4A), a series of y ions (i.e., y3, y4, y5 and y6) and the formation of furylium ion were enough to confirm the identity of the peptide 114ILDKVGINYWLAHK127. Similar results
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were obtained for precursors ions with m/z 491.94 and 638.01 from b-Lg (see Supplementary data 4C and 4D). Again, the loss of galactose, formation of furylium and a series of y ions (i.e., y1–y5) identified the candidate ion with m/z 491.94 from the lactosylated peptide of b-Lg (see Supplementary data 4C). The presence of ions [M 162], [y12-216], b2 and y12 sufficiently confirmed the identity of the ion at m/z 638.01. Thus, the combination of backbone fragments (e.g., mixture of b and y ions) and sugar loss enabled the identity of the candidate lactosylated peptides to be confirmed. 3.1.5. Selection of control peptides One of the challenges of the MRM method is variation in signal intensities from LC/MS analyses. This could be due to differences in efficiency of the digestion, peptide recovery and/or injection volume. As MRM is used to quantify the amount of specific fragments produced from tryptic digests of lactosylated proteins, it is very important to have control peptides. These peptides could also be considered as internal standards; therefore, they can be used to normalise signal intensities of lactosylated peptides for relative quantification. The control peptides included two a-La peptides 134 LDQWLCEK141 and 82DDQNPHSSNICNISCDK98 with m/z 546.27 and 668.63, respectively; and 2 b-Lg peptides 108VLVLDTDYKK117 and 165LSFNPTQLEEQCHI178 with m/z 597.36 and 858.78 (see Supplementary data 5). These peptides were chosen because they were observed consistently in the spectra of both control and stored samples. The control peptides were subjected to MS/MS in order to select the best fragments for MRM transitions. Hence, the two prominent y-ions of each peptide were chosen for Q3. For example, the y5 and y7 fragments were selected from the fragmentation of peptide sequence 134LDQWLCEK141 from a-La (see Supplementary data 5B), so the Q1/Q3 pairs were 546.3/735.4 and 546.3/978.5. Similarly, the y4 and y6 ions were selected for 82DDQNPHSSNICNISCDK98 from a-La (see Supplementary data 5D), so the Q1/Q3 pairs were 668.6/509.2 and 668.6/736.3. For 108VLVLDTDYKK117 from b-Lg (see Supplementary data 5C) the y6 and y8 fragments were selected so the Q1/Q3 pairs were 597.4/769.4 and 597.4/ 981.5. For 165LSFNPTQLEEQCHI178 from b-Lg (see Supplementary data 5E) the y7 and y10 ions were selected so the Q1/Q3 pairs were 858.8/928.4 and 858.8/1254.6. 3.1.6. Linearity and reproducibility of the selected transitions Figs. 3 and 4 demonstrate responses of 20 MRM transitions from candidate peptides in addition to control peptides. The a-La peptide 114ILDKVGINYWLAHK127 could be detected as a triply or quadruply charged ion. The signal-to-noise ratio of the XIC of the triply charged ion (m/z 665.35) was relatively low. Hence, the ion with m/z 449.27 (a quadruply charged ion of the peptide) was included in the MRM list. Generally, only 16 out of 20 MRM transitions showed a reasonably linear response with r2 ranging between 0.89 and 0.97. The results of four other MRM transitions on the Q1/Q3 list 499.27/458.75, 499.27/445.25 (Fig. 3B), 879.79/ 825.78 and 879.79/807.76 (Fig. 4D) varied considerably between samples. MRM transitions of control peptides (Figs. 3A and 4A) had a good reproducibility due to their high signal intensity from the mass spectra. Although the precursor ion with m/z 665.35 (from a-La) (Fig. 3C) and 491.94 (from b-Lg) (Fig. 4B) showed variation between samples, the linearity and reproducibility of precursor ions with m/z 764.86 (from a-Lac) (Fig. 3D) and 638.01 (from b-Lg) (Fig. 4C) were better. The considerable variability of the lactosylated peptides with m/z 665.35 and 491.94 could be due to the lower abundance of these peptides resulting in relatively low signal-to-noise ratio of the extracted ions. From the demonstration of linearity of lactosylation measurement on WPC80, the MRM list was shortened to two control peptides (four transitions) and two lactosylated peptides (four transitions) for
