Milk is a rich source of biologically important proteins and peptides. In addition, milk contains a variety of extracellular vesicles (EVs), including exosomes, that carry their own proteome cargo. EVs are essential for cell-cell communication and modulation of biological processes. They act as nature carriers of bioactive proteins/peptides in targeted delivery during various physiological and pathological conditions. Identification of the proteins and protein-derived peptides in milk and EVs and recognition of their biological activities and functions had a tremendous impact on food industry, medicine research, and clinical applications. Advanced separation methods, mass spectrometry (MS)-based proteomic approaches and innovative biostatistical procedures allowed for characterization of milk protein isoforms, genetic/splice variants, posttranslational modifications and their key roles, and contributed to novel discoveries. This review article discusses recently published developments in separation and identification of bioactive proteins/peptides from milk and milk EVs, including MS-based proteomic approaches.

Institute of Analytical Chemistry of the Czech Academy of Sciences Brno Czech Republic

Zobrazit více v PubMed

Ballard O, Morrow AL. Human milk composition: nutrients and bioactive factors. Pediatr Clin N Am. 2013;60:49-74.

Mohanty DP, Mohapatra S, Misra S, Sahu PS. Milk derived bioactive peptides and their impact on human health-a review. Saudi J Biol Sci. 2016;23:577-83.

Marcone S, Belton O, Fitzgerald DJ. Milk-derived bioactive peptides and their health promoting effects: a potential role in atherosclerosis. Br J Clin Pharmacol. 2017;83:152-62.

Nongonierma AB, O'Keeffe MB, FitzGerald RJ. Milk protein hydrolysates and bioactive peptides. In: McSweeney P, O'Mahony J editors. Advanced dairy chemistry. New York: Springer; 2016. p. 417-82.

Wada Y, lonnerdal B Bioactive peptides derived from human milk proteins-mechanisms of action. J Nutr Biochem. 2014;25:503-14.

Punia H, Tokas J, Malik A, Sangwan S, Baloda S, Singh N, et al. Identification and detection of bioactive peptides in milk and dairy products: remarks about agro-foods. Molecules. 2020;25:3328.

Samtiya M, Samtiya S, Badgujar PC, Puniya AK, Dhewa T, Aluko RE. Health-promoting and therapeutic attributes of milk-derived bioactive peptides. Nutrients. 2022;14:3001.

Wang X, Yu Z, Zhao X, Han R, Huang D, Yang Y, et al. Comparative proteomic characterization of bovine milk containing β-casein variants A1A1 and A2A2, and their heterozygote A1A2. J Sci Food Agric. 2021;101:718-25.

Nguyen DD, Johnson SK, Busetti F, Solah VA. Formation and degradation of beta-casomorphins in dairy processing. Crit Rev Food Sci Nutr. 2015;55:1955-67.

Gomez-Ruiz JA, Miralles B, Aguera P, Amigo L. Quantitative determination of αs2- and αs1-casein in goat's milk with different genotypes by capillary electrophoresis. J Chromatogr A. 2004;1054:279-84.

Buzas H, Szekelyhidi R, Szafner G, Szabo K, Sule J, Bukovics S, et al. Developed rapid and simple RP-HPLC method for simultaneous separation and quantification of bovine milk protein fractions and their genetic variants. Anal Biochem. 2022;658:114939.

Caroli AM, Savino S, Bulgari O, Monti E. Detecting β-casein variation in bovine milk. Molecules. 2016;21:141.

Neyestani TR, Djalali M, Pezeshki M. Isolation of α-lactalbumin, β-lactoglobulin, and bovine serum albumin from cow's milk using gel filtration and anion-exchange chromatography including evaluation of their antigenicity. Protein Exp Purif. 2003;29:202-8.

Ng-Kwai-Hang K, Kroeker E. Rapid separation and quantification of major caseins and whey proteins of bovine milk by polyacrylamide gel electrophoresis. J Dairy Sci. 1984;67:3052-6.

Cavaletto M, Giuffrida MG, Conti A. Milk fat globule membrane components-a proteomic approach. Adv Exp Med Biol. 2008;606:129-41.

Ryskaliyeva A, Henry C, Miranda G, Faye B, Konuspayeva G, Martin P. Alternative splicing events expand molecular diversity of camel CSN1S2 increasing its ability to generate potentially bioactive peptides. Sci Rep. 2019;9:5243.

