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 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.
Kim
Jeongkwon
J
Environmental Molecular Science Laboratory, MSIN K8-98, Pacific Northwest National Laboratory, P.O. Box 999, Richland, WA 99352, USA.
Petritis
Konstantinos
K
Shen
Yufeng
Y
Camp
David G
DG
Moore
Ronald J
RJ
Smith
Richard D
RD
D000595:Amino Acid Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D010766:Phosphorylation; D010768:Phosphoserine / Q000737:chemistry; D010769:Phosphothreonine / Q000737:chemistry; D019000:Phosphotyrosine / Q000737:chemistry; D011237:Predictive Value of Tests; D020543:Proteome / Q000737:chemistry; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D013997:Time Factors; D014269:Trifluoroacetic Acid / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D010455:Peptides; D010748:Phosphopeptides; D020543:Proteome; D010769:Phosphothreonine; D010768:Phosphoserine; D019000:Phosphotyrosine; D014269:Trifluoroacetic Acid; D014357:Trypsin