Phosphopeptide elution times in reversed-phase liquid chromatography

Abstract

Elution time shifts between 33 different peptides and their corresponding phosphopeptides ranging from 4 amino acid residues to 35 amino acids in length were systematically investigated using high-resolution reversed-phase liquid chromatography (RPLC)-tandem mass spectrometry (MS/MS) analysis with trifluoroacetic acid as the ion pairing agent. Observed peptide elution time shifts for a single phosphorylation ranged from -5.28 min (for pYVPML) to +0.59 min (for HRDpSGLLDSLGR). Peptides containing a phosphotyrosine residue displayed a significant decrease in elution time following phosphorylation compared to their similar-sized peptides with phosphoserine or phosphothreonine residues. While peptide phosphorylation generally led to a decrease in the observed elution time, five peptides displayed increased elution times as a result of phosphorylation. For large peptides (> or =18 amino acids), the elution time shifts due to single phosphorylation were limited (ranging between -0.48 and +0.03 min), while the elution time shifts for small peptides (<18 amino acids) were characterized by a larger deviation (ranging between -5.28 and +0.59 min). The predictive capability for the observed RPLC elution time change due to phosphorylation has been suggested, which will aid in assigning confident phosphopeptide identifications and their subsequent confirmation.

Fig. 1

Normalization of elution time applying the segmented conversion of (A) the original basepeak, (B) reconstructed ion chromatogram for the five standard peptides, (C) conversion of (B) to the average elution time from Table 1, and (D) converted basepeak. The values in the parentheses are the average elution times for the five standard peptides that were used for the conversion. Four segmented ranges were used for the conversion. The slope and the y-intercept for each segment were calculated as shown in the figure and then applied to convert other peaks. A mixture of tryptic digest of 2.5 μg chicken MBP and 20 ng of each standard peptide was loaded onto the column. The values in parentheses are the average elution times of the five standard peptides, used for the conversion. For the reconstructed ion chromatogram, scanning of ± 0.5 m/z of the abundant peaks of the standard peptides (singly charged state for Std-2 and Std-3 and doubly charge state for the other standard peptides) were made.

Fig. 2

Elution time confirmation for a peptide of interest. (A) Original basepeak, (B) reconstructed ion chromatogram for of the doubly charged HRDpSGLLDSLGR, theoretical m/z = 703.33, and (C) tandem mass spectrum of the doubly charged HRDpSGLLDSLGR (m/z 703.36) at 46.69 min. The normalized elution time of the peptide of interest is shown in (B). The peak with asterisk is from the result of neutral loss. The intensity around this peak was reduced by half since the peak was highly abundant. For the details of the sample, refer to Fig. 1.

Fig. 3

Plot of the number of amino acids vs. the elution time of non-phosphorylated peptides. Circle or square around a peptide number means the peptide contains a potential phosphotyrosine or phosphothreonine residue, respectively. The other peptides contain potential phosphoserine residues. Potential phosphotyrosine-containing peptides (peptides with circles around the number) have shown higher elution times compared to similar sized peptides. Standard deviations are shown with average elution times. A dark circle (•) indicates that the peptide contains more than one phosphorylated amino acid.

Fig. 4

Effect of the number of amino acids to the normalized elution time shift due to phosphorylation. Circle or square around a peptide number means the peptide contains phosphotyrosine or phosphothreonine residue, respectively. The other peptides contain phosphoserine residues. Standard deviations are shown with average elution times. A dark circle (•) means that the peptide contains more than one phosphorylated amino acid.

Fig. 5

Effect of the normalized elution time of the non-phosphorylated peptides (x-axis) to the normalized elution time shift due to phosphorylation. Circle or square around a peptide number means the peptide contains a phosphotyrosine or phosphothreonine residue, respectively. The other peptides contain phosphoserine residues. Standard deviations are shown with average elution times. A dark circle (•) means that the peptide contains more than one phosphorylated amino acid.

Fig. 6

Dependence of the elution times of non-phosphopeptides upon their GRAVY values. No overall correlation is observed. Circle or square around a peptide number means the peptide contains phosphotyrosine or phosphothreonine residue, respectively. The other peptides contain phosphoserine residues. Standard deviations are shown with average elution times.

Fig. 7

Relation between the GRAVY values and the normalized elution time shifts upon phosphorylation. Circle or square around a peptide number means the peptide contains phosphotyrosine or phosphothreonine residue, respectively. The other peptides contain phosphoserine residues. Standard deviations are shown with average elution times. Linear regression for the hydrophobic phosphotyrosine-containing peptides shows the R² as 0.9934, while linear regression for all the phosphotyrosine-containing peptides shows the R² as 0.9645.

Fig. 8

Dependence of the elution times of non-phosphopeptides upon their hydrophobicity values. Circle or square around a peptide number means the peptide contains phosphotyrosine or phosphothreonine residue, respectively. The other peptides contain phosphoserine residues. Standard deviations are shown with average elution times. Linear regression for all of the peptides and only for the tryptic peptides shows the R² values as 0.9222 and 0.971, respectively.

Fig. 9

Dependence of Δt/t₀ on the number of amino acids (N), where Δt is the normalized elution time shift from phosphorylation and t₀ is the normalized elution time of non-phosphorylated peptide. The linear regression line is calculated based on the phosphotyrosine containing peptides with less than 18 amino acids (peptides 1, 3, 9, 12, and 21). Good correlation has been observed for the regression (R² = 0.9753). Circle or square around a peptide number means the peptide contains phosphotyrosine or phosphothreonine residue, respectively. The other peptides contain phosphoserine residues. Standard deviations are also shown. A dark circle (•) means that the peptide contains more than one phosphorylated amino acid.