Peptidomics Approaches for Authentication of Pork and Water Buffalo Meat Employing Rapid In-solution Fractionation and 2-DE

doi:10.21203/rs.3.rs-2008533/v1

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Peptidomics Approaches for Authentication of Pork and Water Buffalo Meat Employing Rapid In-solution Fractionation and 2-DE

https://doi.org/10.21203/rs.3.rs-2008533/v1

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A rapid and highly reproducible gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) coupled with MALDI-TOF mass spectrometry is explored for the first time for authenticating adulteration of pork with water buffalo meat. The GELFrEE offers novel in-solution protein fractionation with a dynamic range from 3.5–500 kDa and the specificity sufficient to be implemented effectively for the identification of species-specific peptides derived from myoglobin (‘HPGDFGADAQGAMSK’, ‘HPSDFGADAQAAMSK’) and carbonic anhydrase-3 (‘GGPLTAAYR’, ‘GGPLAAPYR’) demonstrating their potential as heat-stable markers for meat authentication including commercial samples. The suitability of 2-dimensional gel electrophoresis (2-DE) coupled with MALDI-TOF MS for efficiently detecting myosin light chain isoforms from contaminating pork in buffalo meat is also demonstrated. The GELFrEE and gel-based 2-DE approaches efficiently authenticate raw and heat-processed meat and meat mixes up to 0.5% (w/w) level; however, quick extraction protocol and rapid fractionation by GELFrEE offer better solutions for routine MS-based approaches to address global meat fraud issues.

GELFrEE

In-solution fractionation

2-dimensional gel electrophoresis MALDI-TOF MS

Meat authentication

Myoglobin

Carbonic anhydrase-3

Food fraud is becoming a global challenge which is estimated to cost $40–50 billion per year (FDA 2021). An increase in international trade with a complex global food supply chain, the nature of animal-derived food products and emerging food frauds, pose challenging tasks for their detection and mitigation. India is a largest producer and exporter of frozen, halal water buffalo meat and it is traded in more than 50 countries mainly to Middle-east and Gulf Nations (APEDA 2022). Authentication of halal meat is a global issue not only for its exclusive religious obligations but also depicts the standard of choice from a non-Muslim perspective. Therefore, development of rapid, sensitive and high-throughput approaches for routine laboratory application for detection buffalo meat adulterated with pork will ensure the integrity of supply chain.

In the last few decades, several techniques for meat authentication have been established targeting proteins, lipids and nucleic acids as biomarkers (Ali et al. 2012). Standard proteomic approaches identify thousands of proteins in complex biological samples, whereas peptidomics provide a deeper insight into the protein sample composition due to the fast analysis of low molecular mass protein fragments at the peptide sequence level. Hence, peptidomic method based on mass spectrometry (MS) combined with bioinformatics tools targeting the specific biomarkers can be developed and validated for assessment of food adulteration and deceptive practices (Gallardo et al. 2013), facilitating the food authentication process. Regardless of the MS strategy, researchers have attempted to develop simple extraction, fractionation, and detection methods, which possess higher resolution to separate many proteins with sufficient sensitivity and dynamic range to detect even low abundance proteins. The 2-dimensional gel electrophoresis (2-DE) is a powerful tool for fractionating individual protein, protein isoforms, or complex protein samples (Jafari et al. 2012). However, the limitations of this in-gel separation method include limited sample loading capacity (max. 1.0 mg/channel), a low dynamic range of separation hindering the detection of low-abundant proteins, and poor chances of automation (Albright et al. 2009). Solution-based separations offer an alternative to the classic gel-based approach for protein fractionation, which is much easier to automate, making them less labor-intensive. The gel-eluted liquid fraction entrapment electrophoresis (GELFrEE) technique fractionates the complex protein samples utilizing their molecular weight in a miniature-gel column and elutes the fractions into solution (Masuoka et al. 1998; Tran and Doucette 2008). Conceptually, GELFrEE is analogous to that of SDS-PAGE but with the collection of protein fractions in the solution phase at the end of the run. The ability to load a relatively large volume of sample (max. 5.0 mg/strip) in GELFrEE yields impressive proteome coverage and is especially valuable during the analysis of low-abundance proteins, which have extremely important functions in various biological processes (Maheswarappa et al. 2021; Banerjee et al. 2021).

Majority of MS methods rely on protein pattern profiling or on the identification of specific peptides or proteins for meat species authentication (Ohana et al. 2016). Many peptides present in raw meat may be destroyed during high-temperature processing, failing to play the role of heat-stable meat marker (Wang et al. 2018). The species-specific electrophoretic mobility of myosin light chain (MLC) isoforms was utilized by Montowska and Pospiech (2012) to differentiate processed meat from cattle, pig, chicken, turkey, duck, and goose using 2-DE and MALDI-TOF MS. An alternative in-solution strategy using OFFGEL electrophoresis, which separates proteins in liquid form according to their isoelectric pH was developed by Sentandreu et al. (2010). These researchers have demonstrated detection of as low as 1.0% w/w contaminating chicken in a pork meat mix using OFFGEL electrophoresis and detection of chicken-specific peptides generated from myosin light chain 3 (MLC-3) by liquid chromatography-tandem mass spectrometry (LC-MS/MS). However, OFFGEL electrophoresis requires a 24–36 h run time and the protein samples need to be purified prior to loading in order to remove the salts that may offer resistance while fractionating (Maheswarappa et al. 2021; Sentandreu et al. 2010)

