Detection of low-copy proteins in complex biological samples is one of the most important issues of modern proteomics. The main reason for inefficient detection of low protein concentrations is the insufficient sensitivity of mass spectrometric detectors and the high dynamic range of protein concentrations. In this study we have investigated the possibilities and limitations of a targeted mass spectrometric analysis using the reconstructed system of standard proteins UPS1 (Universal Proteomic Standard 1) as an example. The study has shown that the sensitivity of the method is affected by the concentration of target proteins of the UPS1 system, as well as by a high level of biological noise modelled by proteins of whole E. coli cell lysate. The limitations of the method have been overcome by concentrating and pre-fractionating the sample peptides in a reversed phase chromatographic system under alkaline elution conditions. Proteomic analysis of the biological sample (proteins of the human hepatocellular carcinoma cell line HepG2 encoded by genes of human chromosome 18) showed an increase in the sensitivity of the method as compared to the standard targeted mass spectrometric analysis. This culminated in registration of 94 proteins encoded by genes located on human chromosome18.
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Keywords: proteomics, mass spectrometry, HepG2, human chromosome 18 gene products
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Archakov A.I., Vavilov N.E., Zgoda V.G. (2024) Detection of low-copy proteins in proteomic studies: issues and solutions. Biomeditsinskaya Khimiya, 70(5), 342-348.
Archakov A.I. et al. Detection of low-copy proteins in proteomic studies: issues and solutions // Biomeditsinskaya Khimiya. - 2024. - V. 70. -N 5. - P. 342-348.
Archakov A.I. et al., "Detection of low-copy proteins in proteomic studies: issues and solutions." Biomeditsinskaya Khimiya 70.5 (2024): 342-348.
Archakov, A. I., Vavilov, N. E., Zgoda, V. G. (2024). Detection of low-copy proteins in proteomic studies: issues and solutions. Biomeditsinskaya Khimiya, 70(5), 342-348.
Hancock W., Omenn G., Legrain P., Paik Y.K. (2011) Proteomics, human proteome project, and chromosomes. J. Proteome Res., 10(1), 210. CrossRef Scholar google search
Kopylov A.T., Zgoda V.G., Lisitsa A.V., Archakov A.I. (2013) Combined use of irreversible binding and MRM technology for low- and ultralow copy-number protein detection and quantitation. Proteomics, 13(5), 727. CrossRef Scholar google search
Anderson N.L., Polanski M., Pieper R., Gatlin T., Tirumalai R.S., Conrads T.P., Veenstra T.D., Adkins J.N., Pounds J.G., Fagan R., Lobley A. (2004) The human plasma proteome: A nonredundant list developed by combination of four separate sources. Mol. Cell. Proteomics, 3(4), 311. CrossRef Scholar google search
Michalski A., Cox J., Mann M. (2011) More than 100,000 detectable peptide species elute in single shotgun proteomics runs but the majority is inaccessible to data-dependent LC-MS/MS. J. Proteome Res., 10(4), 1785. CrossRef Scholar google search
Vavilov N., Ilgisonis E., Lisitsa A., Ponomarenko E., Farafonova T., Tikhonova O., Zgoda V., Archakov A. (2022) Number of detected proteins as the function of the sensitivity of proteomic technology in human liver cells. Curr. Protein Pept. Sci., 23(4), 290. CrossRef Scholar google search
Deinichenko K., Krasnov G., Radko S., Ptitsyn K., Shapovalova V., Timoshenko O., Khmeleva S., Kurbatov L., Kiseleva Y., Ilgisonis E., Pyatnitskiy M., Poverennaya E., Kiseleva O., Vakhrushev I., Tsvetkova A., Buromski I., Markin S., Zgoda V., Archakov A., Lisitsa A., Ponomarenko E. (2021) Human CHR18: “Stakhanovite” genes, missing and uPE1 proteins in liver tissue and HepG2 cells. Biomedical Chemistry: Research and Methods, 4(1), e00144. CrossRef Scholar google search
Furey A., Moriarty M., Bane V., Kinsella B., Lehane M. (2013) Ion suppression: A critical review on causes, evaluation, prevention and applications. Talanta, 115, 104. CrossRef Scholar google search