Kwan SH, Wan-Ibrahim WI, Juvarajah T, Fung SY, Abdul-Rahman PS. Isolation and identification of O- and N-linked glycoproteins in milk from different mammalian species and their roles in biological pathways which support infant growth. Electrophoresis. 2021:42:233-44.

Xiao J, Wang J, Gan R, Wu D, Xu Y, Peng L, et al. Quantitative N-glycoproteome analysis of bovine milk and yogurt. Curr Res Food Sci. 2022:5:182-90.

Buratta S, Urbanelli L, Tognoloni A, Latella R, Cerrotti G, Emiliani C, et al. Protein and lipid content of milk extracellular vesicles: a comparative overview. Life (Basel). 2023;13:401.

Vaswani KM, Peiris H, Koh YQ, Hill RJ, Harb T, Arachchige BJ, et al. A complete proteomic profile of human and bovine milk exosomes by liquid chromatography mass spectrometry. Expert Rev Proteomics. 2021;18:719-35.

Rahman MM, Takashima S, Kamatari YO, Shimizu K, Okada A, Inoshima Y. Comprehensive proteomic analysis revealed a large number of newly identified proteins in the small extracellular vesicles of milk from late-stage lactating cows. Animals (Basel). 2021;11:2506.

Cunsolo V, Miccilli V, Saletti R, Foti S. Review: applications of mass spectrometry techniques in the investigation of milk proteome. Eur J Mass Spectrom. 2011;17:305-20.

Greenwood SL, Honan MC. Symposium review: characterization of the bovine milk protein profile using proteomic techniques. J Dairy Sci. 2019;102:2796-806.

Agregan R, Echegaray N, Lopez-Pedrouso M, Kharabsheh R, Franco D, Lorenzo JM. Proteomic advances in milk and dairy products. Molecules. 2021;26:3832.

Muller L, Bartak P, Bednar P, Frysova I, Sevcik J, Lemr K. Capillary electrophoresis-mass spectrometry-a fast and reliable tool for the monitoring of milk adulteration. Electrophoresis. 2008;29:2088-93.

Calvano CD, De Ceglie C, Monopoli A, Zambonin CG. Detection of sheep and goat milk adulterations by direct MALDI-TOF MS analysis of milk tryptic digests. J Mass Spectrom. 2012;47:1141-9.

de Oliveira LVA, Kleemann CR, Molognoni L, Daguer H, Hoff RB, Prudencio ES. Reference LC-MS/MS method to detect fresh cheese adulteration with whey. Food Res Int. 2022;156:111140.

Ji Z, Zhang J, Deng C, Hu Z, Du Q, Guo T, et al. Identification of mare milk adulteration with cow milk by liquid chromatography-high resolution mass spectrometry based on proteomics and metabolomic approaches. Food Chem. 2023;405(Pt B),134901.

Nardiello D, Natale A, Palermo C, Quinto M, Centonze D. Milk authenticity by ion-trap proteomics following multi-enzyme digestion. Food Chem. 2018;244:317-23.

Masci M, Zoani C, Nevigato T, Turrini A, Jasionowska R, Caproni R, et al. Authenticity assessment of dairy products by capillary electrophoresis. Electrophoresis. 2022;43:340-54.

Correa APF, Daroit DJ, Coelho J, Meira SMM, Lopes FC, Segalin J, et al. Antioxidant, antihypertensive and antimicrobial properties of ovine milk caseinate hydrolyzed with a microbial protease. J Sci Food Agric. 2011;91:2247-54.

Iram D, Kindarle UA, Sansi MS, Meena S, Puniya AK, Vij S. Peptidomics-based identification of an antimicrobial peptide derived from goat milk fermented by Lactobacillus rhamnosus (C25). J Food Biochem. 2022;46:e14450.

Sharma P, Kaur H, Kehinde BA, Chhikara N, Sharma D, Panghal A. Food-derived anticancer peptides: a review. Int J Pept Res Ther. 2021;27:55-70.

Pepe G, Tenore GC, Mastrocinque R, Stusio P, Campiglia P. Potential anticarcinogenic peptides from bovine milk. J Amino Acids. 2013;2013:939804.

Parodi PW. A role for milk proteins and their peptides in cancer prevention. Curr Pharm Des. 2007;13:813-28.

Qian ZY, Jolles P, Migliore-Samour D, Schoentgen F, Fiat AM. Sheep κ-casein peptides inhibit platelet aggregation. Biochim Biophys Acta. 1995;1244:411-7.