The GELFrEE fractionation has been reported by a group of researchers mainly for the separation and characterization of microbial proteins and biological samples (Melani et al. 2016; Catherman et al. 2013; Orton et al. 2013; Kellie et al. 2010; Tran and Doucette 2008, 2009). However, the GELFrEE approach has been minimally explored in meat/food protein fractionation, and recently our laboratory has successfully employed GELFrEE as a quick and rapid pre-fractionation tool prior to MS characterization of total and myofibrillar/sarcoplasmic proteins from water buffalo meat, chicken and pork under raw and heat-processed conditions (Maheswarappa et al. 2021). Our earlier study has encouraged us to explore the potential of GELFrEE for species authentication in complex biological samples like raw and heat-processed meat, and binary meat mixes. Considering the increased demand for a simple extraction and fractionation methods, here, we present for the first time, a unique peptidomics-based strategy involving simple and rapid protein extraction, fractionation of total proteins from water buffalo meat, pork, meat mixes and commercial samples employing GELFrEE and 2-DE, followed by identification of heat-stable, species-specific peptides using MALDI-TOF MS.

Materials

Chemicals associated with 2-dimensional gel electrophoresis and SDS-PAGE were procured from GE Healthcare Life Sciences (Uppsala, Sweden). Modified trypsin (sequencing grade) was purchased from AMRESCO (OH 44139, USA), and the α-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid were sourced from Sigma Aldrich (St. Louis, MO, USA). The GELFrEE 8100 cartridge kits were obtained from Expedeon Ltd. (25 Norman Way, Over, Cambridge CB24 5QE, United Kingdom). All other reagents (salts, acids, alkalis, and organic solvents) were of analytical and chemical purity.

Authentic water buffalo (Bubalus bubalis) meat and pork (Sus scrofa) samples from freshly slaughtered animals were collected hygienically from Municipal slaughterhouse and retail meat shops in Hyderabad, India, and transported to Meat Proteomics laboratory under chilled conditions. The samples were deboned, cleaned for visible fat and connective tissue, divided into aliquots of 500 g each, and stored at -80 °C until use. Ground buffalo meat was substituted for ground pork (w/w) at 100:0, 90:10, 95:5, 99:1, 99.5:0.5 and 0:100 (buffalo: pork). Cooking of meat was carried out in a sealed low-density polyethylene (LDPE) bag, using a boiling water bath at 100 °C for 30 min followed by cooling in ice. The mixed samples were made into 3 aliquots for raw and cooked meat. Similarly, 100 % buffalo meat and 100% pork aliquots were also prepared.

Commercial meat products (pork pepperoni, ham, bacon, sausages, corned buffalo meat) and in-house processed meat products (burger patties, nuggets, sausages) (n = 10) were purchased at supermarkets or manufactured in our own pilot plant. In-house processed products were prepared in a pilot plant of the Indian Council of Agricultural Research-National Research Centre on Meat (India) exclusively from buffalo meat with the addition of spices and condiments and were coarsely minced or emulsified, and cooked. All samples were kept at -80 °C until further analysis.

Protein Extraction and GELFrEE Fractionation

Protein extraction from 200 mg of minced meat was performed using 4 mL of the standardized buffer containing 0.01 M Phosphate buffer (pH 7.4), 0.9% NaCl, and 1% SDS (Tab. S1) by trituration using a pestle and mortar followed by filtration of the extract with Grade 1 filter paper. The GELFrEE 8100 fractionation system consists of a benchtop fractionation instrument and a single-use 8-sample capacity cartridge with four main components viz., a cathode chamber, a specially designed gel column, a collection chamber, and an anode chamber. Gel measures 2.5 cm in length (including stacking and resolving gel) and 8 mm in outer diameter. The 112 µL of protein extract without any purification (~5.0 mg) was mixed with 30 µL tris-acetate sample buffer, 8 µL 1M DTT, and deionized water to make a total volume of 150 µL of the sample. Samples were heated at 95 °C for 5 min prior to loading. Before loading the sample, the storage buffer was removed from the respective channels depending on the number of samples. The anode and cathode reservoir chambers were loaded with 8 mL and 6 mL GELFrEE running buffer (HEPES running buffer), respectively, and 100 µL of running buffer was also added to the collection chamber. Immediately, 150 µL of the sample was loaded onto the sample loading chamber of the cartridge carefully to avoid the formation of bubbles. The cartridge was positioned on the instrument, electrodes were fixed and the lid was closed. In the current study, we used the 5% cartridge with a fractionation range of 3.5-500 kDa. The protocol for fractionation of the sample was selected according to the kit utilized for the study and was edited using central navigation and operating screen depending on the cartridge with different voltage, current, and fraction intervals (Tab. S2). The instrument was paused after each step allowing to collect the fraction from the collection chamber and refilling with 100 µL of running buffer. The cathode reservoir was filled with running buffer to a total volume of 8 mL after the first step was completed. A total of 12 fractions were collected with a complete run time of 196 min.

