Biomedical Chemistry: Research and Methods 2025, 8(2), e00277

PROTOCOLS FOR PROTEOMIC ANALYSIS: ISOLATION, SOLUBILIZATION AND HYDROLYSIS BY PROTEASES

E.N. Obukhova1, E.V. Khryapova2, S.E. Novikova2, L.Sh. Kazieva2, O.V. Tikhonova2, V.G. Zgoda*2

1Skolkovo Institute of Science and Technology, 30c1 Bolshoi Boulevard, Moscow, 121205 Russia
2Institute of Biomedical Chemistry, 10 Pogodinskaya str., Moscow, 119435 Russia; *e-mail: victor.zgoda@gmail.com

Keywords: proteomics; protein solubilization; highly specific protease; protocol

DOI:10.18097/BMCRM00277

The whole version of this paper is available in Russian.

High-throughput studies of protein composition of biological samples have become routine and are used practically in all areas of life sciences. Modern proteomics methods allow reliable identification and quantification of thousands of proteins in a single experiment. The standard procedure for proteomic analysis includes the following steps: 1. isolation and solubilization of proteins, their hydrolysis by proteases; 2. analysis of the resulting peptides by high-performance liquid chromatography with mass spectrometric detection; 3. bioinformatics and statistical processing of the results. This paper presents protocols of the first stage of proteomic analysis, i.e. sample preparation, which are routinely used in the Laboratory of Systems Biology of the Institute of Biomedical Chemistry.

Table 1. The enzymes most commonly used in proteomics.

Enzyme

Cleavage site

рН optimum

Buffer

Trypsin

Arg-X, Lys-X*. -Lys-Pro- and -Arg-Pro- are trypsin-resistant.

7.5**

50 mM ТЕАB***

Chymotrypsin

Chymotrypsinogen A. Cleaves at the C-terminal Tyr, Trp, Phe, partially - C-terminal Leu, Met, Ala, Asp.

7.8 – 8

50 mM ТЕАB

Lys-C

Lysyl endopeptidase. Cleaves at the C-terminus of lysine.

7 – 9

50 mM ТЕАB

Glu-C

Glutamyl peptidase. Specificity depends on buffer composition. In ammonium bicarbonate (pH 7-8) or ammonium acetate (pH 4) it cleaves Glu-X bonds. In phosphate buffers (pH 7.8-8.5) it cleaves Glu-X, Asp-X bonds.

4 or 8

50 mM ТЕАB

Asp-N

Peptidyl-Asp metallopeptidase. Cleaves at the arginine N-terminus.

4 – 9

 

Lys-N

Peptidyl-Lys metalloendopeptidase. Cleaves at the lysine N-terminus.

9.5

50 mM ТЕАB

Pepsin

Non-specific enzyme. Most rapidly hydrolyzes peptide bonds of aromatic amino acids.

1 – 4 ****

40 mN HCl or 100 mM Na-citrate buffer

Arg-C

Clostripain. Cleaves at the arginine C-terminus.

7 – 8

50 mM ТЕАB

Papain

Plant “pepsin”. Hydrolyzes amides, peptides, proteins with unsubstituted amino groups.

Working pH 3-12, optimal pH 5-8

50 mM Na-phosphate buffer, 50 mM L-cysteine, 2  mM EDTA

IdeZ

Recombinant enzyme, recognizes human, sheep, monkey and rabbit antibodies. Specifically cleaves antibodies into Fab and Fc fragments.

6.6

50 mM Na-phosphate buffer, 150 mM NaCl

Note: *X – any amino acid residue; ** pH is adjusted with sodium hydroxide; *** TEAB - triethylammonium bicarbonate; **** pH is adjusted with 1 N hydrochloric acid

Table 2. Recommendations for the selection of protocols for sample preparation in proteomic experiments

Sample

Minimum required sample quantity

Recommended protocols

Ref.

