Phosphopeptide elution times in reversed-phase liquid chromatography
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 utilizing a high resolution reversed-phase liquid chromatography (RPLC) system. 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 the observed elution time generally decreased due to phosphorylation, five peptides displayed increased elution time 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.
- Research Organization:
- Pacific Northwest National Lab. (PNNL), Richland, WA (United States). Environmental Molecular Sciences Lab. (EMSL)
- Sponsoring Organization:
- USDOE
- DOE Contract Number:
- AC05-76RL01830
- OSTI ID:
- 947926
- Report Number(s):
- PNNL-SA-53249; 20496; 400412000
- Journal Information:
- Journal of Chromatography A, 1172(1):9-18, Vol. 1172, Issue 1
- Country of Publication:
- United States
- Language:
- English
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