Two-dimensional Gel Electrophoresis

For two-dimensional gel electrophoresis (2-DE), the sample was extracted from raw and heat-processed meat mixes as per the procedure of Montowska and Pospiech (2013) by homogenizing 100 mg of meat sample with 1 mL of extraction buffer (7 M Urea, 2 M Thiourea, 4% w/v CHAPS, 2% Carrier ampholyte pH 4–7, 40 mM DTT and Protease inhibitor) for 2 x 30 s of 4000 cycles per minute. The extracts were centrifuged, and the supernatant was subjected to TCA/acetone precipitation resulting in a pellet which is finally dissolved in rehydration buffer (7M Urea, 2M Thiourea, and 2%w/v CHAPS). The concentration of extracted protein was performed using the GeNie^TM Protein estimation kit by the BCA method (GeNieTM, Mumbai). Approximately 200 μg of protein was smeared on a 13 cm IPG strip, 3-10 pH (Immobilin^TM Drystrip, GE Health care, Uppsala, Sweden) and subjected to passive rehydration (typically 12-16 h) at room temperature followed by focussing using an Ettan IPGphor 3 System (GE Healthcare, Bio-Sciences). The focused IPG strips were equilibrated using equilibration buffers I and II for 15 minutes each, and proteins were separated in the second dimension (10% SDS PAGE gel), with the Ettan Dalt Six Gel Electrophoresis Unit (GE Healthcare, Uppsala, Sweden) at 100 V with 60 mA/gel until tracking dye reached the lower end of the gel. The gels were produced in triplicates and stained using silver staining. Images were scanned using Image Scanner III, Labscan 6.0 software (GE Healthcare, Bio-Sciences), and analyzed by using ImageMaster2D Platinum 7.0 software (GE Healthcare, Uppsala, Sweeden).

Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis

The SDS-PAGE of GELFrEE fractions was carried out using the method of Laemmli(1970) with midi-electrophoresis apparatus (GE Healthcare, Uppsala, Sweeden, Model: SE-600 Ruby). About 20 μL of GELFrEE protein fractions were mixed with 5 μL of 5X gel loading buffer and heated at 95 °C for 5 min followed by loading of 20 μL sample onto individual lanes of a 12% gel. Electrophoresis was performed at a constant voltage mode of 100 V per slab at 60 mA for 7-8 h. The gel was removed and stained with Coomassie blue for 4-5 h followed by overnight destaining.

In-gel Digestion of Proteins

Gel plugs were washed with Milli-Q water for 5-7 min at room temperature. One mL of destaining solution (25 mM ammonium bicarbonate in acetonitrile) is added to each gel plug and kept for destaining at 24 °C, followed by vortexing at 600 rpm by using Thermomixer vortexer. After proper destaining (3-4 h), the destaining solution was removed, and gel plugs were washed with 100% acetonitrile for 10 min at 24 °C. About 50-100 µL (according to the gel plug size) of reduction solution (10mM Dithiothreitol in 25mM Ammonium bicarbonate solution) was added to the gel plugs and incubated at 56 °C for 1 h. The gel plugs containing tubes were centrifuged for 1 min to settle down the solution, and the reduction solution was removed. About 50-100 µL (according to the gel plug size) of 55 mM Iodoacetamide in 25 mM Ammonium bicarbonate solution was added to the gel plugs and incubated at 24 °C for 45 min. The gel plugs were again washed with 100% acetonitrile for 10 min at 24 °C. Trypsin solution (20-25 ng/µL) was added to each gel plug according to the gel plug volume (10 -25 µl) and kept on ice for 30 min. Gel plugs were incubated at 37 °C for 16h by using a Thermomixer vortexer. After incubation, gel plugs containing tubes were centrifuged for 5 min to settle down the solution. This solution (containing peptides) from each tube was collected in the same corresponding tube. Extraction solution containing 1% trifluoroacetic acid (TFA) in 50% acetonitrile was added to each gel plug (15-25 µL) and kept for 30 min at room temperature with 800 rpm vortexing. The solution was collected and added to the same corresponding tubes. This extraction step was repeated 3 times to collect most of the peptides. Peptides were lyophilized by using a speed vacuum concentrator.

MALDI-TOF/TOF MS Analysis

Lyophilized peptides were dissolved in 5 µl of 1% TFA in 50% acetonitrile solution. An equal volume (1.5 µL) of peptide solution and matrix solution (α-cyano 4- hydroxycinnamic acid) were mixed properly and spotted on the MALDI target plate (MTP AnchorTM chip 384 well plate, Bruker Daltonics, Germany). Spots were allowed to dry completely, and the external peptide calibration standard I (Bruker Daltonics, Germany) was also spotted along with the samples in the calibration spot on the plate. The samples were analyzed in linear, positive ion mode within a mass range of 500 Daltons to 4000 Daltons on the MALDI TOF/TOF ULTRAFLEX III instrument (Bruker Daltonics, Bremen, Germany). An accelerating voltage of 25 kV, 25% laser power with 200 spectra per second speed was used. After laser firing, the peptides were captured and detected in the form of spectra with m/z values on the x-axis and intensity on the y-axis. The resulting spectra (from 600 laser shots) were averaged, noise smoothed, baseline-corrected, and further analysis was done with FLEX ANALYSIS SOFTWARE 3.3 version (Bruker Daltonics, Germany) to extract and process the peptide mass peaks from the spectrum. The post-analysis processed spectrum was used in protein identification.

