Identification of single and double sites of phosphorylation by ECD FT-ICR/MS in peptides related to the phosphorylation site domain of the myristoylated alanine-rich C kinase protein.
A series of phosphorylated test peptides was studied by electron capture dissociation Fourier transform ion cyclotron resonance mass spectrometry (ECD FT-ICR MS). The extensive ECD-induced fragmentation made identification of phosphorylation sites for these peptides straightforward. The site(s) of initial phosphorylation of a synthetic peptide with a sequence identical to that of the phosphorylation site domain (PSD) of the myristoylated alanine-rich C kinase (MARCKS) protein was then determined. Despite success in analyzing fragmentation of the smaller test peptides, a unique site on the PSD for the first step of phosphorylation could not be identified because the phosphorylation reaction produced a heterogeneous mixture of products. Some molecules were phosphorylated on the serine closest to the N-terminus, and others on one of the two serines closest to the C-terminus of the peptide. Although no definitive evidence for phosphorylation on either of the remaining two serines in the PSD was found, modification there could not be ruled out by the ECD fragmentation data.
Woodling
Kellie A
KA
Department of Chemistry, University of Florida, Gainesville, Florida 32611, USA.
Eyler
John R
JR
Tsybin
Yury O
YO
Nilsson
Carol L
CL
Marshall
Alan G
AG
Edison
Arthur S
AS
Al-Naggar
Iman M
IM
Bubb
Michael R
MR
D000595:Amino Acid Sequence; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D017550:Spectroscopy, Fourier Transform Infrared
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D010455:Peptides; D000076250:Myristoylated Alanine-Rich C Kinase Substrate