Discovering and validating unknown phospho-sites from p38 and HuR protein kinases in vitroby Phosphoproteomic and Bioinformatic tools

Table 1 The 3 phosphorylated proteins (HuR, Chain B and p38p) and the 6 phosphopeptides identified and validated (amino acid sequences below the identified proteins) when using SIMAC coupled to MAS by the LTQ ion Trap mass spectrometer are shown in this table.

Proteins	Phosphopeptides
HuR RNA binding protein gi/1022961	DVEDMFSphR (*)
	VLVDQTTphGLSR
	DANLYSphGLPR (*)
	SLFSSIGEVESphAK (*)
Chain B, Structure Of Appbp-1-Uba3-nedd8-Mgatp-Ubc 12 (c111a), A Trapped Ubiquitin-Like Protein Activation Complex gi/126031226	TphAVINAASGR (*)
P38 MAP Kinase gi/1469306	DLSphSIFR (*)

Around ≥ 3 μg of complex peptides mixture were loaded into SIMAC micro-columns and analyzed by nano-LC-ESI-LTQ ion Trap (Thermo).
In addition when using SIMAC coupled to DDNLMS3 3 proteins were identified: (a) HuR RNA binding protein gi/1022961, (b) P38 MAP Kinase gi/1469306 and (c) Change B, A Trapped Ubiquitin like-protein activation gi/126031226.
(a) From the phosphorylated protein HuR RNA binding protein gi/1022961, 3 phosphopeptides were isolated and validated (K.DVEDMFSR.F, K.DANLYISGLPR.T, R.SLFSSIGEVESAK.L).
(b) From the phosphorylated protein p38 MAP Kinase gi/1469306, one phosphopeptide was isolated and validated (K.DLSSIFR.G).
(c) From the phosphorylated protein Change B, A Trapped Ubiquitin like-protein activation gi/126031226, one phosphopeptide was isolated and validated (R.TAVINAASGR.Q).
[Identified and validated phosphopeptides using SIMAC coupled to DDNLMS3 are distinguished by the symbol (*)]