Skip to main content
SpringerLink
Log in

Offline pentafluorophenyl (PFP)-RP prefractionation as an alternative to high-pH RP for comprehensive LC-MS/MS proteomics and phosphoproteomics

  • Research Paper
  • Published:
Analytical and Bioanalytical Chemistry Aims and scope Submit manuscript

Abstract

Technological advances in liquid chromatography and tandem mass spectrometry (LC-MS/MS) have enabled comprehensive analyses of proteins and their post-translational modifications from cell culture and tissue samples. However, sample complexity necessitates offline prefractionation via a chromatographic method that is orthogonal to online reversed-phase high-performance liquid chromatography (RP-HPLC). This additional fractionation step improves target identification rates by reducing the complexity of the sample as it is introduced to the instrument. A commonly employed offline prefractionation method is high pH reversed-phase (Hi-pH RP) chromatography. Though highly orthogonal to online RP-HPLC, Hi-pH RP relies on buffers that interfere with electrospray ionization. Thus, samples that are prefractionated using Hi-pH RP are typically desalted prior to LC-MS/MS. In the present work, we evaluate an alternative offline prefractionation method, pentafluorophenyl (PFP)-based reversed-phase chromatography. Importantly, PFP prefractionation results in samples that are dried prior to analysis by LC-MS/MS. This reduction in sample handling relative to Hi-pH RP results in time savings and could facilitate higher target identification rates. Here, we have compared the performances of PFP and Hi-pH RP in offline prefractionation of peptides and phosphopeptides that have been isolated from human cervical carcinoma (HeLa) cells. Given the prevalence of isobaric mass tags for peptide quantification, we evaluated PFP chromatography of peptides labeled with tandem mass tags. Our results suggest that PFP is a viable alternative to Hi-pH RP for both peptide and phosphopeptide offline prefractionation.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  1. Schwanhausser B, Busse D, Li N, Dittmar G, Schuchhardt J, Wolf J, et al. Global quantification of mammalian gene expression control. Nature. 2011;473:337–42.

    Article  Google Scholar 

  2. Yates JR. Mass spectrometry and the age of the proteome. J Mass Spectrom. 1998;33:1–19.

    Article  CAS  Google Scholar 

  3. Zhang Y, Fonslow BR, Shan B, Moon-Chang B, Yates JR. Protein analysis by shotgun/bottom-up proteomics. Chem Rev. 2013;113(4):2343–94.

    Article  CAS  Google Scholar 

  4. Malmstrom J, Lee H, Aebersold R. Advances in proteomic workflows for systems biology. Curr Opin Biotechnol. 2007;18(4):378–84.

    Article  Google Scholar 

  5. Michalski A, Cox J, Mann M. 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. 2011;10:1785–93.

    Article  CAS  Google Scholar 

  6. McAlister GC, Huttlin EL, Haas W, Ting L, Jedrychowski MP, Rogers JC, et al. Increasing the multiplexing capacity of TMTs using reporter ion isotopologues with isobaric masses. Anal Chem. 2012;84:7469–78.

    Article  CAS  Google Scholar 

  7. Anagnostopoulos AK, Stravopodis DJ, Tsangaris GT. Yield of 6,000 proteins by 1D nLC-MS/MS without pre-fractionation. J Chromatogr B. 2016;1–5.

  8. Gilar M, Olivova P, Daly AE, Gebler JC. Orthogonality of separation in two-dimensional liquid chromatography. Anal Chem. 2005;77:6426–34.

    Article  CAS  Google Scholar 

  9. Paulo JA, Gaun A, Gygi SP. Global analysis of protein expression and phosphorylation levels in nicotine-treated pancreatic stellate cells. J Proteome Res. 2015;14:4246–56.

    Article  CAS  Google Scholar 

  10. Baths TS, Olsen JV. Offline hi pH reversed-phase peptide fractionation for deep phosphoproteome coverage. Methods Mol Biol. 2016;1355:179–92.

    Article  Google Scholar 

  11. Wang Y, Yang F, Gritsenko MA, Wang Y, Clauss T, Liu T, et al. Reversed-phase chromatography with multiple fraction concatenation strategy for proteome profiling of human MCF10A cells. Proteomics. 2011;11:2019–26.

    Article  CAS  Google Scholar 

  12. Magdeldin S, Moresco JJ, Yamamoto T, Yates JR. Off-line multidimensional liquid chromatography and auto sampling result in sample loss in LC/LC-MS/MS. J Proteome Res. 2014;13(8):3826–36.

    Article  CAS  Google Scholar 

  13. Rauniyar N, Gao B, McClatchy DB, Yates JR. Comparison of protein expression ratios observed by sixplex and duplex TMT labeling method. J Proteome Res. 2013;12:1031–9.

