Sequential Fe3O4/TiO2 enrichment for phosphopeptide analysis by liquid chromatography/tandem mass spectrometry.
Protein phosphorylation regulates a wide range of cellular functions and is associated with signaling pathways in cells. Various strategies for enrichment of phosphoproteins or phosphopeptides have been developed. Here, we developed a novel sequential phosphopeptide enrichment method, using magnetic iron oxide (Fe(3)O(4)) and titanium dioxide (TiO(2)) particles, to detect mono- and multi-phosphorylated peptides. In the first step, phosphopeptides were captured on Fe(3)O(4) particles. In a subsequent step, any residual phosphopeptides were captured on TiO(2) particles. The particles were eluted and rinsed to yield phosphopeptide-enriched fractions that were combined and analyzed using liquid chromatography/tandem mass spectrometry (LC/MS/MS). The validity of this sequential Fe(3)O(4)/TiO(2) enrichment strategy was demonstrated by the successful enrichment of bovine alpha-casein phosphopeptides. We then applied the sequential Fe(3)O(4)/TiO(2) enrichment method to the analysis of phosphopeptides in L6 muscle cell lysates and successfully identified mono- and multi-phosphorylated peptides.
Choi
Sunkyu
S
Division of Molecular and Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea.
Kim
Jaeyoon
J
Cho
Kun
K
Park
Gunwook
G
Yoon
Jong Hyuk
JH
Park
Sehoon
S
Yoo
Jong Shin
JS
Ryu
Sung Ho
SH
Kim
Young Hwan
YH
Kim
Jeongkwon
J
D000595:Amino Acid Sequence; D000818:Animals; D002364:Caseins / Q000378:metabolism; D002417:Cattle; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D052203:Ferrosoferric Oxide / Q000737:chemistry*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D010748:Phosphopeptides / Q000032:analysis* / Q000378:metabolism; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*; D014025:Titanium / Q000737:chemistry*
D002364:Caseins; D010446:Peptide Fragments; D010748:Phosphopeptides; C009495:titanium dioxide; D014025:Titanium; D052203:Ferrosoferric Oxide