Cai J, Li, X, Du H, Jiang C, Xu S, Cao Y. Immunomodulatory significance of natural peptides in mammalians: promising agents for medical application. Immunobiology. 2020;225:151936.

Zhu W, Ren L, Zhang L, Qiao Q, Farooq MZ, Xu Q. The potential of food-derived bioactive peptides against chronic intestinal inflammation. Mediat Inflamm. 2020;2020:6817156.

Cavaletto M, Givonetti A, Cattaneo C. The immunological role of milk fat globule membrane. Nutrients. 2022;14:4574.

Gong H, Gao J, Wang Y, Luo QW, Guo KR, Ren FZ, et al. Identification of novel peptides from goat milk casein that ameliorate high-glucose-induced insulin resistance in HepG2 cells. J Dairy Sci. 2020;103:4907-18.

Moreno-Montoro M, Olalla-Herrera M, Rufian-Henares JA, Martinez RG, Miralles B, Bergilos T, et al. Antioxidant, ACE-inhibitory and antimicrobial activity of fermented goat milk: Activity and physicochemical property relationship of the peptide components. Food Funct. 2017;8:2783-91.

Zhang Y, Chen R, Ma H, Chen S. Isolation and identification of dipeptidyl peptidase IV-inhibitory peptides from trypsin/chymotrypsin-treated goat milk casein hydrolysates by 2D-TLC and LC-MS/MS. J Agric Food Chem. 2015;63:8819-28.

Auestad N, Layman DK. Dairy bioactive proteins and peptides: a narrative review. Nutr Rev. 2021;79(Suppl 2):36-47.

Roy D, Ye A, Moughan PJ, Singh H. Composition, structure, and digestive dynamics of milk from different species-a review. Front Nutr. 2020;7:577759.

Recio I, Perez-Rodriquez M, Ramos M, Amigo L. Capillary electrophoretic analysis of genetic variants of milk proteins from different species. J Chromatogr A. 1997;768:47-56.

Hue-Beauvais C, Miranda G, Aujean E, Jaffrezic F, Devinoy E, Martin P, et al. Diet-induced modifications to milk composition have long-term effects on offspring growth in rabbits. J Anim Sci. 2017;95:761-70.

Martin P, Cebo C, Miranda G. Interspecies comparison of milk proteins: quantitative variability and molecular diversity. In McSweeney PIH, Fox PF, editors. Advanced dairy chemistry: volume 1A: Proteins: Basic aspects, 4th ed. New York: Springer Science and Business media; 2013. p. 387-429.

Sun X, Yu Z, Liang C, Xie S, Wang H, Wang J, et al. Comparative analysis of changes in whey proteins of goat milk throughout the lactation cycle using quantitative proteomics. J Dairy Sci. 2023;106:792-806.

Li M, Zheng K, Song W, Yu H, Zhang X, Yue X, et al. Quantitative analysis of differentially expressed milk fat globule membrane proteins between donkey and bovine colostrum based on high-performance liquid chromatography with tandem mass spectrometry proteomics. J Dairy Sci. 2021;104:12207-215.

Bastian ED, Brown RJ. Plasmin in milk and dairy products: an update. Int Dairy J. 1996;6:435-57.

Ismail B, Nielsen SS. Invited review: plasmin protease in milk: current knowledge and relevance to dairy industry. J Dairy Sci. 2010;93:4999-5009.

Cui Q, Duan Y, Zhang M, Liang S, Sun X, Cheng J, et al. Peptide profiles and antioxidant capacity of extensive hydrolysates of milk protein concentrate. J Dairy Sci. 2022;105:7972-85.

Panyayai T, Ngamphiw C, Tongsima S, Mhuantong W, Limsripraphan W, Choowongkomon K, et al. FeptideDB: a web application for new bioactive peptides from food protein. Heliyon. 2019;5:e02076.

Nongonierma AB, FitzGerald RJ. Structure activity relationship modelling of milk protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity. Peptides. 2016:79:1-7.

Lin K, Zhang LW, Han X, Xin L, Meng ZX, Gong PM, et al. Yak milk casein as potential precursor of angiotensin I-converting enzyme inhibitory peptides based on in silico proteolysis. Food Chem. 2018;254:340-47.

Golan-Gerstl R, Reif S. Extracellular vesicles in human milk. Curr Opin Clin Nutr Metab Care. 2022;25:209-15.

Jiang X, You L, Zhang Z, Cui X, Zhong H, Sun X, et al. Biological properties of milk-derived extracellular vesicles and their physiological functions in infant. Front Cell Dev Biol. 2021;9:693534.

