Separation methods for food protein purification and analysis

Abstract

The extraction, separation, and purification of dietary proteins from a variety of food sources are crucial for their targeted use in food applications. To achieve this, proteins should be effectively separated from non-protein components such as cell wall structures, polysaccharides, and lipids. Traditional protein purification methods can be time-consuming, highlighting the need for automated, cost-effective, and sustainable alternatives. This comprehensive review critically assesses various protein purification instruments from an analytical perspective, weighing their advantages and disadvantages. The methods under evaluation include ultrafiltration, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fast protein liquid chromatography (FPLC), high-performance liquid chromatography (HPLC), ultra performance liquid chromatography (UPLC), and microfluidic chips. Among these, FPLC stands out as an affordable and efficient technique that allows for high protein recovery. However, HPLC and UPLC provide faster results but may denature proteins, leading to lower recovery rates. Ultrafiltration is a cost-effective and straightforward method that doesn’t require complex equipment. Microchip-based approaches are emerging as innovative techniques for rapidly analyzing small samples. While SDS-PAGE is user-friendly, it denatures proteins, particularly those linked to other biomolecules. The choice of the most appropriate instrument depends on factors such as cost, energy efficiency, processing time, the characteristics of the target protein, desired outcomes, protein recovery, and resource availability. By critically examining these analytical instruments for protein purification, this review aims to assist researchers and practitioners in selecting the most suitable method for their specific needs, ultimately promoting efficient and successful protein purification endeavors in the field of food science and technology.

Keywords

Protein purification / analytical instruments / food protein fractionation / liquid chromatography / protein separation / ultrafiltration

References

[1] Jiménez—González C, Agrasar AMT, Mallo F, Rúa ML, Fuciños C. Red seaweed proteins: Valuable marine—origin compounds with encouraging applications. Algal Res. 2023; 75: 103262.

[2] Mæhre HK, Dalheim L, Edvinsen GK, Elvevoll EO, Jensen IJ. Protein Determination—Method Matters. Foods. 2018; 7: 5.

[3] Wu Y, Zhang N, Luo K, Liu Y, Bai Z, Tang S. Recent advances of innovative and high—efficiency stationary phases for chromatographic separations. TrAC Trends Anal Chem. 2022; 153: 116647.

[4] Darling AL, Millward DJ, Torgerson DJ, Hewitt CE, Lanham—New SA. Dietary protein and bone health: a systematic review and meta—analysis. Am J Clin Nutr. 2009; 90: 1674—92.

[5] Guasch—Ferré M, Satija A, Blondin SA, Janiszewski M, Emlen E, O’Connor LE, et al. Meta—Analysis of Randomized Controlled Trials of Red Meat Consumption in Comparison With Various Comparison Diets on Cardiovascular Risk Factors. Circulation. 2019; 139: 1828-45.

[6] Jenkins DJ, Wong JM, Kendall CW, Esfahani A, Ng VW, Leong TC, et al. The effect of a plant—based low—carbohydrate (“Eco—Atkins”) diet on body weight and blood lipid concentrations in hyperlipidemic subjects. Arch Intern Med. 2009; 169: 1046-54.

[7] Pan A, Sun Q, Bernstein AM, Schulze MB, Manson JE, Willett WC, et al. Red meat consumption and risk of type 2 diabetes: 3 cohorts of US adults and an updated meta—analysis. Am J Clin Nutr. 2011; 94: 1088-96.

[8] Day L, Cakebread JA, Loveday SM. Food proteins from animals and plants: Differences in the nutritional and functional properties. Trends Food Sci Technol. 2022; 119: 428-42.

[9] Liu S, Li Z, Yu B, Wang S, Shen Y, Cong H. Recent advances on protein separation and purification methods. Adv Colloid Interface Sci. 2020; 284: 102254.

[10] Berg JM, Tymoczko JL, Gatto GJ, Stryler L. Biochemistry. 8th ed. 2024.

[11] Wang YC, Choi MH, Han J. Two—dimensional protein separation with advanced sample and buffer isolation using microfluidic valves. Anal Chem. 2004; 76: 4426-31.