Protein Identification

The measured and calibrated tryptic peptide masses were transferred through MS BioTool (Version 3.2, Bruker Daltonics) as inputs to search against the non-redundant Swiss Prot database using the MASCOT 2.2 (Matrix Science) search engine. Search parameters were as follows: Taxonomy: Mammalia; Trypsin cleavage; allow up to one missed cleavage; peptide mass tolerance is set according to the obtained m/z values; fixed modification: carbamidomethyl (C); variable modification: oxidation (M). Protein identifications were accepted if they had a greater than 95% probability, as represented by the mascot scores on the mascot result page. The protein hit with the highest score was selected to get the sequence that gives information about the protein sequence coverage. Sequence similarity search was done using NCBI BLAST, which compares a protein query to a protein database. BLAST hits with significant sequence alignments are obtained that gives all information about the protein.

Sequence Alignment

The NCBI database (http://www.ncbi.nlm.nih.gov) was searched for amino acid sequences of selected proteins. The alignment of known sequences was constructed using the Clustal Omega program, which calculates the best match for the selected sequences and lines them up so that identities, similarities, and differences can be seen. A pairwise score for each pair of sequences to be aligned is calculated as the number of identities in the best alignment divided by the number of residues compared, and gap positions are excluded (http://www.ebi.ac.uk/Tools/msa/clustalo).

The majority of proteomic-based meat authentication studies have used either 2-dimensional gel electrophoresis (2-DE)/OFFGEL fractionation coupled with mass spectrometry (MS) (Montowska and Pospiech 2012; Sentandreu et al. 2010) or the multiple reaction monitoring (MRM) MS method (Watson et al. 2015). However, considering the unknown food fraud threats emerging globally, rapid extraction and fractionation protocol coupled with MS was developed in the current study for routine laboratory application. Given the enormous complexity of meat proteins, their separation prior to MS is needed to produce fractionated proteins in solution for comprehensive analysis of proteome. The following section describes the simple extraction and novel GELFrEE fractionation technique and MS for authentication of pork mixed with water buffalo meat. The current study also evaluated the robustness of 2-DE, a gold standard protocol used in proteomic studies for authentication of meat species.

Simple Extraction Protocol, GELFrEE Fractionation, and SDS-PAGE Analysis

Choice of muscle protein extraction method is critical for obtaining samples with high protein concentration and free of salt and lipids, which could interfere with further analysis. Researchers have extensively reported extracting muscle proteins with denaturing agents containing urea, thiourea, reducing agents, detergents, and salts (Anderson et al. 2011; Bowker et al. 2008)using in-house laboratory facilities like tissue homogenizers, high-speed refrigerated centrifuge, and sonicator followed by purification of extracted proteins for further characterization. Considering the demand for simple, cost-effective, and quick extraction methods for routine laboratory use, protein extraction from meat samples utilizing simple trituration with the standardized buffer containing a diverse proportion of phosphate-buffered saline (PBS), SDS, and EDTA (Tab. S1, Fig. S1) using pestle and mortar followed by filtration with Grade 1 filter paper was optimized in this study. The 0.01 M phosphate buffer (pH 7.4), 0.9% NaCl, and 1% SDS was found optimal for extraction of proteins from both raw and cooked meats for further authentication.

