Symmetry c18 20 mm 180 μm trapping column

Quantitative LC-MS/MS was performed on 3 μL using an MClass UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm × 180 μm trapping column (5 μL/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 μm Acquity HSS T3 C18 75 μm × 250 mm column (Waters Corp.) with a 90-min linear gradient of 5–30% acetonitrile with 0.1% formic acid at a flow rate of 400 nL/min (nL/min) with a column temperature of 55°C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (−40v, −60v, −80v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with an r = 120,000 (@ m/z 200) full MS scan from m/z 375–1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired in the ion trap in Rapid mode with a target AGC value of 1e4 and max fill time of 35 ms. The total cycle time for each CV was 0.66s, with total cycle times of 2 s between like full MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each injection was approximately 2 h.

Quantitative LC/MS/MS was performed on 2 μL (16.6% of total sample) using an MClass UPLC system (Waters Corp) coupled to a Thermo Orbitrap Fusion Lumos high resolution accurate mass tandem mass spectrometer (Thermo) equipped with a FAIMSPro device via a nanoelectrospray ionization source. Briefly, the sample was first trapped on a Symmetry C18 20 mm × 180 μm trapping column (5 μl/min at 99.9/0.1 v/v water/acetonitrile), after which the analytical separation was performed using a 1.8 μm Acquity HSS T3 C18 75 μm × 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55C. Data collection on the Fusion Lumos mass spectrometer was performed for three difference compensation voltages (−40v, −60v, −80v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a r=120,000 (@ m/z 200) full MS scan from m/z 375 – 1500 with a target AGC value of 4e5 ions was performed. MS/MS scans were acquired in the ion trap in Rapid mode with a target AGC value of 1e4 and max fill time of 35 ms. The total cycle time for each CV was 0.66s, with total cycle times of 2 sec between like full MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The total analysis cycle time for each injection was approximately 2 hours.

PDQ-enriched peptides were reconstituted in 15 μL of 97/2/1 (v/v/v/) H2O/MeCN/TFA, and 4.5 μL was analyzed in duplicate by LC-MS/MS. Unbound fractions were reconstituted in the same buffer at ~1 μg/μL, and 1.5–2.5 μL was analyzed. LC-MS/MS used a Waters M-Class interfaced to a Thermo Fusion Lumos via a NanoSpray Flex source and CoAnn emitter 20 μm ID, 10 μm Tip ID, 6.35 cm length)). Peptides were trapped on a Symmetry C18 180 μm × 20 mm trapping column (Waters) at 5 μL/min with 99.9/0.1 v/v H₂O/MeCN and separated on a 75 μm × 25 cm HSS-T3 analytical column (Waters) at 400 nl/min and 55 °C and a gradient of 5–30% MeCN over 90 min. MS acquisition used a 3 s vendor-supplied synchronous precursor selection (TMTPro-SPS-MS3) template method. Briefly, Precursor scans used 120,000 resolution, standard AGC target and auto maximum injection time (IT). Precursors (scan range 400–1600 m/z, charge state 2–6, intensity threshold 5E3) were selected for collision-induced dissociation using 0.7 m/z, “Turbo” ion trap scan with 25 ms max IT. Finally 10 SPS precursors were subjected to higher-energy collision-induced dissociation with 0.7 m/z isolation, normalized collision energy of 55%, 50,000 resolution, 200% AGC and 200 ms max IT. Analysis of unbound fractions also used a FAIMS-Pro interface with a 3 CV method (−40 CV for 1.2 s, −55 CV for 1.2 s, −70 CV for 0.8 s).