Mei H., Hsieh Y., Nardo C., Xu X., Wang S., Ng K., Korfmacher W.A. (2003) Investigation of matrix effects in bioanalytical high-performance liquid chromatography/ tandem mass spectrometric assays: Application to drug discovery. Rapid Commun. Mass Spectrom., 17(1), 97. CrossRef Scholar google search
Muller L., Fornecker L., van Dorsselaer A., Cianférani S., Carapito C. (2016) Benchmarking sample preparation/ digestion protocols reveals tube-gel being a fast and repeatable method for quantitative proteomics. Proteomics, 16(23), 2953. CrossRef Scholar google search
Ramus C., Hovasse A., Marcellin M., Hesse A.M., Mouton-Barbosa E., Bouyssié D., Vaca S., Carapito C., Chaoui K., Bruley C., Garin J., Cianférani S., Ferro M., van Dorssaeler A., Burlet-Schiltz O., Schaeffer C., Couté Y., Gonzalez de Peredo A. (2016) Benchmarking quantitative label-free LC-MS data processing workflows using a complex spiked proteomic standard dataset. J. Proteomics, 132, 51. CrossRef Scholar google search
Matuszewski B.K., Constanzer M.L., Chavez-Eng C.M. (2003) Strategies for the assessment of matrix effect in quantitative bioanalytical methods based on HPLC-MS/MS. Anal. Chem., 75(13), 3019. CrossRef Scholar google search
Kodo K., Nishizawa T., Furutani M., Arai S., Yamamura E., Joo K., Takahashi T., Matsuoka R., Yamagishi H. (2009) GATA6 mutations cause human cardiac outflow tract defects by disrupting semaphorin-plexin signaling. Proc. Natl. Acad. Sci. USA, 106(33), 13933. CrossRef Scholar google search
Maekawa M., Ishizaki T., Boku S., Watanabe N., Fujita A., Iwamatsu A., Obinata T., Ohashi K., Mizuno K., Narumiya S. (1999) Signaling from Rho to the actin cytoskeleton through protein kinases ROCK and LIM-kinase. Science, 285(5429), 895. CrossRef Scholar google search
Déléris P., Trost M., Topisirovic I., Tanguay P.L., Borden K.L., Thibault P., Meloche S. (2011) Activation loop phosphorylation of ERK3/ERK4 by group I p21-activated kinases (PAKs) defines a novel PAK-ERK3/4-MAPKactivated protein kinase 5 signaling pathway. J. Biol. Chem., 286(8), 6470. CrossRef Scholar google search
Koch C.M., Chiu S.F., Akbarpour M., Bharat A., Ridge K.M., Bartom E.T., Winter D.R. (2018) A beginner's guide to analysis of RNA sequencing data. Am. J. Respir. Cell Mol. Biol., 59(2), 145. CrossRef Scholar google search
Reimegård J., Tarbier M., Danielsson M., Schuster J., Baskaran S., Panagiotou S., Dahl N., Friedlander M.R., Gallant C.J. (2021) A combined approach for single-cell mRNA and intracellular protein expression analysis. Commun. Biol., 4(1), 624. CrossRef Scholar google search
Shyu A.B., Greenberg M.E., Belasco J.G. (1989) The c-fos transcript is targeted for rapid decay by two distinct mRNA degradation pathways. Genes Dev., 3(1), 60. CrossRef Scholar google search
Treisman R. (1985) Transient accumulation of c-fos RNA following serum stimulation requires a conserved 5′ element and c-fos 3′ sequences. Cell, 42(3), 889. CrossRef Scholar google search
Mathieson T., Franken H., Kosinski J., Kurzawa N., Zinn N., Sweetman G., Poeckel D., Ratnu V.S., Schramm M., Becher I., Steidel M., Noh K.M, Bergamini G., Beck M., Bantscheff M., Savitski M.M. (2018) Systematic analysis of protein turnover in primary cells. Nat. Commun., 9(1), 689. CrossRef Scholar google search
Bevilacqua A., Ceriani M.C., Canti G., Asnaghi L., Gherzi R., Brewer G., Papucci L., Schiavone N., Capaccioli S., Nicolin A. (2003) Bcl-2 protein is required for the adenine/ uridine-rich element (ARE)-dependent degradation of its own messenger. J. Biol. Chem., 278(26), 23451. CrossRef Scholar google search
Yang E., van Nimwegen E., Zavolan M., Rajewsky N., Schroeder M., Magnasco M., Darnell J.E. Jr. (2003) Decay rates of human mRNAs: Correlation with functional characteristics and sequence attributes. Genome Res., 13(8), 1863. CrossRef Scholar google search
Ponomarenko E.A., Poverennaya E.V., Ilgisonis E.V., Pyatnitskiy M.A., Kopylov A.T., Zgoda V.G., Lisitsa A.V., Archakov A.I. (2016) The size of the human proteome: The width and depth. Int. J. Anal. Chem., 2016, 7436849. CrossRef Scholar google search
