Peptide Mapping with Ascentis Express Peptide ES-C18

By: Hillel Brandes, Reporter US Volume 28

Hillel Brandes

Peptide mapping is an established technique for assessing changes to the primary structure of a protein. This has applications in areas of quality control or fundamental research in which changes to the protein sequence or to the chemical modification of amino acids is to be monitored. Essentially, the protein(s) is cleaved (digested) in a sequence-dependent manner to generate a finite number of peptide fragments. The mixture of peptide fragments is then resolved chromatographically. The exact pre-treatment protocol of the protein prior to digestion varies and may depend on the sort of questions the researcher is posing. However, a general protein pre-treatment protocol involves denaturation, reduction, and alkylation. Alkylation is performed to control the oxidation state of free sulfhydryls that otherwise can cause undesirable heterogeneity of the resulting chromatograms. Reduction is performed to ensure the sulfhydryls are reduced prior to alkylation. Denaturation is performed to ensure all sulfhydryls are accessible for chemical modification, as well as make the polypeptide chain fully accessible for digestion.

Peptide mapping is generally done by reversed-phase liquid chromatography. Traditionally this was done in conjunction with low-UV detection. A good match with the low-UV detection, is the use of perfluorinated organic acids as ion pairing reagents. TFA was ideal in this regard. As a strong acid, at low levels, it maximizes retention of polypeptides on silica-based columns by keeping the pH well below that of peptidyl carboxyls, and at the same time it ion pairs with basic moieties to optimize peak shape. Thus, TFA-mobile phases became the default method for peptide mapping with UV detection. However, the advent of mass-spectrometry as a preferred method of detection for peptide mapping necessitated the redevelopment of a standard mobile phase; TFA at levels typical for UV-based peptide mapping (0.1%) cause severe reduction in sensitivity as compared to other organic acids, as the mobile phase additive (1). This has been best described as a consequence of higher surface tension of the charged droplet in the ESI source and strong ion pairing in the gas phase, between TFA and peptide basic moieties (1). Acetic acid and formic acid have been used as alternatives to TFA for LC-MS of peptides, but formic acid has become more common. This is likely due, again, to it being a stronger acid, thus minimizing the ionization of peptidyl carboxyls so as to enhance retention. An example of the difference in MS sensitivity with formic acid TFA is shown in Figure 1.

Figure 1. Sensitivity Comparison of Tryptic Digest with Different Ion Pair Reagents

Supelco has recently released a new column on the Fused-Core particle platform designed for polypeptide analysis, Ascentis Express Peptide ES-C18. As such, it exhibits high plate counts by virtue of the uniform column packing of the monodisperse particles, and a possible shorter diffusion path of the porous shell. Table 1 summarizes the major features of the Ascentis Express Peptide particle.

Table 1. Ascentis Express Peptide ES-C18 Fused-Core Particle

The major benefit of this new high-efficiency packing for peptide mapping is the resultant improved peak capacity (Pc), where tg is the gradient time and wave is the average peak width.

Peak capacity provides a theoretical maximum number of peaks that can be chromatographically resolved over the gradient run time. A more efficient column will have lower average peak width, and will therefore exhibit higher peak capacity. An example of this is shown in Figure 2 and Table 2. Figure 2 displays chromatograms of a complex tryptic digest on a typical wide-pore C18 column, versus the Ascentis Express Peptide ES-C18. While visual inspection suggests a greater number of resolved peaks in the case of the Ascentis Express Peptide ES-C18 column, a more convincing quantitative evaluation comes from a sampling of peaks across the chromatogram, as shown in Table 2. Ascentis Express Peptide ES-C18 is able to resolve, statistically, a greater number of peaks.

Figure 2. Comparison of Tryptic Digest with Ascentis Express and Conventional Wide Pore, 5μm Column

Table 2. Comparison of Peak Capacities

While 0.1% formic acid has become a de facto standard for peptide mapping by LC-MS, it is not without some concerns. It has been noted that peak efficiency and peak shape in the presence of 0.1% formic acid are not as good as with 0.1% TFA (2). This is particularly evident with basic peptides as shown in Figure 3. Also note the enhanced retention of peptides in the presence of TFA, presumably due to the greater hydrophobicity of TFA relative to formic acid.

Figure 3. Peak Shape Comparison of Basic Peptides with Different Ion Pair Reagents (53307-U)

McCalley (2) has best explained the poorer peak shape in the presence of formic acid, as reduced peak capacity of the retained analyte (peptides) due to ionic repulsion in the absence of adequate ion pairing, that would otherwise neutralize the analyte charge (2). This can be further understood in reference to Table 3.

Table 3. Ionization of Ion Pair Reagents

As a 0.1% aqueous solution, TFA is 98% ionized while that of formic acid is 7% ionized. Obviously then, there is far greater anion available to ion pair with basic moieties in the case of TFA than in the case of formic acid. This is certainly a plausible explanation consistent with McCalley’s hypothesis. A further approach to testing this idea is to adjust the pH of the formic acid solution higher, such that greater formate anion is available for ion pairing. Adjusting the pH to 3.5 results in a 7-fold increase in the formate anion concentration. Indeed, this minor alteration in the mobile phase has dramatic impact on peak shape of basic peptides as seen in Figure 4.

Figure 4. Peak Shape Comparison of Basic Peptides at Different pH (53307-U)

The improved peak shape at pH 3.5 is best explained as a result of the increase in formate anion concentration, and not an effect of the ammonium cation, because the peak shape with a similar concentration of ammonium formate at pH 3, is not nearly as improved as at pH 3.5 (data not shown). Were the effect similar, it would argue for a more significant role of the ammonium cation in affecting poor peak shape, via mitigation of ion-exchange activity between the peptides and the silica surface.

Ascentis Express Peptide ES-C18, then, is the latest addition to the Ascentis Express product line that can yield state-of-the-art performance for reversed-phase chromatography of polypeptides, be they complex proteomic mixtures or samples derived from peptide drug research and analysis.
 * all concentrations, shown as percentages, are v/v

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  1. Apffel, A. et. al. 1995. Enhanced sensitivity for peptide mapping with electrospray liquid chromatography-mass spectrometry in the presence of signal suppression due to trifluoroacetic acid-containing mobile phases. J. Chrom A 712: 177-190.
  2. McCalley, D.V. 2004. Effect of buffer on peak shape of peptides in reversed-phase high performance liquid chromatography. J. Chrom A 1038: 77-84.

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