Formation of phosphopeptide-metal ion complexes in liquid chromatography/electrospray mass spectrometry and their influence on phosphopeptide detection.
Despite major advances in mass spectrometry, the detection of phosphopeptides by liquid chromatography with electrospray mass spectrometry (LC/ES-MS) still remains very challenging in proteomics analysis. Phosphopeptides do not protonate efficiently due to the presence of one or more acidic phosphate groups, making their detection difficult. However, other mechanisms also contribute to the difficulties in phosphopeptide analysis by LC/ES-MS. We report here on one such undocumented problem: the formation of phosphopeptide-metal ion complexes during LC/ES-MS. It is demonstrated that both synthetic phosphopeptides and phosphopeptides from bovine beta-casein and alpha-casein form phosphopeptide-metal ion complexes containing iron and aluminum ions, resulting in a dramatic decrease in signal intensity of the protonated phosphopeptides. The interaction of phosphopeptides with metal ions on the surface of the C18 stationary phase is also shown to alter their chromatographic behavior on reversed-phase columns such that the phosphopeptides, especially multiply phosphorylated peptides, become strongly retained and very difficult to elute. The sources of iron and aluminum are from the solvents, stainless steel, glassware and C18 material. It was also found that, upon addition of EDTA, the formation of the phosphopeptide-metal ion complex is diminished, and the phosphopeptides that did not elute from the LC column can now be detected efficiently as protonated molecules. The sensitivity of detection was greatly increased such that a tetra-phosphorylated peptide, RELEELNVPGEIVEpSLpSpSpSEESITR from the tryptic digestion of bovine beta-casein, was detected at a limit of detection of 25 fmol, which is 400 times lower than without EDTA.
Liu
Suya
S
Department of Biochemistry, University of Western Ontario, London, Ontario, N6A 5B7, Canada.
Zhang
Cunjie
C
Campbell
J Larry
JL
Zhang
Haixia
H
Yeung
Ken K-C
KK
Han
Victor K M
VK
Lajoie
Gilles A
GA
D000818:Animals; D016477:Artifacts; D002364:Caseins / Q000032:analysis* / Q000737:chemistry*; D002417:Cattle; D002853:Chromatography, Liquid / Q000379:methods*; D007477:Ions; D010748:Phosphopeptides / Q000032:analysis* / Q000737:chemistry*; D011485:Protein Binding; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D002364:Caseins; D007477:Ions; D010748:Phosphopeptides