Mass Spectrometry

HA-LARP6 was expressed and immunoprecipitated from HLFs in presence of phosphatase inhibitors. The immunoprecipitated protein was resolved on SDS PAGE gel and stained with GelCode Blue Stain Reagent (Thermo Scientific, 24590). The HA-LARP6 band was excised and in-gel trypsin digest was done using ProteoExtract All-in-One Trypsin Digestion Kit (Calbiochem, 650212) for 2 hours at 37 °C with shaking. Peptides were eluted in 50 μl 0.1% formic acid, separated on LCMS and the LC eluent was directly nano-sprayed into an LTQ Orbitrap Velos mass spectrometer (Thermo Scientific). The MS data were acquired using the following parameters: 10 data-dependent collisional-induced-dissociation (CID) MS/MS scans per full scan (400 to 2000 m/z) at a mass resolution for MS1 of 60000, minimum signal required to trigger MS2 was 500, MS mass range 0 to 1000000 and dynamic exclusion enabled with following parameters: Repeat count:1, Repeat Duration: 30.00, exclusion list size: 500, exclusion duration: 60.00, exclusion mass width relative to low and high mass: 10 ppm. All measurements were performed at room temperature and three technical replicates per sample were run to allow for statistical comparisons. The raw files were analyzed using Proteome Discoverer (version 1.4) software package with SequestHT and Mascot search nodes using Homo sapiens specific FASTA database and the Percolator peptide validator. Phosphorylation was detected by both SequestHT and Mascot and was verified by inbuilt phosphoRS node in proteome discoverer. Scaffold (version Scaffold_4.3.4, Proteome Software Inc., Portland, OR) was used to validate MS/MS-based peptide and protein identifications. Peptide identifications were accepted if they could be established at greater than 95.0% probability by the Scaffold Local FDR algorithm.