SDS-PAGE coupled to MS was reported to detect a minimum of 10% w/w adulteration of pork with chicken and in order to detect the lower adulteration level (up to 0.5%) an additional proteins/peptides enrichment step was recommended (Sentandreu et al. 2010). To achieve this we used novel Gel-eluted liquid fraction entrapment electrophoresis, a commercial Expedeon’s GELFrEE 8100 unit and their proprietary cartridges. The GELFrEE fractionates the broad mass range of proteins on the basis of molecular weight with high resolution and with >90% recovery of proteins (Tran and Doucette 2008). To assess the feasibility of GELFrEE as an efficient enrichment step, we fractionated 100% buffalo meat, 100% pork and buffalo meat: pork meat mixes at different levels. Fig. 1A provides an overview of the selected fractions obtained following GELFrEE separation of raw (Fig. 1A, a) and heat-processed (Fig. 1A, b) buffalo meat proteins. Similarly, GELFrEE fractionation of raw and heat-processed pork proteins were represented in Fig. 2A. Proteins were fractionated using 5% GELFrEE cartridge with a fractionation range of 3.5 to 500 kDa. This cartridge with an in-built 2.5 cm length gel, separated the meat proteins within 196 min. The rapid separations described were achieved by employing high electric fields (3-11 mA) on short gel columns. The protein fractions from GELFrEE were subjected to separation on 12% SDS-PAGE to visualize the protein bands and to validate the fractionation. The average MW of proteins obtained in the present study displayed a definitive increase as the separation progressed. In the GELFrEE technique, small molecular proteins are eluted first from the gel and subsequently confined in the collection chamber. After the initial run, a similar migration rate for larger proteins was observed. As evident from Figs. 1A and 2A, protein fractions were collected in an approximate “linear” molecular weight (MW) profile and while collecting, proteins of the entire mass range remained focused and recovered in a single fraction with a consistently high yield. The unique entrapment strategy of GELFrEE offers sample enrichment as opposed to dilution, and through automatic pauses at predefined entrapment intervals (longer trapping times at high MW proteins), the probability of trapping given proteins in single fractions is maximized. Our findings also revealed that GELFrEE fractionation did not require any sample purification or removal of salt and this ensured the retaining of even minor or lower abundance proteins. Tran and Doucette (2008) have demonstrated 100% recovery of standard proteins (BSA, cytochrome c, and ubiquitin) from a short GELFrEE column and observed a similar elution pattern and separation with a high degree of reproducibility. In comparison to protein separation using SDS-PAGE, GELFrEE separation trap the proteins and later elute them into a few relatively simple fractions that can be analyzed by MS with greater sensitivity over the whole proteome. These proteins were partitioned according to their size, resulting in 12 clear fractions encompassing total buffalo meat and pork proteins that span the entire mass range in a 2-3 h collection window. For the 5% cartridge used in the current study, an apparent increase in the size of proteins is visualized in SDS-PAGE as the GELFrEE run progresses. A similar logarithmic trend was observed by Orton et al. (2013) in their study on characterizing secreted proteome of a wild-type and mutant strain enteropathogenic Escherichia coli using multiplexed GELFrEE platform.

In the current study, GELFrEE fractions were further subjected to SDS-PAGE in order to validate the linear elution of proteins based on their MW and for the selection of fraction numbers in which the potential marker proteins may be eluted. Separation of GELFrEE fractions for raw and heat-processed (Fig. 1A) meat extracts on SDS-PAGE exhibited elution of low MW proteins in fractions 2-3 and high MW proteins from factions 4-9. As highlighted in Fig. 1A, similar protein band separation pattern/intensity was observed for low MW proteins even after heat-processing. On the contrary, the band intensity corresponding to high MW proteins was reduced to a greater extent and some bands were completely missing after cooking. These results indicated that heat processing or cooking of meat over 100 °C deteriorated the intensity (decrease in intensity and absence of protein bands) of high MW proteins, whereas low MW proteins were more resistant to heat-induced denaturation and suffered minimal damage (Montowska and Spychaj 2018). These low MW proteins might be used as markers for further species identification, as demonstrated by Sentandreu et al. (2010).

Mass Spectrometry Analysis and Identification of Species-specific Peptide Biomarkers

As a logical first step to ensure the authentication of meat species, we identified the species-specific proteins/peptides using 100% water buffalo (Fig. 1) meat and 100% pork (Fig. 2) separately. In-gel digestion and MALDI-TOF MS of low molecular weight protein bands from selected GELFrEE fractions revealed the presence of myoglobin (Mb) and carbonic anhydrase-3 (CA-3) derived peptides (Table 1) that are specific to water buffalo and pork. Myoglobin and CA-3 have been reported as the main proteins of heat-stable peptide markers with high degree of species-specificity (Stachniuk et al. 2019). In the current study, we identified two water buffalo-specific peptides derived from myoglobin ‘TEAEMKASEDLK’ (M+H⁺=1351.78), ‘HPSDFGADAQAAMSK’ (M+H⁺=1532.67), and corresponding peptides from pork myoglobin ‘SEDEMKASEDLK’ (M+H⁺=1381.61), ‘HPGDFGADAQGAMSK’ (M+H⁺=1488.65). Similarly, two specific peptides derived from water buffalo CA-3 ‘GGPLAAPYR’ (M+H⁺= 901.48), ‘GEFQLLLDALDK’ (M+H⁺= 1361.73) and corresponding pork CA-3 peptides ‘GGPLTAAYR’ (M+H⁺= 905.48), ‘GEFQLVLDALDK’ (M+H⁺= 1347.71) were identified. These peptides were detected in both raw and heat-processed meat samples indicating their heat stability. Myoglobin requires less aggressive extraction procedures and Mb derived peptides are heat stable suggesting their suitability as markers for authentication of both raw and heat-processed meat (Taylor et al. 1993). The good heat stability of the ‘HPSDFGADAQAAMSK’ peptide detected in our study was also confirmed by Claydon et al. (2015). The CA-3 is the most abundant cytosolic protein and was reported to discriminate meat species from fresh meat and their mixtures Kim et al. (2017).