Bacteria

1 million cells

Protocol 7 or Protocol 8 

[13], [14]

Eukaryotic cells

0.2 – 1 million cells

Protocol 1 or Protocol 9

[15], [16], [17]

Brain cells, Fat cells

0.2 – 1 million cells

Protocol 3 (or Protocol 5) -> Protocol 7 or Protocol 8

[18], [19]

Tissues

0.2 – 1 mg

Protocol 7 or Protocol 8

[20], [21], [22]

Membrane fractions of cells, nuclei

0.050 – 0.1 mg

Protocol 3 -> Protocol 7 or Protocol 8

[23], [24], [25]

Biological fluids:

 

Plasma / serum

0.01 – 0.05 ml

Protocol 1 or Protocol 9

[26], [27], [28]

Urine

5 ml

Protocol 5 -> Protocol 1

[29]

Spinal fluid

0.1 ml

Protocol 1

[30], [31]

Saliva

0.5 – 1 ml

Protocol 1

[32]

Tears, eye fluid

0.050 – 0.1 ml

Protocol 1

[33]

Food (milk, sausages, meat, fish, invertebrates, eggs, mucus, biofilms, etc.)

Usually present in excess

Protocol 3 (or Protocol 5) -> Protocol 7 or Protocol 8

Protocol 4

[34], [35], [36], [37]

Purified fractions / proteins

10 – 50 µg

Protocol 1

Protocol 6**, 8

[38], [39], [40]

Purified antibody

10 – 50 µg

Protocols 1, 10 – 14

 

Note: *If the preparation contains high concentrations of detergents, glycine, imidazole and other compounds used for protein storage and isolation.

Table 3. Preparation of calibrants for measuring protein concentration using the BCA method on a spectrophotometer CLARIOStar

№ 

Calibrant concentration, µg/mL

Volume of stock solution

Water, µl 

Protein concentration after addition of 1 ml BCA, µg/ml

1 

2 

2 µl of Solution 2

28 

0.066

2 

5 

5 µl of Solution 2

25 

0.166

3 

10 

10 µl of Solution 2

20 

0.33

4 

25 

2.5 µl of Solution 1

27.5 

0.83

5 

50 

5 µl of Solution 1

25 

1.66

6 

75 

7.5 µl of Solution 1

22.5 

2.5

Blank

0 

0 

30 

0

Table 4. Preparation of calibrants for measuring peptide concentration using the Pierce Quantitative Colorimetric Peptide Assay kit

Calibrant

Stock solutions volumes 

Water, µl

Calibrant concentration, µg/µl

1 

Stock 

 

1000

2 

10 µl of Stock 

10

500

3 

10 µl of calibrant 2

10

250

4 

10 µl of calibrant 3

10

125

5 

10 µl of calibrant 4

10

62,5

6 

10 µl of calibrant 5

10

31,3

7

10 µl of calibrant 6

10

15,6

Blank

0 

20

0

 

FUNDING

The work was carried out within the framework of the Russian Federation fundamental research program for the long-term period for 2021-2030 (№-122030100168-2).

