Quantitative protein extraction from biological samples, as well as contaminants removal before LC-MS/MS, is fundamental for the successful bottom-up proteomic analysis. Four sample preparation methods, including the filter-aided sample preparation (FASP), two single-pot solid-phase-enhanced sample preparations (SP3) on carboxylated or HILIC paramagnetic beads, and protein suspension trapping method (S-Trap) were evaluated for SDS removal and protein digestion from Arabidopsis thaliana (AT) lysate. Finally, the optimized carboxylated SP3 workflow was benchmarked closely against the routine FASP. Ultimately, LC-MS/MS analyses revealed that regarding the number of identifications, number of missed cleavages, proteome coverage, repeatability, reduction of handling time, and cost per assay, the SP3 on carboxylated magnetic particles proved to be the best alternative for SDS and other contaminants removal from plant sample lysate. A robust and efficient 2-h SP3 protocol for a wide range of protein input is presented, benefiting from no need to adjust the amount of beads, binding and rinsing conditions, or digestion parameters.

Mendel Centre for Plant Genomics and Proteomics Central European Institute of Technology Masaryk University Brno Czechia

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czechia

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Balliau T., Blein-Nicolas M., Zivy M. (2018). Evaluation of optimized tube-gel methods of sample preparation for large-scale plant proteomics. Proteomes 6:6. 10.3390/proteomes6010006, PMID: PubMed DOI PMC

Basisty N., Meyer J. G., Wei L., Gibson B. W., Schilling B. (2018). Simultaneous quantification of the acetylome and succinylome by “one-pot” affinity enrichment. Proteomics 18:e1800123. 10.1002/pmic.201800123, PMID: PubMed DOI PMC

Bielow C., Mastrobuoni G., Kempa S. (2016). Proteomics quality control: quality control software for MaxQuant results. J. Proteome Res. 15, 777–787. 10.1021/acs.jproteome.5b00780, PMID: PubMed DOI

Cox J., Mann M. (2008). MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372. 10.1038/nbt.1511, PMID: PubMed DOI

Cox J., Neuhauser N., Michalski A., Scheltema R. A., Olsen J. V., Mann M. (2011). Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805. 10.1021/pr101065j, PMID: PubMed DOI

Dagley L. F., Infusini G., Larsen R. H., Sandow J. J., Webb A. I. (2019). Universal solid-phase protein preparation (USP3) for bottom-up and top-down proteomics. J. Proteome Res. 18, 2915–2924. 10.1021/acs.jproteome.9b00217, PMID: PubMed DOI

Erde J., Loo R. R. O., Loo J. A. (2014). Enhanced FASP (eFASP) to increase proteome coverage and sample recovery for quantitative proteomic experiments. J. Proteome Res. 13, 1885–1895. 10.1021/pr4010019, PMID: PubMed DOI PMC

Erde J., Loo R. R. O., Loo J. A. (2017). Improving proteome coverage and sample recovery with enhanced FASP (eFASP) for quantitative proteomic experiments. Methods Mol. Biol. 1550, 11–18. 10.1007/978-1-4939-6747-6_2, PMID: PubMed DOI PMC

Feist P., Hummon A. B. (2015). Proteomic challenges: sample preparation techniques for microgram-quantity protein analysis from biological samples. Int. J. Mol. Sci. 16, 3537–3563. 10.3390/ijms16023537, PMID: PubMed DOI PMC

Finehout E. J., Cantor J. R., Lee K. H. (2005). Kinetic characterization of sequencing grade modified trypsin. Proteomics 5, 2319–2321. 10.1002/pmic.200401268, PMID: PubMed DOI

Glatter T., Ahrné E., Schmidt A. (2015). Comparison of different sample preparation protocols reveals Lysis buffer-specific extraction biases in gram-negative bacteria and human cells. J. Proteome Res. 14, 4472–4485. 10.1021/acs.jproteome.5b00654, PMID: PubMed DOI

Gonzalez-Lozano M. A., Koopmans F., Paliukhovich I., Smit A. B., Li K. W. (2019). A fast and economical sample preparation protocol for interaction proteomics analysis. Proteomics 19:1900027. 10.1002/pmic.201900027, PMID: PubMed DOI