Ong SL, Blenkiron C, Haines S, Acevedo-Fani A, Leite JAS, Zempleni J, et al. Ruminant milk-derived extracellular vesicles: a nutritional and therapeutic opportunity? Nutrients. 2021;13:2505.

Kalra H, Drummen GPC, Mathivanan S. Focus on extracellular vesicles: introducing the next small big thing. Int J Mol Sci. 2016;17:170.

Admyre C, Johansson SM, Qazi KR, Filen J, Lahesmaa R, Norman M, et al. Exosomes with immune modulatory features are present in human breast milk. J Immunol. 2007;179:1969-78.

Benmoussa A, Gotti C, Bourassa S, Gilbert C, Provost P. Identification of protein markers for extracellular vesicle (EV) subsets in cow's milk. J Proteomics. 2019;192:78-88.

Kowal J, Arras G, Colombo M, Jouve M, Morath JP, Primdal-Bengtson B, et al. Proteomic comparison defines novel markers to characterize heterogenous population of extracellular vesicles subtypes. Proc Natl Acad Sci USA. 2016;113:E968-77.

Samuel M, Chisanga D, Liem M, Keerthikumar S, Anand S, Ang CS, et al. Bovine milk-derived exosomes from colostrum are enriched with proteins implicated in immune response and growth. Sci Rep. 2017;7:5933.

Adriano B, Cotto NC, Chauhab N, Jaggi M, Chauhan SC, Yallapu MM. Milk exosomes: nature's abundant nanoplatform for theranostic applications. Bioact Mater. 2021;6:2479-90.

Rasidi M, Bijari S, Khazaei AH, Shojaei-Ghahrizjani F, Rezakhani L. The role of milk-derived exosomes in the treatment of diseases. Front Genet. 2022;13:1009338.

Wolf T, Baier SR, Zempleni J. The intestinal transport of bovine milk exosomes is mediated by endocytosis in human colon carcinoma Caco-2 cells and rat small intestinal IEC-6 cells. J Nutr. 2015;145:2201-6.

Benmoussa A, Lee CHC, Laffont B, Savard P, Laugier J, Boilard E, et al. Commercial dairy cow milk microRNAs resist digestion under simulated gastrointestinal tract conditions. J Nutr. 2016;146:2206-15.

Tian M, Hao D, Liu Y, He J, Zhao Z, Guo T, et al. Milk exosomes: an oral drug delivery system with great application potential. Food Funct. 2023;14:1320-37.

Chutipongtanate S, Morrow AL, Newburg DS. Human milk extracellular vesicles: a biological system with clinical implications. Cells. 2022;11:2345.

Munir J, Ngu A, Wang H, Ramirez DMO, Zempleni J. Review: Milk small extracellular vesicles for use in the delivery of therapeutics. Pharm Res. 2022;40(4):909-15.

Babaker MA, Aljoud FA, Alkhilaiwi F, Algarni A, Ahmed A, Khan MI, et al. The therapeutic potential of milk extracellular vesicles on colorectal cancer. Int J Mol Sci. 2022;23:6812.

Zonneveld MI, Brisson AR, van Herwijnen MJC, Tan S, van de Lest CHA, Redegeld FA, et al. Recovery of extracellular vesicles from human breast milk is influenced by sample collection and vesicle isolation procedures. J Extracell Vesicles. 2014;3:24215. https://doi.org/10.3402/jev.v3.24215

Rahman MM, Shimizu K, Yamauchi M, Takase H, Ugawa S, Okada A, et al. Acidification effects on isolation of extracellular vesicles from bovine milk. PLoS One. 2019;14:e0222613.

Somiya M, Yoskioka Y, Ochiya T. Biocompatibility of highly purified bovine milk-derived extracellular vesicles. J Extracell Vesicles. 2018;7:1440132.

Chaiyasut C, Tsuda T. Isoelectric points estimation of proteins by electroosmotic flow: pH relationship using physically adsorbed proteins on silica gel. Chromatography. 2001;22:91-6.

Morozumi M, Izumi H, Shimizu T, Takeda Y. Comparison of isolation methods using commercially available kits for obtaining extracellular vesicles from cow milk. J Dairy Sci. 2021;104:6463-71.

Tan X, Fang D, Xu Y, Nan T, Song W, Gu Y, et al. Skimmed bovine milk-derived extracellular vesicles isolated via “salting-out”: characterizations and potential functions as nanocarriers. Front Nutr. 2021;8:769223.

