Jupiter c 18 300

Peptide digests (8µL each) were injected onto a 1260 Infinity nHPLC stack (Agilent Technologies), and separated using a 75 micron I.D. x 15 cm pulled tip C-18 column (Jupiter C-18 300 Å, 5 micron, Phenomenex). This system runs in-line with a Thermo Q Exactive HFx mass spectrometer, equipped with a Nanospray Flex™ ion source (Thermo Fisher Scientific), and all data were collected in CID mode. The nHPLC is configured with binary mobile phases that includes solvent A (0.1%FA in ddH₂O), and solvent B (0.1%FA in 15% ddH₂O/85% ACN), programmed as follows; 10min @ 5%B (2µL/min, load), 30min @ 5%-40%B (linear: 0.5nL/min, analyze), 5min @ 70%B (2µL/min, wash), 10min @ 0%B (2µL/min, equilibrate). Following each parent ion scan (300-1200m/z @ 60k resolution), fragmentation data (MS2) were collected on the top most intense 18 ions @7.5K resolution. For data dependent scans, charge state screening and dynamic exclusion were enabled with a repeat count of 2, repeat duration of 30s, and exclusion duration of 90s.

High Performance Liquid Chromatography (HPLC) was performed using a Dionex Ultimate 3000 instrument (Thermo Fisher Scientific; Rockford, IL, USA). To detect total polymer and protein content, polymersomes were disrupted in 0.05% trifluoroacetic acid (TFA) (Sigma-Aldrich). A multi-step gradient of HPLC grade methanol (Sigma-Aldrich) as eluent A and milliQ water (Merck Millipore; Burlington, MA, USA) in 0.05% trifluoroacetic acid as eluent B was used to elute samples over 30 minutes with a C18 column (Jupiter C18 300 Å, 150 × 4.60 mm, 5 μm; Phenomenex®; Torrance, CA,USA) as reported previously, with a mobile phase gradient of 95% mQH2O + 0.05% TFA and 5% MeOH + 0.05% TFA and a flow rate of 1 mL/min^{21 (link)}. Polymer content was detected using fluorescence (λex: 540 nm; λem: 560 nm). Protein content was detected using the UV/Vis channel of 220 nm. Areas under the detected peaks were measured using Chromeleon software (Thermo Fisher Scientific).

Peptide digests (8 μL each) were injected onto a 1260 Infinity nHPLC stack (Agilent Technologies), and separated using a 75 μm inside diameter x 15 cm pulled tip C-18 column (Jupiter C-18 300 Å, 5 micron, Phenomenex). This system runs in-line with a Thermo Orbitrap Velos Pro hybrid mass spectrometer, equipped with a Nanospray Flex^TM ion source (Thermo Fisher Scientific); all data were collected in collision-induced dissociation mode. The nano-High Performance Liquid Chromatography is configured with binary mobile phases that includes solvent A (0.1%FA in ddH₂O), and solvent B (0.1%FA in 15% ddH₂O / 85% ACN), programmed as follows; 10min @ 5%B (2μL/ min, load), 90min @ 5%-40%B (linear: 0.5nL/ min, analyze), 5min @ 70%B (2μL/ min, wash), 10min @ 0%B (2μL/ min, equilibrate). Following each parent ion scan (300-1200m/z @ 60k resolution), fragmentation data (MS2) were collected on the top most intense 15 ions. For data dependent scans, charge state screening and dynamic exclusion were enabled with a repeat count of 2, repeat duration of 30 s, and exclusion duration of 90 s.

Peptide digests (8 μL each) were injected onto a 1260 Infinity nHPLC stack (Agilent) and separated using a 75 micron I.D. × 15 cm pulled tip C-18 column (Jupiter C-18 300 Å, 5 micron, Phenomenex). This system ran in-line with a Thermo Orbitrap Velos Pro hybrid mass spectrometer, equipped with a nanoelectrospray source (Thermo Fisher Scientific), and all data were collected in CID mode. The nHPLC was configured with binary mobile phases that included solvent A (0.1% formic acid in ddH₂O) and solvent B (0.1% formic acid in 15% ddH₂O/85% ACN) programmed as follows: 10 min @ 0%fet alB (2 μL/min, load), 90 min @ 0%−40%B (0.5 nL/min, analyze), 15 min @ 0%B (2 μL/min, equilibrate). Following each parent ion scan (350–1200 m/z @ 60k resolution), fragmentation data (MS2) was collected on the top most intense 15 ions. For data dependent scans, charge state screening and dynamic exclusion were enabled with a repeat count of 2, repeat duration of 30 s, and exclusion duration of 90 s.