Figure 3: LC-MS/MS and SWATH analysis of two HLA-A2 restricted proinsulin peptides eluted from C1R cells transfected with proinsulin and HLA-A2. (A) Total ion chromatogram (TIC) acquired via information dependent data acquisition (IDA) and the extracted ion chromatograms (XIC) of ions corresponding to and m/z value of 484.7454 Da and 528.8080 Da. Shown in (B) are spectra corresponding to these ions identified as insulin B10-18 HLVEALYLV and pre-proinsulin 15-24 AWLGPDPAAA. (C) i depicts the TIC acquired using SWATH-MS and the XIC derived from this SWATH-MS data set corresponding to the HVEALYLV peptide (528.8080 Da) as shown in ii. All ions in the SWATH isolation window of 25 Da width (525-550 Da) containing the B10-18 peptide HLVEALYLV (precursor mass of 528.8080 Da) across the LC are shown in (C) iii. In (C) iv, the overlapping transitions corresponding to the y- and b-ions of HLVEALYLV at identical retention time (21.7 min) are shown confirming detection of this species. For the collection of SWATH data, samples were analyzed by an AB SCIEX TripleTOF® 5600 mass spectrometer by electrospray ionization with the system operating in SWATH™ acquisition mode. Each cycle consisted of an initial MS1 scan of 100-1800 m/z, followed by sequential SWATH™ windows of 25 m/z spanning the range of 300-1000 m/z and within these acquiring MS2 data of 100-1800 m/z. Data were analyzed using the open source software Skyline v1.4. Full scan settings were applied to reflect the SWATH™ acquisition parameters described above. Transition settings were y and b ions with doubly-charged precursor states and 6 product ions with m/z>precursor −2.