HaileMariam M., Eguez R. V., Singh H., Bekele S., Ameni G., Pieper R., et al. . (2018). S-Trap, an ultrafast sample-preparation approach for shotgun proteomics. J. Proteome Res. 17, 2917–2924. 10.1021/acs.jproteome.8b00505, PMID: PubMed DOI

Hartl M., Füßl M., Boersema P. J., Jost J., Kramer K., Bakirbas A., et al. . (2017). Lysine acetylome profiling uncovers novel histone deacetylase substrate proteins in Arabidopsis. Mol. Syst. Biol. 13:949. 10.15252/msb.20177819, PMID: PubMed DOI PMC

Huber M. L., Sacco R., Parapatics K., Skucha A., Khamina K., Müller A. C., et al. . (2014). abFASP-MS: affinity-based filter-aided sample preparation mass spectrometry for quantitative analysis of chemically labeled protein complexes. J. Proteome Res. 13, 1147–1155. 10.1021/pr4009892, PMID: PubMed DOI PMC

Hughes C. S., Foehr S., Garfield D. A., Furlong E. E., Steinmetz L. M., Krijgsveld J. (2014). Ultrasensitive proteome analysis using paramagnetic bead technology. Mol. Syst. Biol. 10:757. 10.15252/msb.20145625, PMID: PubMed DOI PMC

Hughes C. S., Moggridge S., Müller T., Sorensen P. H., Morin G. B., Krijgsveld J. (2019). Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat. Protoc. 14, 68–85. 10.1038/s41596-018-0082-x, PMID: PubMed DOI

Hussein R. A., El-Anssary A. A. (2018). “Plants secondary metabolites: the key drivers of the pharmacological actions of medicinal plants” in Herbal Medicine. ed. P. F. Builders (London, UK: Intechopen; ).

Jorrin-Novo J. V., Komatsu S., Sanchez-Lucas R., Rodríguez de Francisco L. E. (2019). Gel electrophoresis-based plant proteomics: past, present, and future. Happy 10th anniversary journal of proteomics! J. Proteome 198, 1–10. 10.1016/j.jprot.2018.08.016, PMID: PubMed DOI

Komatsu S. (2008). Plasma membrane proteome in Arabidopsis and rice. Proteomics 8, 4137–4145. 10.1002/pmic.200800088, PMID: PubMed DOI

Lewandowska D., Zhang R., Colas I., Uzrek N., Waugh R. (2019). Application of a sensitive and reproducible label-free proteomic approach to explore the proteome of individual meiotic-phase barley anthers. Front. Plant Sci. 10:393. 10.3389/fpls.2019.00393, PMID: PubMed DOI PMC

Li S., Cao X., Wang Y., Zhu Z., Zhang H., Xue S., et al. . (2017). A method for microalgae proteomics analysis based on modified filter-aided sample preparation. Appl. Biochem. Biotechnol. 183, 923–930. 10.1007/s12010-017-2473-9, PMID: PubMed DOI

Lipecka J., Chhuon C., Bourderioux M., Bessard M. -A., van Endert P., Edelman A., et al. . (2016). Sensitivity of mass spectrometry analysis depends on the shape of the filtration unit used for filter aided sample preparation (FASP). Proteomics 16, 1852–1857. 10.1002/pmic.201600103, PMID: PubMed DOI

Liu Y., Lu S., Liu K., Wang S., Huang L., Guo L. (2019). Proteomics: a powerful tool to study plant responses to biotic stress. Plant Methods 15:135. 10.1186/s13007-019-0515-8, PMID: PubMed DOI PMC

Ludwig K. R., Schroll M. M., Hummon A. B. (2018). Comparison of in-solution, FASP, and S-trap based digestion methods for bottom-up proteomic studies. J. Proteome Res. 17, 2480–2490. 10.1021/acs.jproteome.8b00235, PMID: PubMed DOI PMC

Lv B., Yang Q., Li D., Liang W., Song L. (2016). Proteome-wide analysis of lysine acetylation in the plant pathogen Botrytis cinerea. Sci. Rep. 6:29313. 10.1038/srep29313, PMID: PubMed DOI PMC