Benmoussa A, Michel S, Gilbert C, Provost P. Isolating multiple extracellular vesicles subsets, including exosomes and membrane vesicles, from bovine milk using sodium citrate and differential ultracentrifugation. Bio Protoc. 2020;10:e3636.

Sedykh SE, Purvinsh LV, Burkova EE, Dmitrenok PS, Ryabchikova EI, Nevinsky GA. Analysis of proteins and peptides of highly purified CD9+ and CD63+ horse milk exosomes isolated by affinity chromatography. Int J Mol Sci. 2022;23:16106.

Marsh SR, Pridham KJ, Jourdan J, Gourdie RG. Novel protocols for scalable production of high quality purified small extracellular vesicles from bovine milk. Nanotheranostics. 2021;5:488-98.

Morcol T, He Q, Bell SJD. Model process for removal of caseins from milk of transgenic animals. Biotechnol Prog. 2001;17:577-82.

Giancaterino S, Boi C. Alternative biological sources for extracellular vesicles production and purification strategies for process scale-up. Biotechnol Adv. 2023;63:108092.

Li X, Su L, Zhang X, Chen Q, Wang Y, Shen Z, et al. Recent advances on the function and purification of milk exosomes: a review. Front Nutr. 2022;9:871346.

Lin T, Meletharayil G, Kapoor R, Abbaspourrad A. Bioactives in bovine milk: chemistry, technology, and applications. Nutr Rev. 2021;79(Supp l2):48-69.

Sergius-Ronot M, Suwai S, Shama S, Chamberland J, Unger S, O'Connor DL, et al. The ultrafiltration molecular weight cut-off has a limited effect on the concentration and protein profile during preparation of human milk protein concentrates. J Dairy Sci. 2021;104:3820-31.

Wang N, Jiang X, Xu X, Liu Y, Liu L, Lu A, et al. An aptamer affinity column for purification and enrichment of lactoferrin in milk. J Chromatogr B Anal Technol Biomed Life Sci. 2021;1178:122724.

Hernandez-Caravaca I, Cabanas A, Lopez-Ubeda R, Gonzalez-Brusi L, Guillen-Martinez A, Izquierdo-Rico MJ, et al. Analysis of minor proteins present in breast milk by using WGA lectin. Children (Basel). 2022;9:1084.

Wang M, Zhang F, Tang Y, Ali MM, Shen Z, Debrah AA, et al. Boron-doped titania for separation and purification of lactoferrin in dairy products. J Chromatogr B Anal Technol Biomed Life Sci. 2022;1212:123501.

Ghafoori Z, Tehrani T, Pont L, Benavente F. Separation and characterization of bovine milk proteins by capillary electrophoresis-mass spectrometry. J Sep Sci. 2022;45:3614-23.

Sharma N, Sharma R, Rayput SY, 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. 2020;114:198-204.

Nakano T, Ozimek L, Betti M. Separation of bovine κ-casein glycomacropeptide from sweet whey protein products with undetectable level of phenylalanine by protein precipitation followed by anion exchange chromatography. J Dairy Res. 2018;85:110-3.

Baieli MF, Urtasun N, Martinez MJ, Hirsch DB, Pilosof AMR, Miranda MV, et al. Affinity chromatography matrices for depletion and purification of casein glycomacropeptide from bovine whey. Biotechnol Prog. 2017;33:171-80.

Kinghorn NM, Norris CS, Paterson GR, Otter DE. Comparison of capillary electrophoresis with traditional methods to analyse bovine whey proteins. J Chromatogr A. 1995;700:111-23.

Heck JML, Olieman C, Schennink A, van Valenberg HJF, Visker MHPW, Meuldijk RCR, et al. Estimation of variation in concentration, phosphorylation and genetic polymorphism of milk proteins using capillary zone electrophoresis. Int Dairy J. 2008;18:548-55.

Tsakali E, Chatzilazarou A, Houhoula D, Koulouris S, Tsaknis J, Van Impe J. A rapid HPLC method for the determination of lactoferrin in milk of various species. J Dairy Res. 2019;86:238-41.

Bonfatti V, Grigoletto L, Cecchinato A, Gallo L, Carnier P. Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants. J Chromatogr A. 2008;1195:101-6.

Fuerer C, Jenni R, Cardinaux L, Andetsion F, Wagniere S, Moulin J, et al. Protein fingerprinting and quantification of β-casein variants by ultra-performance liquid chromatography-high resolution mass spectrometry. J Dairy Sci. 2020;103:1193-207.