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each whey protein. The peptides with m/z 499.27 and 879.79 were excluded from the final list. 3.2. Experiment 2 3.2.1. MRM quantification of stored MPC80 The lower abundance of whey proteins in MPC80 compared to that in WPC80 could lead to the low signal-to-noise ratio of the transitions in MPC80 samples. Moreover, the presence of abundant non-target peptides obtained from casein could result in the generation of a broad range of fragment ions that could interfere with those from the targeted peptides. Thus, dropping an EPI scan and shortening the MRM list (one control and two lactosylated peptides for each protein) allowed the dwell time to be increased from 50 to 80 ms/transition, while one data point was still acquired every second across the elution profile. In addition, by scheduling MRM transitions, the full cycle time is deployed to detect the peptides expected to elute in a given time window. In this study, 12 transitions were split into two periods. The first period was set up from 1 to 16.5 min. The second period started from 16.5 to 42 min. Therefore, the first period includes precursor ions with m/z 491.94 (from lactosylated b-Lg), 546.38 and 597.34 (control peptides of a-La and b-Lg, respectively). The second period involved precursor ions with m/z 638.01 (from lactosylated b-Lg), 665.35 and 764.86 (from lactosylated a-La). Six transitions were scanned in the full cycle time (little over 1 s). This allowed the dwell time to be increase to 160 ms/transition, resulting in an increase in signal-to-noise ratio. These modifications were incorporated into the assay and it was re-applied to MPC80 samples. Fig. 5 shows the normalised lactosylation levels of precursor ions with m/z 665.35 and 764.86 from a-La in MPC80 stored at 30 °C, 44% and 84% RH. There was a considerable increase in normalised lactosylation levels of these transitions with increasing RH values. The time effect on lactosylation was clearly seen in all transitions; however, the loss of 162 of precursor ion with m/z 665.35 slightly increased (Figs. 5A and B). This could be due to low signal-to-noise ratio of the transition which leads to insignificant changes between MPC80 samples stored at different times. Similarly, the effects of humidity and time on the normalised lactosylation level obtained by MRM were observed on both precursor ions with m/z at 491.94 and 638.01 from b-Lg (see Supplementary data 6); however, the loss of 162 of m/z 491.94 was not considerably changed with time. This could be explained by a low signalto-noise ratio of the transition. The normalised lactosylation levels in both a-La and b-Lg seem to reach a plateau at 12 weeks of storage for samples stored at 84% RH. 3.2.2. Correlation with furosine content Normalised lactosylation levels were correlated with furosine levels to test the applicability of the MRM assay. Fig. 6 shows a good linear response (r2 = 0.98–0.99) of precursor ion with m/z 764.86 at two transitions (the loss of 162 and 216 Da). The precursor ion with m/z 764.86 also gave higher signal-to-noise ratio compared to the ion with m/z 665.35 which only showed a reasonable linear response (r2 = 0.89–0.92). For b-Lg, the precursor ions with m/z 638.01 and m/z 491.94 showed good correlations (r2 = 0.950.99) with furosine content, at both transitions (see Supplementary data 7). The variation in slope of the lines reflects the differences in ionisation and fragmentation efficiency of the peptides. These differences would be overcome by using matched isotopically-labelled internal standards for normalisation as they should have the same ionisation and fragmentation behaviour as the peptides of interest. As mentioned in the Introduction, the Amadori products have mostly been quantified by indirect ways (e.g., furosine). This hydrolysed compound cannot be considered as the absolute
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Fig. 3. Demonstration of linearity of lactosylation measurement. Peak areas of extracted ions from 10 MRM transitions of a-La were calculated by using Analyst 1.5 quantification module software. (A) d, m/z 546.38/735.35; s, 546.38/978.43; ., 668.61/509.20; D, 668.61/736.33; (B) d, 499.27/458.75; s, 499.27/445.25; (C) d, 665.35/ 611.34; s, 665.35/593.33; (D) d, 764.86/683.83; s 764.86/656.82.