REFERENCES

  1. Kahn, P. (1995) From genome to proteome: looking at a cell’sproteins. Science (New York, N.Y.), 270(5235), 369–370. DOI
  2. Shevchenko, A., Tomas, H., Havlis, J., Olsen, J.V., Mann, M. (2006) In-geldigestion for mass spectrometric characterization of proteins and proteomes.Nature Protocols, 1(6), 2856–2860. DOI
  3. Thanou, E., Koopmans, F., Pita-Illobre, D., Klaassen, R.V., Özer, B.,Charalampopoulos, I., Smit, A.B., Li, K.W. (2023) Suspension TRAPping Filter(sTRAP) Sample preparation for quantitative proteomics in the low μg inputrange using a plasmid DNA micro-spin column: analysis of the hippocampusfrom the 5xFAD Alzheimer’s disease mouse model. Cells, 12(9), 1242. DOI
  4. Wiśniewski, J.R., Zougman, A., Nagaraj, N., Mann, M. (2009) Universalsample preparation method for proteome analysis. Nature Methods, 6(5),359–362. DOI
  5. Ji, Y., Liu, M., Bachschmid, M. M., Costello, C. E., Lin, C. (2015). Surfactant-Induced Artifacts during Proteomic Sample Preparation. Analytical chemistry,87(11), 5500–5504. DOI
  6. Pirmoradian, M., Budamgunta, H., Chingin, K., Zhang, B., Astorga-Wells,J., Zubarev, R.A. (2013) Rapid and deep human proteome analysis by singledimensionshotgun proteomics. Molecular & Cellular Proteomics: MCP, 12(11),3330–3338. DOI
  7. Saveliev, S.V., Woodroofe, C.C., Sabat, G., Adams, C.M., Klaubert, D., Wood,K., Urh, M. (2013) Mass spectrometry compatible surfactant for optimizedin-gel protein digestion. Analytical Chemistry, 85(2), 907–914. DOI
  8. Danko, K., Lukasheva, E., Zhukov, V.A., Zgoda, V., Frolov, A. (2022)Detergent-assisted protein digestion-on the way to avoid the key bottleneck ofshotgun bottom-up proteomics. International Journal of Molecular Sciences,23(22), 13903. DOI
  9. Tsiatsiani, L., Heck, A.J. (2015) Proteomics beyond trypsin. The FEBSJournal, 282(14), 2612–2626. DOI
  10. Kazieva, L. S., Farafonova, T. E., Zgoda, V. G. (2023) Proteomika antitel[Antibody proteomics]. Biomeditsinskaya Khimiya, 69(1), 5–18. DOI
  11. PEPTIDEMASS - a World-Wide Web accessible tool for peptidecharacterization. Retrieved May 7, 2025, from: https://web.expasy.org/peptide_mass
  12. Fragment Ion Calculator. Retrieved May 7, 2025, from:http://db.systemsbiology.net:8080/proteomicsToolkit/FragIonServlet.html
  13. Karneyeva, K., Kolesnik, M., Livenskyi, A., Zgoda, V., Zubarev,V., Trofimova, A., Artamonova, D., Ispolatov, Y., Severinov, K. (2024)Interference requirements of type III CRISPR-Cas systems from thermusthermophilus. Journal of Molecular Biology, 436(6), 168448. DOI
  14. Demina, I.A., Serebryakova, M. V., Ladygina, V.G., Rogova, M.A., Zgoda,V.G., Korzhenevskyi, D.A., Govorun, V.M. (2009) Proteome of the bacteriummycoplasma gallisepticum. Biochemistry. Biokhimiia, 74(2), 165–174. DOI
  15. Vavilov, N.E., Zgoda, V.G., Tikhonova, O.V., Farafonova, T.E., Shushkova,N.A., Novikova, S.E., Yarygin, K.N., Radko, S.P., Ilgisonis, E.V., Ponomarenko,E.A., Lisitsa, A.V., Archakov, A.I. (2020) Proteomic analysis of Chr 18 proteinsusing 2D fractionation. Journal of Proteome Research, 19(12), 4901–4906. DOI
  16. Novikova, S., Tikhonova, O., Kurbatov, L., Farafonova, T., Vakhrushev,I., Lupatov, A., Yarygin, K., Zgoda, V. (2021) Omics technologies to decipherregulatory networks in granulocytic cell differentiation. Biomolecules, 11(6),907. DOI
  17. Shkrigunov, T., Kisrieva, Y., Samenkova, N., Larina, O., Zgoda, V., Rusanov,A., Romashin, D., Luzgina, N., Karuzina, I., Lisitsa, A., Petushkova, N. (2022)Comparative proteoinformatics revealed the essentials of SDS impact onHaCaT keratinocytes. Scientific Reports, 12(1), 21437. DOI