Meyer S, Clases D, de Vega RG, Padula MP, Doble PA. Separation of intact proteins by capillary electrophoresis. Analyst. 2022;147:2988-96.

Horka M, Salplachta J, Karasek P, Roth M. Sensitive identification of milk protein allergens using on-line combination of transient isotachophoresis/micellar electrokinetic chromatography and capillary isoelectric focusing in fused silica capillary with roughened part. Food Chem. 2022;377:131986.

Oberckal J, Liaqat H, Matijasic BB, Rozman V, Treven P. Quantification of lactoferrin in human milk using monolithic cation exchange HPLC. J Chromatogr B Anal Technol Biomed Life Sci. 2023;1214:123548.

Seker S, Alharthi S, Aydogan C. Open tubular nano-liquid chromatography with a new polylysine grafted on graphene oxide stationary phase for the separation and determination of casein protein variants in milk. J Chromatogr A. 2022;1667:462885.

Schulmeister U, Hochwallner H, Swoboda I, Focke-Tejkl M, Geller B, Nystrand M, et al. Cloning, expression, and mapping of allergenic determinants of alphaS1-casein, a major cow's milk allergen. J Immunol. 2009;182:7019-29.

Maehashi K, Huang L. Bitter peptides and bitter taste receptors. Cell Mol Life Sci. 2009;66:1661-71.

Ewert J, Schlierenkamp F, Nesensohn L, Fischer L, Stressler T. Improving the colloidal and sensory properties of a caseinate hydrolysate using particular exopeptidases. Food Funct. 2018;9:5989-98.

Raksakulthai R, Haard NF. Exopeptidases and their application to reduce bitterness in food: a review. Crit Rev Food Sci Nutr. 2003;43:401-45.

FitzGerald RJ, O'Cuinn G. Enzymatic debittering of food protein hydrolysates. Biotechnol Adv. 2006;24:234-7.

Hinnenkamp C, Ismail BP. A proteomics approach to characterizing limited hydrolysis of whey protein concentrate. Food Chem. 2021;350:129235.

Liu B, Li N, Chen F, Zhang J, Sun X, Xu L, et al. Review on the release mechanism and debittering technology of bitter peptides from protein hydrolysates. Compr Rev Food Sci Food Saf. 2022;21:5153-70.

Hohme L, Fischer C, Kleinschmidt T. Characterization of bitter peptides in casein hydrolysates using comprehensive two-dimensional liquid chromatography. Food Chem. 2023;404(Pt A),134527.

Alu'datt MH, Al-U'datt DGF, Alhamad MN, Tranchant CC, Rababah T, Gammoh S, et al. Characterization and biological properties of peptides isolated from dried fermented cow milk products by RP-HPLC: amino acid composition, antioxidant, antihypertensive, and antidiabetic properties. J Food Sci. 2021;86:3046-60.

Aslebagh R, Whitham D, Channaveerappa D, Mutsengi P, Pentecost BT, Arcaro KF, et al. Mass-spectrometry-based proteomics of human milk to identify differentially expressed proteins in women with breast cancer versus controls. Proteomes. 2022;10:36.

Calvano CD, Bianco M, Losito I, Cataldi TRI. Proteomic analysis of food allergens by MALDI TOF/TOF mass spectrometry. Methods Mol Biol. 2021;2178:357-76.

Di Francesco L, Di Girolamo F, Mennini M, Masotti A, Salvatori G, Rigon G, et al. A MALDI-TOF MS approach for mammalian, human, and formula milks’ profiling. Nutrients. 2018;10:1238.

Mansor M, Al-Obaidi JR, Jaafar NN, Ismail IH, Zakaria AF, Abidin MAZ, et al. Optimization of protein extraction method for 2DE proteomics of goat's milk. Molecules. 2020;25:2625.

Piovesana S, Capriotti AL, Cavaliere C, La Barbera G, Samperi R, Zenezini Chiozzi R, et al. Peptidome characterization and bioactivity analysis of donkey milk. J Proteomics. 2015;119:21-9.

Segl M, Stutz H. Bottom-up analysis of proteins by peptide mass fingerprinting with tCITP-CZE-ESI-TOF MS after tryptic digest. Methods Mol Biol. 2022;2531:93-106.

