0 with neat TFA, incubated at 60 C for 2 hrs and spun at 14,000 rpm for 5 min to re move hydrolyzed ALS 1. Samples have been either subjected to LC MS analysis following a 10X dilution into mobile phase A or subjected to a TiO2 primarily based phosphopeptide enriched protocol. To enrich for phosphorylated peptides prior to LC MS evaluation, 1,125 ug of selleck chemical total digested protein from RBC ghosts had been brought to near dryness applying vacuum cen trifugation after which resuspended in 200 uL of 80% acetonitrile, 1% TFA, 50 mg ml MassPrep Enhancer. Samples had been loaded onto an in property packed TiO2 spin column having a 562 ug binding capacity pre equilibrated with 80% acetonitrile, 1% TFA. For all loading, washing, and elution actions, the centrifuge was set to attain a flow price of no more quickly than one hundred uL min.
Samples were washed twice with 200 uL 80% aceto P005091 882257-11-6 nitrile, 1% TFA, 50 mg ml MassPrep Enhancer followed by two washes with 200 uL 80% acetonitrile, 1% TFA. Retained peptides were eluted twice with one hundred uL 20% acetonitrile, 5% aqueous ammonia, acidified to pH 3 with neat formic acid and then brought to dryness working with vacuum centrifugation. Prior to LC MS evaluation, each sample was resuspended in 20 uL 2% acetonitrile, 0. 1% TFA, 25 mM citric acid. LC MS MS evaluation of RBC membrane ghosts Chromatographic separation of phosphopeptide enriched or non enriched samples was performed on a Waters NanoAquity UPLC equipped using a 1. 7 um BEH130 C18 75 um I. D. X 250 mm reversed phase column. The mo bile phase consisted of 0. 1% formic acid in water and 0. 1% formic acid in acetonitrile.
5 uL injec tions of each and every sample had been trapped for five min on a five um Symmetry C18 180 um I. D. X 20 mm column at 20 ul min in 99. 9% A. The analytical column was then switched in line and the mobile phase was held for five min at 5% B before applying a linear elution gradient of 5% B to 40% B over 90 min at 300 nL min. The analyt ical column was connected to fused silica PicoTip emit ter with a 10 um tip orifice and coupled towards the mass spectrometer by means of an electrospray interface. MS was acquired on a Thermo LTQ Orbitrap XL mass spectrometer operating in positive ion mode with an electrospray voltage of 2. 0 kV with genuine time lock mass correction on ambient polycyclodimethylsiloxane enabled. The instrument was set to obtain a precursor MS scan from m z 400 2000 with r 60,000 at m z 400 as well as a target AGC setting of 1e6 ions. MS MS spectra have been acquired in the linear ion trap for the major five most abundant precursor ions above a threshold of 500 counts. Maximum fill occasions had been set to 1000 ms for full MS scans acquired inside the OT and 250 ms for MS MS acquired inside the linear ion trap, using a CID power setting of 35% in addition to a dynamic exclusion of 60 s for previously frag mented precursor ions.