  18. Buneeva, O.A., Kapitsa, I.G., Kazieva, L.S., Vavilov, N.E., Zgoda, V.G.(2023) Quantitative changes of brain isatin-binding proteins of rats with therotenone-induced experimental parkinsonism. Biomeditsinskaya Khimiya,69(3), 188–192. DOI
  19. Medvedev, A., Kopylov, A., Buneeva, O., Kurbatov, L., Tikhonova, O.,Ivanov, A., Zgoda, V. (2020) A Neuroprotective dose of isatin causes multilevelchanges involving the brain proteome: prospects for further research.International Journal of Molecular Sciences, 21(11), 4187. DOI
  20. Anselm, V., Novikova, S., Zgoda, V. (2017) Re-adaption on earth afterspaceflights affects the mouse liver proteome. International Journal ofMolecular Sciences, 18(8), 1763. DOI
  21. Vavilov, N.E., Ilgisonis, E.V., Lisitsa, A.V., Ponomarenko, E.A., Farafonova,T.E., Tikhonova, O.V., Zgoda, V.G., Archakov, A.I. (2022) Deep proteomicdataset of human liver samples obtained by two-dimensional samplefractionation coupled with tandem mass spectrometry. Data in Brief, 42,108055. DOI
  22. Kurochkina, N.S., Orlova, M.A., Vigovskiy, M.A., Zgoda, V.G., Vepkhvadze,T.F., Vavilov, N.E., Makhnovskii, P.A., Grigorieva, O.A., Boroday, Y.R.,Philippov, V.V., Lednev, E.M., Efimenko, A. Y., Popov, D. V. (2024) Age-relatedchanges in human skeletal muscle transcriptome and proteome are moreaffected by chronic inflammation and physical inactivity than primary aging.Aging Cell, 23(4), e14098. DOI
  23. Davydov, D.R., Dangi, B., Yue, G., Ahire, D. S., Prasad, B., Zgoda, V.G.(2022) Exploring the interactome of cytochrome P450 2E1 in human livermicrosomes with chemical crosslinking mass spectrometry. Biomolecules,12(2), 185. DOI
  24. Novikova, S., Tolstova, T., Kurbatov, L., Farafonova, T., Tikhonova, O.,Soloveva, N., Rusanov, A., Archakov, A., Zgoda, V. (2022) Nuclear proteomicsof induced leukemia cell differentiation. Cells, 11(20), 3221. DOI
  25. Zgoda, V.G., Moshkovskii, S.A., Ponomarenko, E.A., Andreewski, T.V.,Kopylov, A.T., Tikhonova, O.V., Melnik, S.A., Lisitsa, A.V., Archakov, A.I. (2009)Proteomics of mouse liver microsomes: performance of different proteinseparation workflows for LC-MS/MS. Proteomics, 9(16), 4102–4105. DOI
  26. Kopylov, A.T., Ponomarenko, E.A., Ilgisonis, E.V., Pyatnitskiy, M.A., ,A.V., Poverennaya, E.V., Kiseleva, O.I., Farafonova, T.E., Tikhonova, O.V.,Zavialova, M.G., Novikova, S.E., Moshkovskii, S.A., Radko, S.P., Morukov, B.V.,Grigoriev, A.I., Paik, Y.K., Salekdeh, G.H., Urbani, A., Zgoda, V.G., Archakov,A.I. (2019) 200+ Protein concentrations in healthy human blood plasma:targeted quantitative SRM SIS screening of chromosomes 18, 13, Y, and themitochondrial chromosome encoded proteome. Journal of Proteome Research,18(1), 120–129. DOI
  27. Zgoda, V.G., Kopylov, A.T., Tikhonova, O.V., Moisa, A.A., Pyndyk,N.V., Farafonova, T.E., Novikova, S.E., Lisitsa, A.V., Ponomarenko, E.A.,Poverennaya, E.V., Radko, S.P., Khmeleva, S.A., Kurbatov, L.K., Filimonov,A.D., Bogolyubova, N.A., Ilgisonis, E.V., Chernobrovkin, A.L., Ivanov, A.S.,Medvedev, A.E., Mezentsev, Y.V., Archakov, A.I. (2013) Chromosome 18transcriptome profiling and targeted proteome mapping in depleted plasma, livertissue and HepG2 cells. Journal of Proteome Research, 12(1), 123–134. DOI
  28. Smirnova, L., Seregin, A., Boksha, I., Dmitrieva, E., Simutkin, G.,Kornetova, E., Savushkina, O., Letova, A., Bokhan, N., Ivanova, S., Zgoda, V.(2019) The difference in serum proteomes in schizophrenia and bipolar disorder.BMC Genomics, 20(Suppl 7), 535. DOI
  29. Pastushkova, L.H., Rusanov, V.B., Goncharova, A.G., Brzhozovskiy, A.G.,Kononikhin, A.S., Chernikova, A.G., Kashirina, D.N., Nosovsky, A.M., Baevsky,R.M., Nikolaev, E.N., Larina, I.M. (2019) Urine proteome changes associatedwith autonomic regulation of heart rate in cosmonauts. BMC Systems Biology,13(Suppl 1), 17. DOI