Monopoli A, Nacci A, Cataldi TRI, Calvano CD. Synthesis and matrix properties of α-cyano-5-phenyl-2,4-pentadienic acid (CPPA) for intact proteins analysis by matrix-assisted laser desorption/ionization mass spectrometry. Molecules. 2020;25:6054.

Lu Y, Dai J, Zhang S, Qiao J, Lian H, Mao L. Identification of characteristic peptides of casein in cow milk based on MALDI-TOF MS for direct adulteration detection of goat milk. Foods. 2023;12:1519.

Guo D, Deng X, Gu S, Chen N, Zhang X, Wang S. Online trypsin digestion coupled with LC-MS/MS for detecting of A1 and A2 types of β-casein proteins in pasteurized milk using biomarker peptides. J Food Sci Technol. 2022;59:2983-91.

Miranda G, Bianchi L, Krupova Z, Trossat P, Martin P. An improved LC-MS method to profile molecular diversity and quantify the six main bovine milk proteins, including genetic and splicing variants as well as post-translationally modified isoforms. Food Chemistry: X. 2020;5:100080.

Li W, Huang J, Zheng L, Liu W, Fan L, Sun B, et al. A fast stop-flow two-dimensional liquid chromatography tandem mass spectrometry and its application in food-derived protein hydrolysates. Food Chem. 2023;406:135000.

Minkiewicz P, Iwaniak A, Darewicz M. BIOPEP-UWM database of bioactive peptides: current opportunities. Int J Mol Sci. 2019;20:5978.

Cui L, Yang G, Lu S, Zeng X, He J, Guo Y, et al. Antioxidant peptides derived from hydrolyzed milk proteins by Lactobacillus strains: A BIOPEP-UWM database-based analysis. Food Res Int. 2022;156:111339.

Ashokbhai JK, Basaiawmoit B, Sakure A, Das S, Patil GB, Mankad M, et al. Purification and characterization of antioxidative and antimicrobial peptides from lactic-fermented sheep milk. J Food Sci Technol. 2022;59:4262-72.

Nielsen SD, Beverly RL, Qu Y, Dallas DC. Milk bioactive peptide database: a comprehensive data-base of milk protein-derived bioactive peptides and novel visualization. Food Chem. 2017;232:673-82.

Minkiewicz P, Iwaniak A, Darewitz M. BIOPEP-UWM database of bioactive peptides: current opportunities. Int J Mol Sci. 2019;20:5978.

Cui Q, Sun Y, Cheng J, Guo M. Effect of two-step enzymatic hydrolysis on the antioxidant properties and proteomics of hydrolysates of milk protein concentrate. Food Chem. 2022;366:130711.

Cui Q, Zhang Z, Li M, Zhou M, Sun X. Peptide profiles and allergy-reactivity of extensive hydrolysates of milk protein. Food Chem. 2023;411:135544.

Xiao H, Jiang H, Tu H, Jia Y, Wang H, Lu X, et al. Extraction, isolation and identification of low molecular weight peptides in human milk. Foods. 2022;11:1836.

Dingess KA, Gazi I, van den Toorn HWP, Mank M, Stahl B, Reiding KR, et al. Monitoring human milk β-casein phosphorylation and O-glycosylation over lactation reveals distinct differences between the proteome and endogenous peptidome. Int J Mol Sci. 2021;22:8140.

Kumar BSG, Lijina P, Jinesh P, Anagha SM. N-glycoprofiling of immunoglobulin G and lactoferrin with site-specificity from goat milk using RP-UHPLC MS/MS. Food Chem. 2022;383:132376.

Valk-Weeber RL, Eshuis-de Ruiter T, Dijkhuizen L, van Leeuwen SS. Dynamic temporal variations in bovine lactoferrin glycan structures. J Agric Food Chem. 2020;68:549-60.

Kumar BSG, Lijina P, Akshata SH. N-glycoprofiling of lactoferrin with site-specificity from buffalo colostrum. Int Dairy J. 2022;127:105215.

Nwosu CC, Aldredge DL, Lee H, Lerno LA, Zivkovic AM, German JB, et al. Comparison of the human and bovine milk N-glycome via high-performance microfluidic chip liquid chromatography and tandem mass spectrometry. J Proteome Res. 2012;11:2912-24.

Sudarshan M, Shree VD, Jyothibai P, Kumar BSG. N-glycoprofiling of immunoglobulin G and lactoperoxidase from sheep milk using LC-MS/MS. Int Dairy J. 2023;140:105582.