Min L., Choe L. H., Lee K. H. (2015). Improved protease digestion conditions for membrane protein detection. Electrophoresis 36, 1690–1698. 10.1002/elps.201400579, PMID: PubMed DOI PMC

Mo R., Yang M., Chen Z., Cheng Z., Yi X., Li C., et al. . (2015). Acetylome analysis reveals the involvement of lysine acetylation in photosynthesis and carbon metabolism in the model cyanobacterium Synechocystis sp. PCC 6803. J. Proteome Res. 14, 1275–1286. 10.1021/pr501275a, PMID: PubMed DOI

Moggridge S., Sorensen P. H., Morin G. B., Hughes C. S. (2018). Extending the compatibility of the SP3 paramagnetic bead processing approach for proteomics. J. Proteome Res. 17, 1730–1740. 10.1021/acs.jproteome.7b00913, PMID: PubMed DOI

Müller T., Kalxdorf M., Longuespée R., Kazdal D. N., Stenzinger A., Krijgsveld J. (2020). Automated sample preparation with SP3 for low-input clinical proteomics. Mol. Syst. Biol. 16:e9111. 10.15252/msb.20199111, PMID: PubMed DOI PMC

Nel A. J. M., Garnett S., Blackburn J. M., Soares N. C. (2015). Comparative reevaluation of FASP and enhanced FASP methods by LC-MS/MS. J. Proteome Res. 14, 1637–1642. 10.1021/pr501266c, PMID: PubMed DOI

Ni M. -W., Wang L., Chen W., Mou H. -Z., Zhou J., Zheng Z. -G. (2017). Modified filter-aided sample preparation (FASP) method increases peptide and protein identifications for shotgun proteomics. Rapid Commun. Mass Spectrom. 31, 171–178. 10.1002/rcm.7779, PMID: PubMed DOI

Niu L., Yuan H., Gong F., Wu X., Wang W. (2018). Protein extraction methods shape much of the extracted proteomes. Front. Plant Sci. 9:802. 10.3389/fpls.2018.00802, PMID: PubMed DOI PMC

Perez-Riverol Y., Csordas A., Bai J., Bernal-Llinares M., Hewapathirana S., Kundu D. J., et al. . (2019). The PRIDE database and related tools and resources in 2019: improving support for quantification data. Nucleic Acids Res. 47, D442–D450. 10.1093/nar/gky1106, PMID: PubMed DOI PMC

Potriquet J., Laohaviroj M., Bethony J. M., Mulvenna J. (2017). A modified FASP protocol for high-throughput preparation of protein samples for mass spectrometry. PLoS One 12:e0175967. 10.1371/journal.pone.0175967, PMID: PubMed DOI PMC

Scheerlinck E., Dhaenens M., Van Soom A., Peelman L., De Sutter P., Van Steendam K., et al. . (2015). Minimizing technical variation during sample preparation prior to label-free quantitative mass spectrometry. Anal. Biochem. 490, 14–19. 10.1016/j.ab.2015.08.018, PMID: PubMed DOI

Sielaff M., Kuharev J., Bohn T., Hahlbrock J., Bopp T., Tenzer S., et al. . (2017). Evaluation of FASP, SP3, and iST protocols for proteomic sample preparation in the low microgram range. J. Proteome Res. 16, 4060–4072. 10.1021/acs.jproteome.7b00433, PMID: PubMed DOI

Song G., Hsu P. Y., Walley J. W. (2018). Assessment and refinement of sample preparation methods for deep and quantitative plant proteome profiling. Proteomics 18:e1800220. 10.1002/pmic.201800220, PMID: PubMed DOI PMC

Stoychev S., Naicker P., Mamputha S., Buthelezi S., Gerber I., Westhuyzen C., et al. . (2018). Magnetic HILIC: An enabling & versatile tool for robust automated MS sample preparation workflows. Available at: https://resynbio.com/hilic/ (Accessed February 25, 2021).

Stoychev S., Naicker P., Mamputha S., Khumalo F., Gerber I., Westhuyzen C., et al. . (2017). Universal Unbiased pre-MS Clean-Up Using Magnetic HILIC Microparticles for SPE. Available at: https://resynbio.com/hilic/ (Accessed February 25, 2021).