The Mb and CA-3 peptides exhibited differences in sequence between buffalo meat and pork, which can be detected by mass spectrometry analysis. The differences in peptide sequence between Mb and CA-3 of buffalo meat and pork were compared using Clustal O (1.2.4) multiple sequence alignment (Figs. S2 and S3). The percent identity score for pairs of CA-3 and Mb sequences from buffalo meat and pork was calculated using Clustal 1.2. It was revealed that the amino acid sequences of CA-3 and Mb between buffalo meat and pork differed by 7% and 12%, respectively. The species-specific peptides derived from CA-3 and Mb were further subjected to sequence scoring and validated through NCBI BLAST. The representative MS/MS spectra of selected marker peptides from buffalo meat carbonic-anhydrase-3 (‘GGPLAAPYR’) (Fig. 1C) and pork myoglobin (‘HPGDFGADAQGAMSK’) (Fig. 2C) were presented. The spectra under each chromatographic peak were analyzed, and the ratio was determined between molecular weight to molecular ions. Sequence score and NCBI BLAST were used to identify the species-specific peptides as signature peptides. All of the peptide markers were found to be present in one of the two species under investigation, but not in the other, according to the peptide detection analysis. In each of the chosen peptides, there was a relatively low error mass (1.0073 Da) between theoretical and observed m/z, indicating a good match percentage. Previously, the myoglobin peptide HPGDFGADAQGAMTK was used to identify horse meat in fresh meat (Watson et al. 2015).

Establishing Limit of Detection from Binary Meat Mixture

Identification of buffalo meat and pork-specific peptides has prompted us to detect contaminating pork in water buffalo meat at a proportion as low as 0.5%. Different proportions of ground pork (0.5%, 1.0%, 5.0%, and 10%) were mixed with ground buffalo meat, cooked at 100 °C for 30 min followed by extraction of proteins from raw and heat-processed samples, as described earlier using optimized buffer, and finally, protein fractionation was performed using GELFrEE electrophoresis. All the 12 fractions from GELFrEE electrophoresis were collected separately and characterized on 12% polyacrylamide gel. Selected protein bands from the 1st GELFrEE fraction (Fig. 3) were subjected to trypsin digestion followed by protein identification using MALDI-TOF MS analysis. Buffalo meat and pork-specific peptides, that were identified as potential biomarkers in 100% raw and cooked buffalo meat and pork were also detected in 0.5% pork adulterated with buffalo meat under raw as well as in cooked conditions. Our repeated experiments (n = 6) with raw and cooked buffalo meat and pork mix revealed the elution of species-specific peptides in GELFrEE fraction 1 that can be used for meat authentication. We could successfully detect (Fig. 3) both water buffalo-specific peptides (‘TEAEMKASEDLK’, m/z 1352.31; ‘HPSDFGADAQAAMSK’, m/z 1532.63) as well as pork-specific peptides (‘SEDEMKASEDLK’, m/z 1381.24; ‘HPGDFGADAQGAMSK’, m/z 1488.66) derived from Mb in meat mixes (as low as 0.5% contaminating pork in buffalo meat mix). Similarly, water buffalo (‘GGPLAAPYR’ m/z 901.48; ‘EPITVSSDQIAK’, m/z 1287.20) and pork-specific (‘GGPLTAAYR’, m/z 905.48; ‘EPITVSSDQMAK’ m/z 1306.24) peptides derived from CA-3 in meat mixes were also detected. Detection of beef-specific Mb peptide ‘HPSDFGADAQAAMSK’ in the sample containing 5% (w/w) beef in the pork and horse meat matrix using label-free quantification combined with the infusion of high-resolution MS was also reported by Montowska and Spychaj (2018). A highly sensitive and specific method using liquid chromatography-tandem mass spectrometry was established by Li et al. (2018) for the simultaneous determination of heat-stable species-specific peptides from beef Mb (‘HPSDFGADAQAAMSK’), beef CA-3 (‘GEFQLLLDALDK’), pork myoglobin (‘HPGDFGADAQGAMSK’, ‘YLEFISEAIIQVLQSK’) and pork CA-3 (‘HDPSLLPWTASYDPGSAK, EPITVSSDQMAK’).

Application of GELFrEE for Authentication of Commercial Meat Products

We examined the applicability of the peptidomic approach adopted in this study (simple extraction, GELFrEE fractionation, and MALDI-TOF MS) using various commercially processed meat products including ham, sausage, frankfurter, bacon, etc. purchased from supermarkets, as well as in-house processed meat products as reference samples (Fig. S4). All the products were screened for the presence of species-specific Mb and CA-3 derived peptides, which were detected in 100% buffalo meat or pork and their mixes. The list of peptide markers identified in this study for the 10 different processed meat products is presented in Table 2. Most of the observed peptides identified as heat-stable markers belonging to Mb and CA-3 were unique to both species, confirming that the selected peptides are not only highly specific but that they have good thermal stability. In the commercial and in-house processed samples, we identified the most abundant Mb and CA-3 as previously mentioned in Table 1. The canned corned buffalo meat (sample no. 4), which was produced under high temperature and high pressure, retained at least two CA-3 peptides, thereby fully meeting qualitative requirements. Although the pork cocktail sausage (sample no. 3) was marked with chicken ingredients, the specific chicken meat content was not labelled and none of the specific peptides of chicken were detected. All the tested commercial products were in compliance with their label descriptions. The results confirmed that Mb and CA-3 were both heat-stable proteins and could be used as screening targets of species-specific markers. Li et al. (2021) also utilized pork-specific peptides, ‘EPITVSSDQMAK’, ‘GGPLTAAYR’, ‘HDPSLLPWTASYDPGSAK’ from CA-3 to accurately quantify pork content in different types of meat products. Pork-specific CA-3 derived processing resistant marker ‘HDPSLLPWTASYDPGSAK’ was reported by Kotecka-Majchrzak et al. (2021) in different meat and vegan products. Our results confirm the simple protein extraction coupled with GELFrEE fractionation as a robust and efficient enrichment step for low MW proteins, which can be successfully explored in the development of a peptidomic-based approach for meat authentication.

