Nanospray 3 source

Samples for co-IP were prepared as described previously with minor modification (Wang et al., 2015 (link)). In brief, anti-CD3/CD28-stimulated Jurkat T cells were lysed and followed by IP with anti-PKC-θ or anti-IgG. IPs immobilized on protein G beads and 1 μg recombinant GST-RanGAP1 protein immobilized on GSH Sepharose beads were separately washed 2× with kinase buffer and mixed to initiate the kinase assay. Reactions were terminated by Laemmli sample buffer, boiled, and resolved on SDS–PAGE. Gel bands of interest were excised and subjected to tryptic digestion. After desalting, the peptides were analyzed by tandem MS. A splitless Ultra 2D Plus system (Eksigent) coupled to the TripleTOF 5600 System (AB SCIEX) with a Nanospray III source (AB SCIEX) were performed to analyze immunoprecipitated proteins and identify posttranscriptional modification sites of RanGAP1. The search engine ProteinPilot V4.5 was used to assigned potential modification sites with high confidence.

The protocol for MS analysis was as described previously by [22 (link)] with slight modifications. Firstly, each fraction was resuspended in buffer A (5% ACN, 0.1% FA) and centrifuged at 20000 × g for 10 min, and the final concentration of the peptide was about 0.5 μg/μl. Subsequently, 10 μl of the supernatant was loaded onto a 2 cm C18 trap of the column LC-20 AD nanoHPLC pump system (Shimadzu, Kyoto, Japan). The peptides were eluted onto a 10 cm analytical C18 column (inner diameter 75 μm) packed in-house. The samples were loaded at 8 μL/min for 4 min, then the gradient started at 300 nL/min, from 2 to 35% B (95% ACN, 0.1% FA) for 35 min, linear gradient to 60% for 5 min, 80% for 2 min, maintenance for 4 min, and finally returned to 5% for 1 min. The fractions were analyzed using a TripleTOF 5600 System (AB SCIEX, Concord, ON) with a Nanospray III source (AB SCIEX, Concord, ON) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA). Data were acquired in 250 ms, and as many as 30 product ion scans were collected if a threshold of 120 counts per second (counts/s) was exceeded, with a 2+ to 5+ charge-state and a 15-s dynamic exclusion setting. Each fraction was analyzed by the nano LC-MS/MS as described previously by [23 (link)].

The iTRAQ-labeled peptides were fractionated by RP chromatography using the Shimadzu LC-20AB HPLC Pump system. Each fraction was resuspended in buffer A (5% CAN, 0.1% FA). Centrifuging the mixture at 20,000 × g for ten minutes, we obtained the supernatant, of which the peptide final concentration was approximately 0.5 g/l. Finally, according to the experimental protocol, we can acquire the results. The data were acquired with a 2.5 kV ion spray voltage, 30 psi curtain gas, 15 psi nebulizer gas and interface heater temperature of 150 °C. In addition, the data were obtained with a Triple TOF5600 System (AB SCIEX, Concord, ON) fitted with a Nanospray III source (AB SCIEX, Concord, ON) and a pulled quartz tip as the emitter (New Objectives, Woburn, MA) and controlled with Analyst 1.6 software (AB SCIEX, Concord, ON).