Fig. 4. Demonstration of linearity of lactosylation measurement. Peak areas of extracted ions from 10 MRM transitions of b-Lg were calculated by using Analyst 1.5 quantification module software. (A) d, m/z 597.34/769.37; s, 597.34/981.53; ., 858.41/928.42; D, 858.41/1254.58; (B) d, 491.94/437.92; s, 491.94/419.91; (C) d, 638.01/ 583.99; s, 638.01/565.98; (D) d, 879.79/825.78; s 879.79/807.76.
concentration of the Amadori products. The MS approaches such as LC–ESI-MS and MALDI-TOF-MS allows direct detection of lactulosyllysine (Losito et al., 2007; Meltretter et al., 2009). The advantage of this MS analysis of lactosylation is minimal sample workup, without protein hydrolysis resulting in minimal artifact formation and fast analysis. In fact, MALDI-TOF-MS is even faster and easier than LC–ESI-MS as the milk sample can be directly loaded onto the MALDI target for MS analysis without chromatography and other sample preparation steps (e.g., protein extraction or tryptic digestion). It was first optimized for relative quantifica-
tion of lactosylation in milk samples by Meltretter et al. (2009). That study failed to quantify lactosylated whey proteins in severely heated samples (e.g., condensed milk) as whey protein are largely aggregated. However, LC–ESI-MS was shown to be a sensitive method for detecting and quantifying lactosylated whey proteins in pasteurised milk with a low degree of lactosylation. The method has not been applied on severely heated samples. Since, under high temperature of processing and storage, milk proteins can undergo many modifications such as denaturation and lactosylation, these changes could affect multiple extracted ion chromatograms of both
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Fig. 5. MRM quantification of lactosylated peptides from a-La in stored MPC80. Normalised lactosylation levels were calculated from the peak areas of two selected precursor ions including loss of 162 (d) and 216 Da (s). (A and B) m/z 665.35 from a-La of MPC80 stored at 30 °C, (A) 44% and (B) 84% RH; (C and D) 764.86 from a-La of MPC80 stored at 30 °C, (C) 44% and (D) 84% RH.
Fig. 6. Correlation of normalised lactosylation level (from MRM results) with furosine content (Le, Bhandari, et al., 2011; Le, Deeth, et al., 2001). MPC80 samples were stored at different temperatures and humidities before tryptic digestion for MRM analysis: (A) 25 °C, 44% RH; (B) 25 °C, 84% RH; (C) 30 °C, 44% RH; (D) 30 °C, 84% RH. Two selected precursor ions from a-La with m/z 665.35 with the loss of 162 Da (d) and 216 Da (s); and m/z 764.86 with the loss of 162 (.) and 216 Da (D).
