Highly efficient and selective enrichment of peptide subsets combining fluorous chemistry with reversed-phase chromatography

Cardiovascular Proteomics Center, Boston University School of Medicine, Boston, MA 02118-2543, USA.
Rapid Communications in Mass Spectrometry (Impact Factor: 2.25). 11/2009; 23(24):4019-30. DOI: 10.1002/rcm.4343
Source: PubMed

ABSTRACT The selective capture of target peptides poses a great challenge to modern chemists and biologists, especially when enriching them from proteome samples possessing extremes in concentration dynamic range and sequence diversity. While approaches based on traditional techniques such as biotin-avidin pairing offer versatile tools to design strategies for selective enrichment, problems are still encountered due to sample loss or poor selectivity of enrichment. Here we show that the recently introduced fluorous chemistry approach has attractive properties as an alternative method for selective enrichment. Through appending a perfluorine group to the target peptide, it is possible to dramatically increase the peptide's hydrophobicity and thus enable facile separation of labeled from non-labeled peptides. Use of reversed-phase chromatography allowed for improved peptide recovery in comparison with results obtained using the formerly reported fluorous bonded phase methods. Furthermore, this approach also allowed for on-line separation and identification of both labeled and unlabeled peptides in a single experiment. The net result is an increase in the confidence of protein identification by tandem mass spectrometry (MS2) as all peptides and subsequent information are retained. Successful off-line and on-line enrichment of cysteine-containing peptides was obtained, and high quality MS2 spectra were obtained by tandem mass spectrometry due to the stability of the tag, allowing for facile identification via standard database searching. We believe that this strategy holds great promise for selective enrichment and identification of low abundance target proteins or peptides.

6 Reads
  • [Show abstract] [Hide abstract]
    ABSTRACT: Continuing from the foundation laid by our previous work in the field, we present here an examination of the effects of monolith density and overall composition on the efficacy of performance in the realm of fluorous separations. By variation of the proportions of monomer and cross-linking agent relative to a static porogenic solvent composition, it was found that a composition of 30% polymer-forming material provides the optimal results in terms of resolution and peak shape for fluorous chromatography of a mixture of similarly labeled benzylamines. The presence of so-called "secondary interactions" that can compete with fluorous specificity in columns of this type were also examined and discussed, with similar results to those observed for commercial fluorous columns being noted. We suggest that these effects may actually be positive if they can be properly harnessed, as the ability to provide a second dimension for fluorous separations based on polarity may allow more complex analyses of labeled proteomic samples to be effectively undertaken. Finally, we present some initial results on the effectiveness of our optimized fluorous monoliths in a series of tagging and separation experiments using a custom-synthesized peptide. With successful resolution of labeled biological samples from their nonfluorous counterparts achieved, we discuss the potential expansion and further applicability of fluorous monoliths of this type in proteomic avenues, as well as their amenability to the greater analytical community.
    Analytical Chemistry 02/2011; 83(5). DOI:10.1021/ac102827t · 5.64 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Protein chemical derivatization has emerged as an invaluable bioanalytical approach in mass spectrometry-based proteomics with nearly unlimited potential. To date, derivatization strategies in proteomics have primarily focused on improving mass spectral identification and relative quantification of proteins, as well as increasing enrichment yield from complex mixtures. However, there is a great opportunity to develop and exploit front-end chemical processes to enhance the ability to detect low-abundant peptides and proteins for a large number of applications. The content of this article focuses on improvements in targeted, mass spectrometry-based proteomic strategies, achieved by taking advantage of the mechanism of ESI through the use of hydrophobic chemical derivatization.
    Expert Review of Proteomics 06/2011; 8(3):317-23. DOI:10.1586/epr.11.24 · 2.90 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: In this work, the interior-walls decyl-perfluorinated functionalized magnetic mesoporous microspheres (F(17) -Fe(3) O(4) @mSiO(2) ) were synthesized for the first time, and applied as adsorbents to extract and concentrate perfluorinated compounds (PFCs) from water samples. The fluorous functionalized interior pore-walls contributed to the high-selective preconcentration of PFCs due to fluorous affinity; and abundant silanol groups on the exterior surface of microspheres contributed to the good dispersibility in water sample. Four kinds of PFCs were selected as model analytes, including perfluorooctanoic acid, perfluorononanoic acid, perfluorododecanoic acid, and perfluorooctane sulphonate. In addition, UHPLC-ESI/MS/MS was introduced to the fast and sensitive detection of the analytes after sample pretreatment. Important parameters of the extraction procedure were optimized, including salinity, eluting solvent, the amount of F(17) -Fe(3) O(4) @mSiO(2) microspheres, and extraction time. The optimized procedure took only 10 min to extract analytes with high recoveries and merely 800-μL acetonitrile to elute analytes from the magnetic adsorbents. Validation experiments showed good linearity (0.994-0.998), precision (2.6-7.6%), high recovery (93.4-105.7%) of the proposed method, and the limits of detection were from 0.008 to 0.125 μg/L. The F(17) -Fe(3) O(4) @mSiO(2) magnetic microspheres have the advantages of great dispersibility in aqueous solution, high specificity of extraction, large surface area, and efficient separation ability. The results showed that the proposed method based on F(17) -Fe(3) O(4) @mSiO(2) microspheres is a simple, fast, and sensitive tool for the analysis of PFCs in water sample.
    Journal of Separation Science 10/2012; 35(19):2629-36. DOI:10.1002/jssc.201200300 · 2.74 Impact Factor
Show more