Two-Dimensional Gel Electrophoresis and MS Approach for Meat Authentication

Unlike simple extraction followed in GELFrEE, for 2-DE total proteins were extracted using the procedures of Montowska and Pospiech (2011) and subjected to TCA/acetone precipitation followed by fractionation of 100% buffalo meat, 100% pork and buffalo: pork meat mix (95.5:0.5). The 2-DE gels from raw and cooked meat mix containing buffalo: pork (95.5:0.5) are represented in Fig. 4. Based on the pioneering work reported by Montowska and Pospiech(2012, 2013) and our earlier research findings (Naveena et al. 2017), cluster of two to three protein spots as indicated in Fig. 4 was reported to provide species-specific information. From our laboratory, we have consistently obtained these spots and we anticipate that these spots will correspond to species-specific marker peptides derived from myosin light chain isoforms.

The MALDI-TOF MS analysis and species-specific peptides identified are listed in Tab. S3 with their accession number, mass, protein score, sequence coverage, and the number of matched peptides. The results showed the presence of both buffalo and pork-specific peptides in meat mix containing buffalo meat and pork. Buffalo-specific myosin light chain-3 (MLC-3) (spot No. R1) and pork-specific myosin light chain-4 (MLC-4) (spot No. R2) were detected in the raw meat mix. In cooked meat mix, buffalo-specific MLC-3 (spot No. C1) and pork-specific myosin light polypeptide 6 (spot no. C2) were detected. On 2-DE gels, MLC isoforms form a characteristic pattern, which is typical of each of the species, and on the basis of which it is possible to identify those species in meat mixtures. The MLC isoforms retain their species-specific electrophoretic mobility even after processing, including minced meat and various meat products (Montowska and Pospiech 2012).

The water buffalo-specific MLC-3 derived tryptic peptide ‘AAAAPAPAPAPPPAPEPSK’ detected in both raw and cooked meat mix and ‘EPEFDPSKIK’ in raw meat mix in the current study were also reported by Naveena et al. (2017, 2018) in protein extracts of meat mix identified by peptide mass fingerprinting using MALDI-TOF MS. The inter-species differences in MLC isoforms in raw meat of six species, namely cattle, pig, chicken, turkey, duck, and goose, have been observed by Montowska and Pospiech (2011). The study demonstrated the robustness of 2-DE for authentication of pork in buffalo meat mix up to 0.5% level in both raw and cooked conditions; however, 2-DE fractionation is laborious and consumes almost 48-72 h.

In the current study, we proposed the Gel Eluted Liquid Fraction Entrapment Electrophoresis (GELFrEE) and 2-dimensional gel electrophoresis (2-DE) fractionation coupled to MALDI-TOF-MS based peptidomic approach for authentication of water buffalo meat adulterated with pork. The study identified a set of heat-stable, species-specific peptide markers derived from myoglobin, carbonic anhydrase-3, and myosin light chain isoforms. The advantage of GELFrEE as a front-end separation approach, over other existing extraction and fractionation techniques, including 2-DE, are their methodological simplicity, minimal sample preparation without any protein purification, and reduction in extraction and fractionation time to 2–3 h. Regardless of sample complexity, accurate identification of myoglobin and carbonic anhydrase-3 derived peptides by GELFrEE enrichment step followed by MALDI-TOF MS analysis will greatly expand the utility of this strategy in meat authentication. The study has also proved the suitability of the developed method that can detect contaminating pork in water buffalo meat mix even after cooking down to 0.5% (w/w). Implementing this easy-to-use, rapid extraction, and GELFrEE fractionation platform coupled with mass spectrometry-based peptidomics will help to identify novel proteins/peptides of low abundance and address food fraud issues. With the current innovation, we demonstrate effective coupling of GELFrEE to MALDI-TOF MS, establishing that the platform yields nearly analogous protein/peptide identifications relative to the ‘classic’ 2-DE/MS approach.

Author Contribution

Conceptualization: Rituparna Banerjee and Naveena B. Maheswarappa. Methodology: Rituparna Banerjee and Naveena B. Maheswarappa. Formal analysis and investigation: Rituparna Banerjee, Naveena B. Maheswarappa and Sowmya Dasoju. Writing - original draft preparation: Rituparna Banerjee. Writing - review and editing: Naveena B. Maheswarappa and Ranjith Ramanathan Funding acquisition: Naveena B. Maheswarappa. Resources: Sowmya Dasoju and Kiran Mohan. Supervision: Subhasish Biswas.

Data Availability Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author on request.