native or lactosylated forms of proteins. Thus, the MRM assay used in this study could become a useful method for a direct quantification of lactosylation in low-, medium- and high-heat-treated milk samples. In addition to the advantages of MS-based quantification methods (as listed above), there is less interference of structural modifications of proteins as only specific fragment ions of targeted lactosylated peptides are detected and quantified. However, the method requires further optimisation to become an applicable method for assessing the quality of dairy products. To assess the quantitative performance of the MRM assay, the
limits of detection and quantification (LOD and LOQ, respectively) should be determined using an internal standard. Aliquots of isotopically labelled lactosylated-peptides can be spiked into the tryptic digests of stored MPC80 samples at a range of concentrations, allowing determination of LOD and LOQ. 4. Conclusion Lactosylation in stored MPC80 can be quantified by the MRM assay. A good correlation between normalised lactosylation levels
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(obtained by MRM) and furosine content proves the feasibility of the MRM assay for quantification of protein changes during storage of milk powders. The approach of MRM in quantification of lactosylation could lead to a new method of detecting and quantifying other modifications of milk protein such as phosphorylation, deamidation, lactosylated casein and protein cross-linking. However, in order to replace traditional methods of quantifying lactosylation, an internal standard needs to be used to obtain absolute quantification of lactose adducts by MRM. Acknowledgements We thank Alun Jones from IMB Mass Spectrometry Facility for expert technical assistance and Dairy Innovation Australia Ltd. and the Australian Research Council for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2013. 03.073. References Arena, S., Renzone, G., Novi, G., Paffetti, A., Bernardini, G., Santucci, A., et al. (2010). Modern proteomic methodologies for the characterization of lactosylation protein targets in milk. Proteomics, 10, 3414–3434. Cox, D. M., Zhong, F., Du, M., Duchoslav, E., Sakuma, T., & McDermott, J. C. (2005). Multiple reaction monitoring as a method for identifying protein posttranslational modifications. Journal of Biomolecular Techniques, 16, 83–90.
Erbersdobler, H. F., & Somoza, V. (2007). Forty years of furosine-forty years of using Maillard reaction products as indicators of the nutritional quality of foods. Molecular Nutrition and Food Research, 51, 423–430. Gadgil, H. S., Bondarenko, P. V., Treuheit, M. J., & Ren, D. (2007). Screening and sequencing of glycated proteins by neutral loss scan LC/MS/MS method. Analytical Chemistry, 79, 5991–5999. Holland, J. W., Gupta, R., Deeth, H. C., & Alewood, P. F. (2011). Proteomic analysis of temperature-dependent changes in stored UHT milk. Journal of Agricultural and Food Chemistry, 59, 1837–1846. Krause, R., Knoll, K., & Henle, T. (2003). Studies on the formation of furosine and pyridosine during acid hydrolysis of different Amadori products of lysine. European Food Research and Technology, 216, 277–283. Le, T. T., Bhandari, B. R., & Deeth, H. C. (2011). Chemical and physical changes of milk protein concentrate (MPC80) powder during storage. Journal of Agricultural and Food Chemistry, 59, 5465–5473. Le, T. T., Deeth, H. C., Bhandari, B., Alewood, P. F., & Holland, J. W. (2011). A proteomic approach to detect lactosylation and other chemical changes in milk protein concentrate. Food Chemistry, 132, 655–662. Losito, I., Carbonara, T., Monaci, L., & Palmisano, F. (2007). Evaluation of the thermal history of bovine milk from the lactosylation of whey proteins: An investigation by liquid chromatography–electrospray ionization mass spectrometry. Analytical and Bioanalytical Chemistry, 389, 2065–2074. Meltretter, J., Birlouez Aragon, I., Becker, C., & Pischetsrieder, M. (2009). Assessment of heat treatment of dairy products by MALDI TOF MS. Molecular Nutrition & Food Research, 53, 1487–1495. Molle, D., Morgan, F., Bouhallab, S., & Leonil, J. (1998). Selective detection of lactolated peptides in hydrolysates by liquid chromatography/electrospray tandem mass spectrometry. Analytical Biochemistry, 259, 152–161. Morgan, F., Leonil, J., Molle, D., & Bouhallab, S. (1997). Nonenzymatic lactosylation of bovine beta-lactoglobulin under mild heat treatment leads to structural heterogeneity of the glycoforms. Biochemical and Biophysical Research Communications, 236, 413–417. Thomas, M. E. C., Scher, J., Desobry-Banon, S., & Desobry, S. (2004). Milk powders ageing: Effect on physical and functional properties. Critical Reviews in Food Science and Nutrition, 44, 297–322.