  30. Novikova, S., Soloveva, N., Farafonova, T., Tikhonova, O., Shimansky,V., Kugushev, I., Zgoda, V. (2023) Proteomic shotgun and targeted massspectrometric datasets of cerebrospinal fluid (liquor) derived from patients withvestibular schwannoma. Data, 8(4), 71. DOI
  31. Zavialova, M.G., Shevchenko, V.E., Nikolaev, E.N., Zgoda, V.G. (2017)Is myelin basic protein a potential biomarker of brain cancer? EuropeanJournal of Mass Spectrometry (Chichester, England), 23(4), 192–196. DOI
  32. Baskova, I.P., Zavalova, L.L., Basanova, A.V., Moshkovskii, S.A., Zgoda,V.G. (2004) Protein profiling of the medicinal leech salivary gland secretion byproteomic analytical methods. Biochemistry. Biokhimiia, 69(7), 770–775. DOI
  33. Kliuchnikova, A.A., Samokhina, N. ., Ilina, I.Y., Karpov, D.S., Pyatnitskiy,M.A., Kuznetsova, K.G., Toropygin, I.Y., Kochergin, S.A., Alekseev, I.B., Zgoda,V.G., Archakov, A.I., Moshkovskii, S.A. (2016) Human aqueous humor proteomein cataract, glaucoma, and pseudoexfoliation syndrome. Proteomics, 16(13),1938–1946. DOI
  34. Odorskaya, M.V., Mavletova, D.A., Nesterov, A.A., Tikhonova, O.V.,Soloveva, N.A., Reznikova, D.A., Galanova, O.O., Vatlin, A.A., Slynko,N.M., Vasilieva, A.R., Peltek, S.E., Danilenko, V.N. (2024) The use of omicstechnologies in creating LBP and postbiotics based on the limosilactobacillusfermentum U-21. Frontiers in Microbiology, 15, 1416688. DOI
  35. Pakharukova, M.Y., Savina, E., Ponomarev, D.V., Gubanova, N.V., Zaparina,O., Zakirova, E.G., Cheng, G., Tikhonova, O.V., Mordvinov, V.A. (2023)Proteomic characterization of opisthorchis felineus exosome-like vesicles andtheir uptake by human cholangiocytes. Journal of Proteomics, 283-284, 104927. DOI
  36. Gerringer, M.E., Yancey, P.H., Tikhonova, O.V., Vavilov, N.E., Zgoda, V.G.,Davydov, D.R. (2020) Pressure tolerance of deep-sea enzymes can be evolvedthrough increasing volume changes in protein transitions: a study with lactatedehydrogenases from abyssal and hadal fishes. The FEBS Journal, 287(24),5394–5410. DOI
  37. Fedulova, L., Vasilevskaya, E., Tikhonova, O., Kazieva, L., Tolmacheva,G., Makarenko, A. (2022) Proteomic markers in the muscles and brain ofpigs recovered from hemorrhagic stroke. Genes, 13(12), 2204. DOI
  38. Filatov, A.V., Krotov, G.I., Zgoda, V.G., Volkov, Y. (2007) Fluorescentimmunoprecipitation analysis of cell surface proteins: a methodologycompatible with mass-spectrometry. Journal of Immunological Methods, 319(1-2), 21–33. DOI
  39. Kopylov, A.T., Zgoda, V.G., Lisitsa, A.V., Archakov, A.I. (2013) Combineduse of irreversible binding and MRM technology for low- and ultralow copynumberprotein detection and quantitation. Proteomics, 13(5), 727–742. DOI
  40. Laptev, I., Shvetsova, E., Levitskii, S., Serebryakova, M., Rubtsova, M.,Zgoda, V., Bogdanov, A., Kamenski, P., Sergiev, P., Dontsova, O. (2020)METTL15 interacts with the assembly intermediate of murine mitochondrialsmall ribosomal subunit to form m4C840 12S rRNA residue. Nucleic AcidsResearch, 48(14), 8022–8034. DOI
  41. Ludwig, K. R., Schroll, M. M., Hummon, A. B. (2018). Comparison of In-Solution, FASP, and S-Trap Based Digestion Methods for Bottom-Up ProteomicStudies. Journal of proteome research, 17(7), 2480–2490. DOI
  42. Wiśniewski, J. R., Ostasiewicz, P., Mann, M. (2011). High recovery FASPapplied to the proteomic analysis of microdissected formalin fixed paraffinembedded cancer tissues retrieves known colon cancer markers. Journal ofproteome research, 10(7), 3040–3049. DOI
  43. Wiśniewski, J.R., Zielinska, D.F., Mann, M. (2011) Comparison ofultrafiltration units for proteomic and N-glycoproteomic analysis by the filteraidedsample preparation method. Analytical Biochemistry, 410(2), 307–309. DOI