Ultimate 3000 nano uplc system

Digested serum samples were first cleaned using a C18 spin plate (Nest Group, Southborough, MA, USA) and then analyzed using an Orbitrap Fusion tribrid mass spectrometer (Thermo Scientific, Waltham, MA, USA) coupled with an Ultimate 3000 nano-UPLC system (Thermo Scientific). Two micrograms of reconstituted peptide (1 µg/mL) was first trapped and washed on a Pepmap100 C18 trap (5 μm, 0.3 × 5 mm) at 20 μL/min using 2% acetonitrile in water (with 0.1% formic acid) for 10 min and then separated on a Pepman100 RSLC C18 column (2.0 μm, 75 μm × 150 mm) using a gradient of 2 to 40% acetonitrile with 0.1% formic acid over 120 min at a flow rate of 300 nL/min and a column temperature of 40 °C. Eluted peptides were introduced into an Orbitrap Fusion MS via nano-electrospray ionization source with a temperature of 300 °C and spray voltage of 2000 V. The peptides were analyzed by data-dependent acquisition in positive mode using an Orbitrap MS analyzer for a precursor scan at 120,000 FWHM from 400 to 2000 m/z and an ion-trap MS analyzer for MS/MS scans in top speed mode (3 s cycle time) with dynamic exclusion settings (repeat count 1 and exclusion duration 15 s). Higher-energy collisional dissociation (HCD) was used as a fragmentation method with a normalized collision energy of 32%.

Peptides were reconstituted in solvent A (2% ACN/0.1% FA) and approximately, two µg samples (2/12 µL) were injected on a 50 cm long EASY-Spray C18 column (Thermo Fisher Scientific) connected to an Ultimate 3000 nanoUPLC system (Thermo Fisher Scientific) using a 90 min long gradient: 4-26% of solvent B (98% ACN/0.1% FA) in 90 min, 26-95% in 5 min, and 95% of solvent B for 5 min at a flow rate of 300 nL/min. Mass spectra were acquired on a Q Exactive HF hybrid quadrupole orbitrap mass spectrometer (Thermo Fisher Scientific) ranging from m/z 375 to 1700 at a resolution of R=120,000 (at m/z 200) targeting 5x10⁶ ions for a maximum injection time of 80 ms, followed by data-dependent higher-energy collisional dissociation (HCD) fragmentations of precursor ions with a charge state 2+ to 8+, using 45 s dynamic exclusion. The tandem mass spectra of the top 18 precursor ions were acquired with a resolution of R=60,000, targeting 2x10⁵ ions for a maximum injection time of 54 ms, setting quadrupole isolation width to 1.4 Th and normalized collision energy to 33%.

20 μg of recombinant PKD, incubated with and without ATP for 60 min, were precipitated 37 and digested with trypsin (0.4 μg, overnight, 37 °C). The resulting peptide mixture was desalted on C18 Micro Spin Columns (Harvard Apparatus) before being subjected to anti‐pTyr IP (PY99, Santa Cruz Biotechnology, Dallas, TX, USA) in TBS/1% n‐octyl glucoside (overnight, 4 °C). Beads were eluted with 50% acetonitrile/1% formic acid (10 min, RT). Subsequently, samples were prepared for MS by using C18 ZipTips. The resulting peptide mixture was submitted to high resolution LC‐MS/MS using an Ultimate 3000 nano UPLC system interfaced with an Orbitrap Q‐Exactive MS via an EASY‐spray (C18, 15 cm) column (Thermo Fisher Scientific). The Q‐Exactive MS was operated in data‐dependent mode selecting the top ten precursors for MS/MS. Protein identifications were obtained from the mascot (Matrix science, version 2.2.2) search engine using UniProt/SwissProt (Homo sapiens, 20202 entries) as a database, allowing up to three missed tryptic cleavages, phosphorylation of STY and oxidation of Met as variable modifications. Peptide abundances were determined using the progenesis qi software package (Nonlinear Dynamics, Newcastle upon Tyne, UK).