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 highresolution 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 (= 18 amino acids), the elution time shifts due to single phosphorylation were limited (ranging between −0.48 min 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 min 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.

Application of the broadband collision‐induced dissociation (bbCID) mass spectrometry approach for protein glycosylation and phosphorylation analysis

“…The presence of a phospho group is generally expected to decrease the hydrophobicity of a peptide and lower its retention time during reversed‐phase chromatography. However, an increased phosphopeptide hydrophobicity has also been previously reported and attributed to polar interactions between the phospho group and the side chains of neighbouring amino acids . This is consistent with the results observed in the present study.…”

Chromatographic behavior of peptides containing oxidized methionine residues in proteomic LC–MS experiments: Complex tale of a simple modification

“…Systematic large-scale observations on chromatographic behavior of PTM peptides including Met oxidation appeared following rapid developments in LC-MS proteomics applications [8,10,[20][21][22]. Literature indicates that peptides containing oxidized methionine are more hydrophilic under RP LC conditions than their unmodified analogs.…”

Separation of multiphosphorylated peptide isomers by CZE

“…Although RP-HPLC is well suited for separating many peptides from a digest of one or a few proteins, it is difficult to separate longer phosphopeptides from the homologous unmodified peptides, even for shallow gradients. Several reports have shown that for a given mixture of a few peptides and phosphopeptides it is possible to optimize the oeluent system, ion pair reagent and stationary phase to achieve at least a partial separation of most components up to 15mer peptides [3][4][5]. Separation of phosphopeptide isomers, i.e.…”