    Article  CAS  Google Scholar 

  14. Edwards A, Haas W. Multiplexed quantitative proteomics for high-throughput comprehensive proteome comparisons of human cell lines. Methods Mol Biol. 2016;1394:1–13.

    Article  Google Scholar 

  15. Cao JY, Xu YP, Cai XZ. TMT-based quantitative proteomics analyses reveal novel defense mechanisms of Brassica napus against the devastating necrotrophic pathogen Sclerotinia scleriotorum. J Proteome. 2016;143:265–77.

    Article  CAS  Google Scholar 

  16. Kettenbach AN, Gerber SA. Rapid and reproducible single stage phosphopeptide enrichment of complex peptide mixtures: application to general and phosphotyrosine-specific phosphoproteomics experiments. Anal Chem. 2011;83:7635–44.

    Article  CAS  Google Scholar 

  17. Paulo JA, O’Connell JD, Everley RA, O’Brien J, Gygi MA, Gygi SP. Quantitative mass spectrometry-based multiplexing compares the abundance of 5000 S. cerevisiae proteins across 10 carbon sources. J Proteome. 2016;148:85–93.

    Article  CAS  Google Scholar 

  18. Chick JM, Munger SC, Simecek P, Huttlin EL, Choi K, Gatti DM, et al. Defining the consequences of genetic variation on a proteome-wide scale. Nature. 2016;534:500–5.

    Article  CAS  Google Scholar 

  19. Xie R, Oleschuk R. Photoinduced polymerization for entrapping of octadecylsilane microsphere columns for capillary electrochromatography. Anal Chem. 2007;79(4):1529–35.

    Article  CAS  Google Scholar 

  20. Eng JK, Jahan TA, Hoopmann MR. Comet: an open-source MS/MS sequence database search tool. Proteomics. 2013;13:22–4.

    Article  CAS  Google Scholar 

  21. Arike L, Valgepea K, Peil L, Nahku R, Adamberg K, Vilu R. Comparison and applications of label-free absolute proteome quantification methods on Escherichia coli. J Proteome. 2012;75:5437–48.

    Article  CAS  Google Scholar 

  22. Kyte J, Doolittle R. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982;157(1):105–32.

    Article  CAS  Google Scholar 

  23. Link AJ, Eng J, Schieltz DM, Carmack E, Mize GJ, Morris DR, et al. Direct analysis of protein complexes using mass spectrometry. Nat Biotechnol. 1999;17(7):676–92.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors would like to thank Hildreth R. Frost for help with statistical considerations and Richard Avonti, Jason Gilmore, and Sameh Magdeldin for help in generating Venn diagrams. The authors would like to acknowledge funding from the National Institutes of Health (R01-CA155260 and S10-OD016212) to S.A.G.

Author information

Authors and Affiliations

  1. Departments of Molecular & Systems Biology, and Biochemistry & Cell Biology, Geisel School of Medicine, Lebanon, NH, 03756, USA

    Andrew V. Grassetti, Rufus Hards & Scott A. Gerber

  2. Norris Cotton Cancer Center, One Medical Center Drive HB-7937, Lebanon, NH, 03756, USA

    Scott A. Gerber

Authors
  1. Andrew V. Grassetti
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rufus Hards
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Scott A. Gerber
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Scott A. Gerber.

Ethics declarations

The authors declare no conflicts of interest.

Electronic supplementary material

ESM 1

(PDF 656 kb)

ESM 3

Spreadsheet of Peptides Identified in pH 8 and pH 10 Hi-pH RP Analyses (XLSX 13.1 mb)

ESM 4

Spreadsheet of Proteins Identified in pH 8 and pH 10 Hi-pH RP Analyses (XLSX 1.41 mb)

ESM 6

Spreadsheet of Peptides Identified in PFP RP Analyses (XLSX 13.2 mb)

ESM 7

Spreadsheet of Proteins Identified in PFP RP Analyses (XLSX 1.24 mb)

ESM 8

Spreadsheet of Peptides Identified in Hi-pH RP Analyses (XLSX 9.41 mb)

ESM 9

Spreadsheet of Proteins Identified in Hi-pH RP Analyses (XLSX 1.23 mb)

ESM 10

Spreadsheet of Phosphorylated Peptides Identified in PFP and Hi-pH RP Analyses (XLSX 4.34 mb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Grassetti, A.V., Hards, R. & Gerber, S.A. Offline pentafluorophenyl (PFP)-RP prefractionation as an alternative to high-pH RP for comprehensive LC-MS/MS proteomics and phosphoproteomics. Anal Bioanal Chem 409, 4615–4625 (2017). https://doi.org/10.1007/s00216-017-0407-6

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00216-017-0407-6

Keywords

Search

Navigation