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

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.

Figure 1

Representative MALDI-TOF mass spectra of the cysteine-containing peptide Cys-Kemptide (CLRRASLG): (A) before and (B) after labeling with the fluorous reagent. The peak corresponding to the unlabeled [M+H]⁺ peptide ion at m/z 875.60 was observed to shift by the mass of the fluorous group (517.043 Da) to m/z 1392.47. Peaks in the mass region below the peptide ion peak correspond to impurities in the synthetic peptide which were still observed post-labeling. Peaks in the higher mass region can be attributed to salt adducts. To confirm these results, ESI-Q-TOF-MS analyses were performed with the addition of an internal standard, as indicated in Supplemental Fig. 1 (see Supporting Information); the labeling efficiency reached higher than 95%.

Figure 2

Representative MALDI-TOF mass spectra showing a comparison among three methods for enrichment of fluorous-labeled peptides from a peptide mixture using fluorous NuTips™. (A), (B), (C) correspond to methods A, B, C, respectively, in Table 1. Roughly equivalent recovery was shown between A and B, while limited recovery was observed for method C. (D) The flow-through fraction from method A was subjected to C18 ZipTip™ column enrichment. The results indicated that a significant portion of the fluorous-labeled peptide did not bind to the NuTip™ and could be recovered on the C18 material. Based on the peak intensities in (A) and (D), we estimate that a 50% recovery efficiency for NuTip™ can be accomplished. To confirm these results, ESI-Q-TOF-MS analyses performed with the addition of an internal standard indicated an approximate recovery of 42%, as indicated in Supplemental Fig. 2 (see Supporting Information).

Figure 3

Representative MALDI-TOF mass spectra showing the selective enrichment of a fluorous-labeled peptide from a peptide mixture using a C18 ZipTip™. A tryptic peptide mixture of myoglobin was mixed with fluorous-labeled Cys-Kemptide with a molar ratio of 1:1 and subjected to following analyses. (A) Reference spectrum from 1 pmol of the peptide mixture. (B) The peptide mixture was dissolved in 20% ACN/0.1% TFA and a ZipTip™ column was used to extract the peptide. The unbound fraction was detected by MALDI-TOF-MS. Peptide concentration, 1 pmol/μL. (C) or (D) First-step elution of the fraction in 35% ACN/0.1%TFA or in 40% ACN/0.1%TFA. (E) Second-step elution of the fraction in 60% ACN/0.1% TFA after the 35% ACN/0.1%TFA wash. (F) Third-step elution of the fraction in 80% ACN/0.1% TFA after the 60% ACN/0.1%TFA wash. The gray bar denotes the fluorous-labeled Cys-Kemptide [M+H]⁺ ion at m/z 1392.47.

Figure 4

Representative MALDI-TOF mass spectra showing selective enrichment of fluorous-labeled peptides from tryptic digests of bovine serum albumin. (A) The labeled BSA peptide mixture was desalted with 0.1% TFA and eluted with 80% ACN/0.1% TFA. (B) Peptide mixture eluted from a ZipTip™ with 80% ACN/0.1% TFA after selective removal of non-fluorous-labeled peptides via washing with 35% ACN/0.1% TFA. In experiments (C) and (D), fluorous-labeled peptides were bound to the ZipTip™ column as in (B), but eluted in a stepwise mode with (C) 50% ACN/0.1% TFA and (D) 80% ACN/0.1% TFA, respectively. Peaks correspond to *, non-cysteine-containing peptides; +, peptides containing one fluorinated cysteine; ^, peptides containing two fluorinated cysteines; @, peptides containing three fluorinated cysteines; #, peaks which were observed with a 42-Da increase from the fluorous peptides, which may have resulted from acetylation of the peptide N-terminus during the reaction procedure. Peak assignments can be found in Table 2.

Figure 5

Base peak chromatograms showing separation and identification of BSA peptides by nano-LC/MS/MS. (A) IAA-labeled peptides or (B) fluorous-labeled peptides were separated under the same conditions. The elution profiles from 38–50 min are magnified by 3× to aid the comparison. The increased delay in retention time for two representative peptides, T139–155 and T483–489, are indicated; each of these contains one cysteine. The MS/MS spectra of the fluorinated peptides are shown in Fig. 7. Peak assignments can be found in Table 2.

Figure 6

Influence of charge state and label incorporation on peptide retention times. RT was affected by solution charge state and the hydrophobicity change that resulted from labeling of fluorous groups. The dashed line shows the change of the gradient, as scaled on the right y-axis. Peptides observed with higher charge states in ESI-MS tended to elute earlier than those with fewer charges, and peptides with incorporation of more than one fluorous group conjugated to the peptide eluted later. Peak assignments can be found in Table 2.

Figure 7

Examples of tandem mass spectra of fluorous-labeled peptides after CID. (A) Product ion spectrum of [M+2H]²⁺ precursor at m/z 827.19; (B) product ion spectrum of [M+2H]²⁺ precursor at m/z 679.64. Both spectra contain distinct and abundant b and y ion series. Internal fragment ions+are also observed, as labeled in (B). As can be seen from the two tandem mass spectra, the fluorous groups were very stable. The position of the cysteine, either in the center or close to one end of the peptide, did not affect the production of ion series. The collision energy was automatically set by the MassLynx software using the charge-state-recognition module.