Reinhardt TA, Sacco RE, Nonnecke BJ, Lippolis JD. Bovine milk proteome: quantitative changes in normal milk exosomes, milk fat globule membranes and whey proteomes resulting from Staphylococcus aureus mastitis. J Proteom. 2013;82:141-54.

Delosiere M, Pires JAA, Bernard L, Cassar-Malek I, Bonnet M. Dataset reporting 4654 cow milk proteins listed according to lactation stages and milk fractions. Data Brief. 2020;29:105105.

Ferreira RF, Blees T, Shakeri F, Buness A, Sylvester M, Savoini G, et al. Comparative proteome profiling in exosomes derived from porcine colostrum versus mature milk reveals distinct functional proteomes. J Proteomics. 2021;249:104338.

Koh YQ, Peiris HN, Vaswani K, Meier S, Burke CR, Macdonald KA, et al. Characterization of exosomes from body fluids of dairy cows. J Anim Sci. 2017;95:3893-904.

Chen W, Wang R, Li D, Zuo C, Wen P, Liu H, et al. Comprehensive analysis of the glycome and glycoproteome of bovine milk-derived exosomes. J Agric Food Chem. 2020;68:12692-701.

Rahman MM, Takashima S, Kamatari YO, Badr Y, Kitamura Y, Shimizu K, et al. Proteomic profiling of milk small extracellular vesicles from bovine leukemia virus-infected cattle. Sci Rep. 2021;11:2951.

Vahkal B, Kraft J, Ferretti E, Chung M, Beaulie J, Altosaar I. Review of methodological approaches to human milk small extracellular vesicle proteomics. Biomolecules. 2021;11:833.

Maity S, Bhat AH, Giri K, Ambatipudi K. BoMiProt: a database of bovine milk proteins. J Proteom. 2020;215:103648.

Das A, Giri K, Behera RN, Maity S, Ambatipudi K. BoMiProt 2.0: an update of the bovine milk protein database. J Proteomics. 2022;267:104696.

Shtatland T, Guettler D, Kossodo M, Pivovarov M, Weissleder R. PepBank-a database of peptides based on sequence text mining and public peptide data sources. BMC Bioinform. 2007;8:280.

Li Q, Zhang C, Chen H, Xue J, Guo X, Liang M, et al. BioPepDB: an integrated data platform for food-derived bioactive peptides. Int J Food Sci Nutr. 2018;69:963-8.

Wang G, Li X, Wang Z. APD3: the antimicrobial peptide database as a tool for research and education. Nucleic Acids Res. 2016;44:D1087-93.

Thomas S, Karnik S, Barai RS, Jayaraman VK, Idicula-Thomas S. CAMP: a useful resource for research on antimicrobial peptides. Nucleic Acids Res. 2010;38:D774-80.

Gawde U, Chakraborty S, Waghu FH, Barai RS, Khanderkar A, Indraguru R, et al. CAMPR4: a database of natural and synthetic antimicrobial peptides. Nucleic Acids Res. 2023;51:D377-83.

Waghu FH, Idicula-Thomas S. Collection of antimicrobial peptides database and its derivatives: applications and beyond. Protein Sci. 2020;29:36-42.

Keerthikumar S, Chisanga D, Ariyaratne D, Saffar HA, Anand S, Zhao K, et al. ExoCarta: a web-based compendium of exosomal cargo. J Mol Biol. 2016;428:688-92.

Pathan M, Fonseka P, Chitti SV, Kang T, Sanwlani R, Van Deun J, et al. Vesiclepedia 2019: a compendium of RNA, proteins, lipids and metabolites in extracellular vesicles. Nucleic Acids Res. 2019;47(D1):D516-9.

Mohan A, McClements DJ, Udenigwe CC. Encapsulation of bioactive whey peptides in soy lecithin-derived nanoliposomes: influence of peptide molecular weight. Food Chem. 2016;213:143-8.

Liu W, Ye A, Liu W, Liu C, Singh H. Stability during in vitro digestion of lactoferrin-loaded liposomes prepared from milk fat globule membrane-derived phospholipids. J Dairy Sci. 2013;96:2061-70.

Santos-Coquillat A, Gonzales MI, Clemente-Moragon A, Gonzales-Arjona M, Albaladejo-Garcia V, Peinado H, et al. Goat milk exosomes as natural nanoparticles for detecting inflammatory processes by optical imaging. Small. 2022;18:e2105421.

The Role of Proteomics in Identification of Key Proteins of Bacterial Cells with Focus on Probiotic Bacteria