[12] Smith DM. Protein Separation and Characterization Procedures. In: Nielsen SS, editor. Food Analysis. Food Science Text Series. Springer, Cham; 2017. pp. 431-53.

[13] Rathore AS, Shirke A. Recent developments in membrane—based separations in biotechnology processes: review. Prep Biochem Biotechnol. 2011; 41: 398-421.

[14] Becht NO, Malik DJ, Tarleton ES. Evaluation and comparison of protein ultrafiltration test results: Dead—end stirred cell compared with a cross—flow system. Sep Purif Technol. 2008; 62: 228-39.

[15] Burns DB, Zydney AL. Effect of solution pH on protein transport through ultrafiltration membranes. Biotechnol Bioeng. 1999; 64: 27-37.

[16] LaRue RJ, Kazemi AS, Latulippe DR. Microscale stirred—cell filtration for high—throughput evaluation of separation performance. Biochem Eng J. 2018; 130: 34-8.

[17] Jones KL, O’Melia CR. Ultrafiltration of protein and humic substances: effect of solution chemistry on fouling and flux decline. J Membr Sci. 2001; 193: 163-73.

[18] Denis C, MasséA, Fleurence J, Jaouen P. Concentration and pre—purification with ultrafiltration of a R—phycoerythrin solution extracted from macro—algae Grateloupia turuturu: Process definition and up—scaling. Sep Purif Technol. 2009; 69: 37-42.

[19] Kammakakam I, Lai Z. Next—generation ultrafiltration membranes: A review of material design, properties, recent progress, and challenges. Chemosphere. 2023; 316: 137669.

[20] Nakatsuka S, Michaels AS. Transport and separation of proteins by ultrafiltration through sorptive and non—sorptive membranes. J Membr Sci. 1992; 69: 189-211.

[21] Wan Y, Cui Z, Ghosh R. Fractionation of Proteins Using Ultrafiltration: Developments and Challenges. Dev Chem Eng Miner Process. 2008; 13: 121—36.

[22] Zhang L, Spencer HG. Selective separation of proteins by microfiltration with formed—in—place membranes. Desalination. 1993; 90: 137-46.

[23] Rabiller—Baudry M, Chaufer B, Lucas D, Michel F. Ultrafiltration of mixed protein solutions of lysozyme and lactoferrin: role of modified inorganic membranes and ionic strength on the selectivity. J Membr Sci. 2001; 184: 137—48.

[24] Zydney AL. Protein Separations Using Membrane Filtration: New Opportunities for Whey Fractionation. Int Dairy J. 1998; 8: 243-50.

[25] Kumar P, Sharma N, Ranjan R, Kumar S, Bhat ZF, Jeong DK. Perspective of membrane technology in dairy industry: a review. Asian—Australas J Anim Sci. 2013; 26: 1347—58.

[26] Bleakley S, Hayes M. Algal Proteins: Extraction, Application, and Challenges Concerning Production. Foods. 2017; 6: 33.

[27] Boi C, Malavasi A, Carbonell RG, Gilleskie G. A direct comparison between membrane adsorber and packed column chromatography performance. J Chromatogr A. 2020; 1612: 460629.

[28] Christy C, Adams G, Kuriyel R, Bolton G, Seilly A. High—performance tangential flow filtration: a highly selective membrane separation process. Desalination. 2002; 144: 133-6.

[29] Roy S, Kumar V. A Practical Approach on SDS PAGE for Separation of Protein. Int J Sci Res. 2014; 3: 955-60.

[30] Anderson BL, Berry RW, Telser A. A sodium dodecyl sulfate—polyacrylamide gel electrophoresis system that separates peptides and proteins in the molecular weight range of 2500 to 90,000. Anal Biochem. 1983; 132: 365-75.

[31] Lázaro—Silva DN, De Mattos JC, Castro HC, Alves GG, Amorim LM. The use of DNA extraction for molecular biology and biotechnology training: a practical and alternative approach. Creative Education. 2015; 6: 762.

[32] Goetz H, Kuschel M, Wulff T, Sauber C, Miller C, Fisher S, et al. Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination. J Biochem Biophys Methods. 2004; 60: 281-93.