Funding

This research was funded by the Department of Biotechnology (DBT), Government of India (Grant No. BT/PR27198/PFN/20/1333/2017).

Conflict of Interest Rituparna Banerjee declares that she has no conflict of interest. Naveena B. Maheswarappa declares that he has no conflict of interest. Subhasish Biswas declares that he has no conflict of interest. Sowmya Dasoju declares that she has no conflict of interest. Kiran Mohan declares that she has no conflict of interest. Ranjith Ramanathan declares that he has no conflict of interest.

Ethical Approval This study does not contain experiments that involve human or animals and the authors have procured the meat from the slaughterhouse or retail shops for the work.

Informed Consent All authors have read the manuscript and agreed upon its submission to the journal.

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Table 1 Species-specific peptides derived from GELFrEE fractionation and MALDI-TOF MS analysis of buffalo meat and pork proteins

Species

Protein identified/

accession No.

pI/MW (Da)^a

Peptide position

m/z value

MC^b

Peptide Sequence

Buffalo

(Bubalus bubalis)

Carbonic anhydrase-3

NP_001029609.1

pI: 7.71/MW: 29637

81-89

154-165

213-224

901.48

1361.73

1287.67

GGPLAAPYR

GEFQLLLDALDK

EPITVSSDQIAK

Myoglobin

Accession no.

NP_776306.1

pI: 6.90/MW: 17067

52-63

89-97

120-134

1351.78

1006.50

1532.67

TEAEMKASEDLK

HLAESHANK

HPSDFGADAQAAMSK

Pork

(Sus scrofa)

Carbonic anhydrase-3

NP_001008688.1

pI: 7.72/MW: 29678

40-57

81-89

154-165

213-224

1941.93

905.48

1347.71

1305.63

HDPSLLPWTASYDPGSAK

GGPLTAAYR

GEFQLVLDALDK

EPITVSSDQMAK

Myoglobin

Accession no.

NP_999401.1

pI: 6.75/MW: 17074

36-43

52-63

104-119

120-134

135-146

149-154

1300.65

1381.61

1881.03

1488.65

1378.71

650.32

GHPETLEK

SEDEMKASEDLK

YLEFISEAIIQVLQSK

HPGDFGADAQGAMSK

ALELFRNDMAAK

ELGFQG

Peptides highlighted with bold letters were further validated through MS/MS sequencing

^apI and MW recorded in NCBI database

^bMissed cleavages

Table 2 GELFrEE fractionation and MALDI-TOF MS of commercial meat products obtained from retail markets and in-house processed meat products

Samples	Labelled ingredients	Detected species	Accession No.*	Identified protein	Detected peptides
1. Pork pepperoni	Pork meat, pork fat, water, spices & condiments, paprika oleoresin, food additives	Sus scrofa	NP_999401.1	Myoglobin	ELGFQG SEDEMKASEDLK HPGDFGADAQGAMSK
2. Premium ham	Pork meat (74%), water, edible starch, food additives	Sus scrofa	NP_001008688.1	Carbonic anhydrase-3	GGPLTAAYR EPITVSSDQMAK
3. Pork cocktail sausage	Pork meat (50%), chicken, water, wheat flour, soya, common salt, spices and condiments, food additives	Sus scrofa	NP_999401.1	Myoglobin	HPGDFGADAQGAMSK
4. Corned buffalo meat (canned)	Buffalo meat, binder, salt, preservatives	Bubalus bubalis	XP_006077475.1	Carbonic anhydrase-3	EPITVSSDQIAK GGPLAAPYR
5. Pork frankfurter	Pork meat (79%), water, iodized salt, mixed spices, food additives	Sus scrofa	NP_999401.1 NP_001008688.1	Myoglobin Carbonic anhydrase-3	HPGDFGADAQGAMSK EPITVSSDQMAK
6. Premium bacon bits	Pork meat (80%), water, common salt, edible starch, dextrose, food additives	Sus scrofa	NP_999401.1	Myoglobin	GHPETLEK HPGDFGADAQGAMSK YLEFISEAIIQVLQSK
7. Breakfast bacon	Pork meat, water, spices, condiments, food additives	Sus scrofa	NP_001008688.1	Carbonic anhydrase-3	GGPLTAAYR GEFQLVLDALDK EPITVSSDQMAK
8. In-house sausages	Buffalo meat (76%), water, salt, spice, condiments, additives	Bubalus bubalis	XP_006074486.1	Myoglobin	TEAEMKASEDLK HPSDFGADAQAAMSK
9. In-house ground meat patties	Buffalo meat (76%), water, salt, spice, condiments, additives	Bubalus bubalis	XP_006077475.1	Carbonic anhydrase-3	GGPLAAPYR GEFQLLLDALDK
10. In-house emulsion nuggets	Buffalo meat (76%), water, salt, spice, condiments, additives	Bos taurus	NP_776306.1	Myoglobin	ALELFR TEAEMKASEDLK HLAESHANK HPSDFGADAQAAMSK

*NCBI accession no.

No competing interests reported.

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Peptidomics Approaches for Authentication of Pork and Water Buffalo Meat Employing Rapid In-solution Fractionation and 2-DE

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