Table 2.

Identified NHERF1 phosphorylation sites

Overexpressed TAP–NHERF1 was purified as described under “Experimental procedures” and digested by GluC (cleavage site, Asp or Glu) or trypsin (cleavage site, Arg or Lys). Samples were loaded on an LTQ Orbitrap XL mass spectrometer. Data were searched using Sequest (99) against a human sequence database. Xcorr values indicate the proximity of the observed spectrum to an ideal spectrum for the matched peptide. Parts/million (ppm) indicate the difference between the observed peptide mass and the expected peptide mass calculated from the peptide sequence. The confidence of phosphorylation site localization was evaluated with phosphoRS 2.0 (44) implemented in Proteome Discoverer (ThermoFisher Scientific). The phosphoRS score is based on the cumulative binomial probability (0–100%) that the observed match is a random event. Values >75% are confidence scores, good evidence of true phosphorylation sites.

Position	Identified peptide	Protease	Charge	Xcorr	ppm	PhosphoRS site probability
46	R.LVEPGSPAEK.A	Trypsin	2	1.972	1.13	100
162	K.KGPSGYGFNLHSDK.S	Trypsin	2	2.057	9.69	92.0
181	R.SVDPDSPAEASGLR.A	Trypsin	2	2.192	0.72	100
269	E.NSREALAE.A	GluC	2	2.446	−4.64	100
280	R.EALAEAALESPRPALVR.S	Trypsin	2	2.565	−2.66	100
290	R.SASSDTSEELNSQDSPPK.Q	Trypsin	2	4.616	3.73	92.4
291	R.SASSDTSEELNSQDSPPK.Q	Trypsin	2	4.529	−0.06	92.9
293	R.SASSDTSEELNSQDSPPK.Q	GluC	2	4.711	−1.30	86.4
299	E.LNSQDSPPKQDSTAPSSTSSSDPILD.F	GluC	2	4.052	−3.21	87.2
302	E.LNSQDSPPKQD.S	GluC	2	3.453	0.12	100