[33] Cermeño M, Kleekayai T, Amigo—Benavent M, Harnedy—Rothwell P, FitzGerald RJ. Current knowledge on the extraction, purification, identification, and validation of bioactive peptides from seaweed. ELECTROPHORESIS. 2020; 41: 1694-717.

[34] Rogowska—Wrzesinska A, Le Bihan MC, Thaysen—Andersen M, Roepstorff P. 2D gels still have a niche in proteomics. J Proteomics. 2013; 88: 4-13.

[35] Westermeier R. Looking at proteins from two dimensions: a review on five decades of 2D electrophoresis. Arch Physiol Biochem. 2014; 120: 168-72.

[36] Pomastowski P, Buszewski B. Two—dimensional gel electrophoresis in the light of new developments. TrAC Trends Anal Chem. 2014; 53: 167-77.

[37] Postu PA, Ion L, Drochioiu G, Petre BA, Glocker MO. Mass spectrometric characterization of the zein protein composition in maize flour extracts upon protein separation by SDS—PAGE and 2D gel electrophoresis. Electrophoresis. 2019; 40: 2747-58.

[38] Harcum S. 2—Purification of protein solutions. In: Abbott A, Ellison M, editors. Biologically Inspired Textiles. Biologically Inspired Textiles; 2008. pp. 26-43.

[39] Malairaj S, Muthu S, Gopal VB, Perumal P, Ramasamy R. Qualitative and quantitative determination of R—phycoerythrin from Halymenia floresia (Clemente) C. Agardh by polyacrylamide gel using electrophoretic elution technique. J Chromatogr A. 2016; 1454: 120-6.

[40] Pathak M, Dutta D, Rathore A. Analytical QbD: development of a native gel electrophoresis method for measurement of monoclonal antibody aggregates. Electrophoresis. 2014; 35: 2163—71.

[41] Zhu D, Dai W, Srinivasan P, McClellan H, Braden D, Allee—Munoz A, et al. Characterization of AMA1—RON2L complex with native gel electrophoresis and capillary isoelectric focusing. Electrophoresis. 2022; 43: 509—15.

[42] Sharma N, Sharma R, Rajput YS, Mann B, Singh R, Gandhi K. Separation methods for milk proteins on polyacrylamide gel electrophoresis: Critical analysis and options for better resolution. Int Dairy J. 2021; 114: 104920.

[43] Leeb E, Haller N, Kulozik U. Effect of pH on the reaction mechanism of thermal denaturation and aggregation of bovine β—lactoglobulin. Int Dairy J. 2018; 78: 103—11.

[44] Booth PPM, Lamb DT, Anderson JP, Furtaw MD, Kennedy RT. Capillary electrophoresis Western blot using inkjet transfer to membrane. J Chromatogr A. 2022; 1679: 463389.

[45] Dawod M, Arvin NE, Kennedy RT. Recent advances in protein analysis by capillary and microchip electrophoresis. Analyst. 2017; 142: 1847-66.

[46] Kašička V. Peptide mapping of proteins by capillary electromigration methods. J Sep Sci. 2022; 45: 4245-79.

[47] Štěpánová S, Kašička V. Applications of capillary electromigration methods for separation and analysis of proteins (2017—mid 2021)—A review. Anal Chim Acta. 2022; 1209: 339447.

[48] Kasicka V. From micro to macro: conversion of capillary electrophoretic separations of biomolecules and bioparticles to preparative free—flow electrophoresis scale. Electrophoresis. 2009; 30: S40-52.

[49] Stastna M. Continuous flow electrophoretic separation—Recent developments and applications to biological sample analysis. Electrophoresis. 2020; 41: 36-55.

[50] Madadlou A, O’Sullivan S, Sheehan D. Fast protein liquid chromatography. Methods Mol Biol. 2011; 681: 439-47.

[51] Léonil J, Gagnaire V, Mollé D, Pezennec S, Bouhallab S. Application of chromatography and mass spectrometry to the characterization of food proteins and derived peptides. J Chromatogr A. 2000; 881: 1-21.

[52] Shinde S, Rajurkar V. Advances in High—Performance Liquid Chromatography (HPLC) Method Development and Validation: A Comprehensive Review. J Drug Deliv Biotherapeutics. 2024; 2: 16-26.

