. 2012 Feb 24;442(Pt 3):681–692. doi: 10.1042/BJ20111530

Table 1. MS analysis of peptides from HPLC peaks and TLC spots.

Labelled fractions from autophosphorylated GST–eEF2K wild-type and mutant HPLC runs (Figure 2A) were analysed by nanospray ESI–tandem MS or LC–tandem MS as described in the Experimental section. Also, spots from TLC plates obtained after autophosphorylation and trypsin digestion of wild-type eEF2K, comparable with the separation pattern seen in Figure 3(A), were taken and eluted for LC–tandem MS analysis. Phosphopeptides were identified either by multistage activation or by loss of 98 Da upon collision-induced dissociation. The phosphorylated residue (bold) was further identified by fragmentation in MS³ mode. Residue numbering refers to the sequence of human eEF2K (Uniprot O00418) without the GST tag.

Peak	Phosphopeptide sequence(s) from HPLC peaks	Phosphorylation sites identified in HPLC peaks	Phosphorylation sites identified in TLC spots^*
I	K⁴⁶⁷YESDEDS⁴⁷⁴LGSSGR⁴⁸⁰	Ser⁴⁷⁴	Ser⁴⁷⁴ in spot E
II	Y⁵⁹YSNLTKS⁶⁶ER⁶⁸ and Y⁵⁹YS⁶¹NLTKS⁶⁶ER⁶⁸	Ser⁶⁶ and Ser⁶¹	Ser⁶¹ in spot B
III	E⁴³⁴SENSGDSGYPS⁴⁴⁵EKRGELDDPEPR⁴⁵⁷	Ser⁴⁴⁵	Ser⁴⁴⁵ in spots G and l
IV	L³⁴²LQSAKT³⁴⁸ILRGT³⁵³EEK³⁵⁶ and Y⁶⁹SSSGSPANS⁷⁸FHFK⁸² and L³⁴²LQSAKT³⁴⁸ILR³⁵¹	Thr³⁴⁸, Thr³⁵³ and Ser⁷⁸	Thr³⁴⁸ in spot A
V	T³⁶⁴LS³⁶⁶GSRPPLLR³⁷⁴	Ser³⁶⁶
VI	W⁴⁸⁶NLLNS⁴⁹¹SR⁴⁹³	Ser⁴⁹¹
VII	No identification
VIII	T³⁶⁴LS³⁶⁶GSRPPLLRPLSENSGDENMSDVTFDSLPSSPSSATPHSQK⁴⁰⁶	Ser³⁶⁶
IX	No identification

*See Figure 3(B) for the lettering of spots.