Pal autosampler

The dimethylamino acid (1.0 equivalent), NHS (1.2 equivalents), and DIC (1.0 equivalent) were first dissolved in DMSO (10 μL), and then diluted with anhydrous DCM (30 μL). The solution was incubated overnight at room temperature. A solution containing five peptides, NSILTETLHR, LSLVPDSEQGEAILPR, LSEPAELTDAVK, YGGFLR, and SVILLGR dissolved in 10 μL of 10% DIEA/DMF, was added to the activated dimethylamino acid. The solution was incubated overnight at room temperature. The coupling was then quenched with 20% formic acid/H₂O on ice. The solution was first dried with a speed-vac (Savant SC100, Thermo Fisher) then lyophilized (Labconco FreeZone Plus). After drying, the sample was desalted via an empty spin column (Thermo Fisher) packed with hydrophilic-lipophilic-balanced (HLB, Oasis^®) reversed-phase sorbent. The sample was then dried and reconstituted with FA/H₂O for liquid chromatography (LC)-MS analysis (Shimadzu HPLC pumps/controller, Kyoto, KYT, Japan and HTC PAL autosampler, Carrboro, NC).

Standards were prepared in 50% MeOH. Samples and standards were loaded onto a 1 mm × 75 mm C12 column (ACE 3 C18 PFP, Mac-Mod) using a Shimadzu HPLC system and PAL auto-sampler (20 μL per injection) at a flow rate of 70 μL min⁻¹. The column was maintained at 45°3 using a column oven. The column was connected inline to an electrospray source coupled to an LTQ-Orbitrap XL mass spectrometer (Thermo). Caffeine (2 pM μL⁻¹ in 50% acetonitrile with 0.1% formic acid) was injected as a lock mass through a tee at the column outlet using a syringe pump at 45 μL min⁻¹ (Harvard PHD 2000). Chromatographic separation was achieved with a linear gradient from 1% to 45% B in 6 min (A: 0.1% formic acid, B: 0.1% formic acid in acetonitrile) with an initial 1 min hold at 1% B and followed by 4 min wash at 100% B and equilibration for 8 min with 1% B (total 20 min program). Electrospray ionization was achieved using a spray voltage of 4.50 kV aided by sheath gas (Nitrogen) at a flow rate of 12 (arbitrary units) and auxiliary gas (Nitrogen) flow rate of 1 (arbitrary units). Full scan MS data were acquired in the Orbitrap at a resolution of 60,000 in profile mode from the m/z range of 110-220. Raw files were imported into Skyline v4.1 (MacCoss Lab) and peak areas for DPO were extracted using the small molecule workflow.