[53] Liu H, Finch JW, Lavallee MJ, Collamati RA, Benevides CC, Gebler JC. Effects of column length, particle size, gradient length and flow rate on peak capacity of nano—scale liquid chromatography for peptide separations. J Chromatogr. 2007; 1147: 30-6.

[54] Sheehan D. Physical Biochemistry: Principles and Applications. John Wiley & Sons; 2013.

[55] Cabrera K. Applications of silica—based monolithic HPLC columns. J Sep Sci. 2004; 27: 843-52.

[56] Kremmer T, Paulik E, Boldizsár M, Holényi I. Application of the fast protein liquid chromatographic system and MonoQ HR 5/5 anion exchanger to the separation of nucleotides. J Chromatogr. 1989; 493: 45-52.

[57] Tangvarasittichai S, Tangvarasittichai O, Jermnim N. Comparison of fast protein liquid chromatography (FPLC) with HPLC, electrophoresis & microcolumn chromatography techniques for the diagnosis of beta—thalassaemia. Indian J Med Res. 2009; 129: 242-8.

[58] Plank J, Andres PR, Krause I, Winter C. Gram scale separation of casein proteins from whole casein on a Source 30Q anion—exchange resin column utilizing fast protein liquid chromatography (FPLC). Protein Expr Purif. 2008; 60: 176-81.

[59] Riaz M, Perveen R, Javed MR, Nadeem H, Rashid MH. Kinetic and thermodynamic properties of novel glucoamylase from Humicola sp . Enzyme Microb Technol. 2007; 41: 558-64.

[60] Moon PG, Hwang HH, Boo YC, Kwon J, Cho JY, Baek MC. Identification of rat urinary glycoproteome captured by three lectins using gel and LC—based proteomics. ELECTROPHORESIS. 2008; 29: 4324—31.

[61] Miyazaki O, Fukamachi I, Mori A, Hashimoto H, Kawashiri M, Nohara A, et al. Formation of preβ1—HDL during lipolysis of triglyceride—rich lipoprotein. Biochem Biophys Res Commun. 2009; 379: 55-9.

[62] Santos MJ, Teixeira JA, Rodrigues LR. Fractionation and recovery of whey proteins by hydrophobic interaction chromatography. J Chromatogr. 2011; 879: 475—9.

[63] Kunz C, Lönnerdal B. Human milk proteins: separation of whey proteins and their analysis by polyacrylamide gel electrophoresis, fast protein liquid chromatography (FPLC) gel filtration, and anion—exchange chromatography. Am J Clin Nutr. 1989; 49: 464-70.

[64] Ozturkoglu—Budak S, Akal HC, Bereli N, Cimen D, Akgonullu S. Use of antimicrobial proteins of donkey milk as preservative agents in Kashar cheese production. Int Dairy J. 2021; 120: 105090.

[65] Gautam P, Sharma R, Ranvir S, Gandhi K, Mann B. Soymilk protein detection in milk—A review. Indian J Dairy Sci. 2019; 72: 231-40.

[66] Garber DW, Kulkarni KR, Anantharamaiah GM. A sensitive and convenient method for lipoprotein profile analysis of individual mouse plasma samples. J Lipid Res. 2000; 41: 1020—6.

[67] Aguilar MI. HPLC of Peptides and Proteins. In: Aguilar MI, editor. HPLC of Peptides and Proteins: Methods and Protocols. Totowa, NJ: Springer New York; 2004.

[68] Reeves R. HMGA proteins: isolation, biochemical modifications, and nucleosome interactions. Methods Enzymol. 2004; 375: 297-322.

[69] Kariluoto MS, Vahteristo LT, Piironen VI. Applicability of microbiological assay and affinity chromatography purification followed by high—performance liquid chromatography (HPLC) in studying folate contents in rye. J Sci Food Agric. 2001; 81: 938-42.

[70] Kadlecová Z, Kozlík P, Tesařová E, Gilar M, Kalíková K. Characterization and comparison of mixed—mode and reversed—phase columns; interaction abilities and applicability for peptide separation. J Chromatogr A. 2021; 1648: 462182.

