Development of a novel chemical probe for the selective enrichment of phosphorylated serine- and threonine-containing peptides.
Gaining insight into phosphoproteomes is of the utmost importance for understanding regulation processes such as signal transduction and cellular differentiation. While the identification of phosphotyrosine-containing amino acid sequences in peptides and proteins is now becoming possible, mainly because of the availability of high-affinity antibodies, no general and robust methodology allowing the selective enrichment and analysis of serine- and threonine-phosphorylated proteins and peptides is presently available. The method presented here involves chemical modification of phosphorylated serine or threonine residues and their subsequent derivatization with the aid of a multifunctional probe molecule. The designed probe contains four parts: a reactive group that is used to bind specifically to the modified phosphopeptide, an optional part in which heavy isotopes can be incorporated, an acid-labile linker, and an affinity tag for the selective enrichment of modified phosphopeptides from complex mixtures. The acid-cleavable linker allows full recovery from the affinity-purified material and removal of the affinity tag prior to MS analysis. The preparation of a representative probe molecule containing a biotin affinity tag and its applicability in phosphoproteome analysis is shown in a number of well-defined model systems of increasing degrees of complexity. Amounts of phosphopeptide as low as 1 nmol can be modified and enriched from a mixture of peptides. During the development of the beta-elimination/nucleophilic addition protocol, special attention was paid to the different experimental parameters that might affect the chemical-modification steps carried out on phosphorylated residues.
van der Veken
Pieter
P
Department of Medicinal Chemistry, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands.
Dirksen
Eef H C
EH
Ruijter
Eelco
E
Elgersma
Ronald C
RC
Heck
Albert J R
AJ
Rijkers
Dirk T S
DT
Slijper
Monique
M
Liskamp
Rob M J
RM
D000345:Affinity Labels; D015335:Molecular Probes / Q000138:chemical synthesis*; D010455:Peptides / Q000302:isolation & purification*; D010750:Phosphoproteins / Q000302:isolation & purification*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D012694:Serine; D013912:Threonine
D000345:Affinity Labels; D015335:Molecular Probes; D010455:Peptides; D010750:Phosphoproteins; D013912:Threonine; D012694:Serine