Takáč T., Šamajová O., Šamaj J. (2017). Integrating cell biology and proteomic approaches in plants. J. Proteome 169, 165–175. 10.1016/j.jprot.2017.04.020, PMID: PubMed DOI

Tubaon R. M., Haddad P. R., Quirino J. P. (2017). Sample clean-up strategies for ESI mass spectrometry applications in bottom-up proteomics: trends from 2012 to 2016. Proteomics 17:1700011. 10.1002/pmic.201700011, PMID: PubMed DOI

Tyanova S., Cox J. (2018). Perseus: a bioinformatics platform for integrative analysis of proteomics data in cancer research. Methods Mol. Biol. 1711, 133–148. 10.1007/978-1-4939-7493-1_7, PMID: PubMed DOI

Wang W. -Q., Jensen O. N., Møller I. M., Hebelstrup K. H., Rogowska-Wrzesinska A. (2018). Evaluation of sample preparation methods for mass spectrometry-based proteomic analysis of barley leaves. Plant Methods 14:72. 10.1186/s13007-018-0341-4, PMID: PubMed DOI PMC

Wang W., Tai F., Chen S. (2008). Optimizing protein extraction from plant tissues for enhanced proteomics analysis. J. Sep. Sci. 31, 2032–2039. 10.1002/jssc.200800087, PMID: PubMed DOI

Wiśniewski J. R. (2017). “Filter-aided sample preparation: the versatile and efficient method for proteomic analysis” in Methods in enzymology: Proteomics in Biology, Part A. Vol. 585. ed. Shukla A. K. (Cambridge, Massachusetts, USA: Academic Press, Elsevier: ). PubMed

Wiśniewski J. R. (2018). Filter-aided sample preparation for proteome analysis. Methods Mol. Biol. 1841, 3–10. 10.1007/978-1-4939-8695-8_1, PMID: PubMed DOI

Wiśniewski J. R. (2019). Filter aided sample preparation - a tutorial. Anal. Chim. Acta 1090, 23–30. 10.1016/j.aca.2019.08.032, PMID: PubMed DOI

Wiśniewski J. R., Gaugaz F. Z. (2015). Fast and sensitive total protein and peptide assays for proteomic analysis. Anal. Chem. 87, 4110–4116. 10.1021/ac504689z, PMID: PubMed DOI

Wiśniewski J. R., Mann M. (2012). Consecutive proteolytic digestion in an enzyme reactor increases depth of proteomic and phosphoproteomic analysis. Anal. Chem. 84, 2631–2637. 10.1021/ac300006b, PMID: PubMed DOI

Wiśniewski J. R., Ostasiewicz P., Mann M. (2011). High recovery FASP applied to the proteomic analysis of microdissected formalin fixed paraffin embedded cancer tissues retrieves known colon cancer markers. J. Proteome Res. 10, 3040–3049. 10.1021/pr200019m, PMID: PubMed DOI

Wiśniewski J. R., Zougman A., Nagaraj N., Mann M. (2009). Universal sample preparation method for proteome analysis. Nat. Methods 6, 359–362. 10.1038/nmeth.1322, PMID: PubMed DOI

Yeung Y. -G., Stanley E. R. (2010). Rapid detergent removal from peptide samples with ethyl acetate for mass spectrometry analysis. Curr. Protoc. Protein Sci. 59, 16.12.1–16.12.5. 10.1002/0471140864.ps1612s59, PMID: PubMed DOI PMC

Zhang Z., Dubiak K. M., Huber P. W., Dovichi N. J. (2020). Miniaturized filter-aided sample preparation (MICRO-FASP) method for high throughput, ultrasensitive proteomics sample preparation reveals proteome asymmetry in Xenopus laevis embryos. Anal. Chem. 92, 5554–5560. 10.1021/acs.analchem.0c00470, PMID: PubMed DOI PMC

Zougman A., Selby P. J., Banks R. E. (2014). Suspension trapping (STrap) sample preparation method for bottom-up proteomics analysis. Proteomics 14, 1006–1000. 10.1002/pmic.201300553, PMID: PubMed DOI

Benchmarking of Two Peptide Clean-Up Protocols: SP2 and Ethyl Acetate Extraction for Sodium Dodecyl Sulfate or Polyethylene Glycol Removal from Plant Samples before LC-MS/MS