[71] Josic D, Kovac S. Reversed—phase High Performance Liquid Chromatography of proteins. Curr Protoc Protein Sci. 2010; 61: 8.7.1-8.7.22.

[72] Samuelsson J, Eiriksson FF, Åsberg D, Thorsteinsdóttir M, Fornstedt T. Determining gradient conditions for peptide purification in RPLC with machine—learning—based retention time predictions. J Chromatogr A. 2019; 1598: 92-100.

[73] Soares NP, Magalhaes GC, Mayrink PH, Verano—Braga T. Omics to Unveil Diabetes Mellitus Pathogenesis and Biomarkers: Focus on Proteomics, Lipidomics, and Metabolomics. In: Verano—Braga T, editor. Mass Spectrometry—Based Approaches for Treating Human Diseases and Diagnostics. Cham: Springer International Publishing; 2024. pp. 211—20.

[74] Dong M. HPLC and UHPLC for Practicing Scientists. John Wiley & Sons; 2019.

[75] Lin Y, Wang Y, Ji Z, Le X. Isolation, Purification, and Identification of Coconut Protein through SDS—PAGE, HPLC, and MALDI—TOF/TOF—MS. Food Anal Methods. 2020; 13: 1246-54.

[76] Admassu H, Gasmalla MAA, Yang R, Zhao W. Identification of Bioactive Peptides with α—Amylase Inhibitory Potential from Enzymatic Protein Hydrolysates of Red Seaweed (Porphyra spp). J Agric Food Chem. 2018; 66: 4872—82.

[77] Ko JY, Lee JH, Samarakoon K, Kim JS, Jeon YJ. Purification and determination of two novel antioxidant peptides from flounder fish (Paralichthys olivaceus) using digestive proteases. Food Chem Toxicol. 2013; 52: 113—20.

[78] Nollet LML. Food Analysis by HPLC, Second Edition. CRC Press; 2000.

[79] Call L, Kapeller M, Grausgruber H, Reiter E, Schoenlechner R, D’Amico S. Effects of species and breeding on wheat protein composition. J Cereal Sci. 2020; 93: 102974.

[80] Swartz ME. UPLC^TM: An Introduction and Review. J Liq Chromatogr Relat Technol. 2005; 28: 1253-63.

[81] Sroga GE, Vashishth D. UPLC methodology for identification and quantitation of naturally fluorescent crosslinks in proteins: a study of bone collagen. J Chromatogr B Analyt Technol Biomed Life Sci. 2011; 879: 379-85.

[82] Boxi A, Parikh I, Sivaprasad R, Shreekar SK. Current Trends in Protein Purification: A Review. Int J Sci Res Sci Technol. 2020; 7: 279-310.

[83] De Vos J, Broeckhoven K, Eeltink S. Advances in Ultrahigh—Pressure Liquid Chromatography Technology and System Design. Anal Chem. 2016; 88: 262-78.

[84] Guillarme D, Nguyen DT, Rudaz S, Veuthey JL. Method transfer for fast liquid chromatography in pharmaceutical analysis: application to short columns packed with small particle. Part II: gradient experiments. Eur J Pharm Biopharm. 2008; 68: 430—40.

[85] Guillarme D, Grata E, Glauser G, Wolfender JL, Veuthey JL, Rudaz S. Some solutions to obtain very efficient separations in isocratic and gradient modes using small particles size and ultra—high pressure. J Chromatogr A. 2009; 1216: 3232-43.

[86] Li L, Wang J, Li M, Yang Y, Wang Z, Miao J, et al. Detection of the adulteration of camel milk powder with cow milk by ultra—high performance liquid chromatography (UPLC). Int Dairy J. 2021; 121: 105117.

[87] Francisco CL, Jorge AM, Dal—Pai—Silva M, Carani FR, Cabeço LC, Silva SR. Muscle fiber type characterization and myosin heavy chain (MyHC) isoform expression in Mediterranean buffaloes. Meat Sci. 2011; 88: 535—41.

[88] Lee SY, Liu BL, Wu JY, Chang YK. Egg white lysozyme purification by a stirred cell contactor equipped with a weak ion—exchange nanofiber membrane: Process development and scale—up. Food Chem. 2021; 338: 128144.

