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Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry

Nature Biotechnology volume 23, pages 463–468 (2005)Cite this article

Abstract

Although mass spectrometry has become a powerful tool for the functional analysis of biological systems, complete proteome characterization cannot yet be achieved. Instead, the sheer complexity of living organisms demands fractionation of cellular extracts to enable more targeted analyses. Here, we introduce the concept of 'fluorous proteomics,' whereby specific peptide subsets from samples of biological origin are tagged with perfluorinated moieties and subsequently enriched by solid-phase extraction over a fluorous-functionalized stationary phase. This approach is extremely selective, yet can readily be tailored to enrich different subsets of peptides. Additionally, this methodology overcomes many of the limitations of traditional bioaffinity-based enrichment strategies, while enabling new affinity enrichment schemes impossible to implement with bioaffinity reagents. The potential of this methodology is demonstrated by the facile enrichment of peptides bearing particular side-chain functionalities or post-translational modifications from tryptic digests of individual proteins as well as whole cell lysates.

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Figure 1: Fluorous proteomics strategy for the isolation of specific classes of peptides.
Figure 2: Isolation of fluorous-tagged peptides from a highly complex peptide mixture.
Figure 3: MS/MS spectra corresponding to (a) MPcF17TEDYLSLILNR (cF17 represents a cysteine residue after reaction with N-[(3-perfluorooctyl)-propyl] iodoacetamide), and (b) MPcTEDYLSLILNR (c represents a cysteine residue after reaction with iodoacetamide); CI*, immonium ion of the fluorous-labeled cysteine residue; *, loss of water/ammonia.
Figure 4: Separation of peptides based on fluorine content.

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Authors and Affiliations

  1. Genomics Institute of the Novartis Research Foundation, 10675 John Jay Hopkins Drive, San Diego, 92121, California, USA

    Scott M Brittain, Scott B Ficarro, Ansgar Brock & Eric C Peters

Authors
  1. Scott M Brittain
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  2. Scott B Ficarro
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  3. Ansgar Brock
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  4. Eric C Peters
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Correspondence to Eric C Peters.

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Supplementary information

Supplementary Fig. 1

Recovery of a fluorous-labeled synthetic peptide from a complex mixture by FSPE (PDF 197 kb)

Supplementary Fig. 2

MASCOT search results (PDF 51 kb)

Supplementary Fig. 3

MS/MS spectrum of doubly charged SLHTLFGDELcF17K (PDF 62 kb)

Supplementary Fig. 4

MS/MS spectrum of doubly charged QLsF17SGVSEIR. (PDF 22 kb)

Supplementary Fig. 5

MS/MS spectrum of doubly charged F17VTQHFAK. (PDF 26 kb)

Supplementary Table 1

Peptide sequences with localized sites of modification. (PDF 12 kb)

Supplementary Table 2

Peptide sequences with candidate phosphorylation sites. (PDF 13 kb)

Supplementary Methods (PDF 17 kb)

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Brittain, S., Ficarro, S., Brock, A. et al. Enrichment and analysis of peptide subsets using fluorous affinity tags and mass spectrometry. Nat Biotechnol 23, 463–468 (2005). https://doi.org/10.1038/nbt1076

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