[89] Giaretta N, Di Giuseppe AM, Lippert M, Parente A, Di Maro A. Myoglobin as marker in meat adulteration: a UPLC method for determining the presence of pork meat in raw beef burger. Food Chem. 2013; 141: 1814-20.

[90] Cermeño M, Stack J, Tobin PR, O’Keeffe MB, Harnedy PA, Stengel DB, et al. Peptide identification from a Porphyra dioica protein hydrolysate with antioxidant, angiotensin converting enzyme and dipeptidyl peptidase IV inhibitory activities. Food Funct. 2019; 10: 3421—9.

[91] Losacco GL, Wang H, Haidar Ahmad IA, DaSilva J, Makarov AA, Mangion I, et al. Enantioselective UHPLC Screening Combined with In Silico Modeling for Streamlined Development of Ultrafast Enantiopurity Assays. Anal Chem. 2022; 94: 1804—12.

[92] Xiang Y, Maynes DR, Lee ML. Safety concerns in ultrahigh pressure capillary liquid chromatography using air—driven pumps. J Chromatogr A. 2003; 991: 189-96.

[93] Štěpánová S, Kašička V. Recent developments and applications of capillary and microchip electrophoresis in proteomics and peptidomics (mid—2018—2022). J Sep Sci. 2023; 46: e2300043.

[94] Phan NVH, Sussitz HF, Ladenhauf E, Pum D, Lieberzeit PA. Combined Layer/Particle Approaches in Surface Molecular Imprinting of Proteins: Signal Enhancement and Competition. Sensors (Basel). 2018; 18: 180.

[95] Vitorino R, Guedes S, da Costa JP, Kašička V. Microfluidics for Peptidomics, Proteomics, and Cell Analysis. Nanomaterials. 2021; 11: 1118.

[96] Hisey CL, Dorayappan KDP, Cohn DE, Selvendiran K, Hansford DJ. Microfluidic affinity separation chip for selective capture and release of label—free ovarian cancer exosomes. Lab Chip. 2018; 18: 3144-53.

[97] Rho HS, Hanke AT, Ottens M, Gardeniers HJGE. A microfluidic chip with a staircase pH gradient generator, a packed column and a fraction collector for chromatofocusing of proteins. Electrophoresis. 2018; 39: 1031—9.

[98] Chun H. Electropreconcentration, gate injection, and capillary electrophoresis separation on a microchip. J Chromatogr A. 2018; 1572: 179-86.

[99] Yamauchi KA, Tentori AM, Herr AE. Arrayed isoelectric focusing using photopatterned multi—domain hydrogels. Electrophoresis. 2018; 39: 1040-7.

[100] Nielsen AV, Nielsen JB, Sonker M, Knob R, Sahore V, Woolley AT. Microchip electrophoresis separation of a panel of preterm birth biomarkers. Electrophoresis. 2018; 39: 2300—7.

[101] Blazek V, Caldwell RA. Comparison of SDS gel capillary electrophoresis with microfluidic lab—on—a—chip technology to quantify relative amounts of 7S and 11S proteins from 20 soybean cultivars. Int J Food Sci Technol. 2009; 44: 2127-34.

[102] Monti G, De Napoli L, Mainolfi P, Barone R, Guida M, Marino G, et al. Monitoring food quality by microfluidic electrophoresis, gas chromatography, and mass spectrometry techniques: effects of aquaculture on the sea bass (Dicentrarchus labrax). Anal Chem. 2005; 77: 2587—94.

[103] Ueno H, Wang J, Kaji N, Tokeshi M, Baba Y. Quantitative determination of amino acids in functional foods by microchip electrophoresis. J Sep Sci. 2008; 31: 898-903.

[104] Atalay YT, Vermeir S, Witters D, Vergauwe N, Verbruggen B, Verboven P, et al. Microfluidic analytical systems for food analysis. Trends Food Sci Technol. 2011; 22: 386-404.

[105] Convery N, Gadegaard N. 30 years of microfluidics. Micro Nano Eng. 2019; 2: 76-91.