Symmetry c18 trapping column

The protein samples were digested in Amicon Ultra-0.5 centrifugal filters using modified FASP method^{50 (link)}. The peptides were separated with the nanoAcquity UPLC system (Waters) equipped with a 5-µm Symmetry C18 trapping column, 180 µm×20 mm, reverse-phase (Waters), followed by an analytical 1.7-µm, 75 µm×250 mm BEH-130 C18 reversed-phase column (Waters), in a single-pump trapping mode. The parameters of the HD-MSE runs were described previously^{51 (link)}. Protein identifications were performed with ProteinLynx Global Server (PLGS v3.0) as described^{51 (link)}. Database searches were carried out against UniProt human protein database (release_07072015, 71907 entries) with Ion Accounting algorithm and using the following parameters: peptide and fragment tolerance: automatic, maximum protein mass: 500 kDa, minimum fragment ions matches per protein: 7, minimum fragment ions matches per peptide: 3, minimum peptide matches per protein: 1, primary digest reagent: trypsin, missed cleavages allowed: 2, fixed modification: carbamidomethylation C, variable modifications: deamidation (N, Q), oxidation of Methionine (M) and FDR < 4%.

Tryptic Digests: ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nano-Ultra Performance Liquid Chromatography (10 kpsi nanoAcquity; Waters, Milford, MA, USA). The mobile phase was: A) H2O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed-phase Symmetry C18 trapping column (180 μm internal diameter, 20 mm length, 5 μm particle size; Waters). The peptides were then separated using a HSS T3 nano-column (75 μm internal diameter, 250 mm length, 1.8 μm particle size; Waters) at 0.35 μL/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4–30%B in 163 min, 30–90%B in 5 min, maintained at 90% for 5 min and then back to initial conditions. The nanoUPLC was coupled online through a nanoESI emitter (10 μm tip; New Objective; Woburn, MA, USA) to a quadrupole orbitrap mass spectrometer (Q Exactive Plus, Thermo Scientific) using a FlexIon nanospray apparatus (Thermo). Data was acquired in data dependent acquisition (DDA) mode, using a Top10 method. MS1 resolution was set to 70,000 (at 400 m/z), mass range of 300–1,650m/z, AGC of 3e6 and maximum injection time was set to 50 ms. MS2 resolution was set to 17,500, quadrupole isolation 1.7 m/z, AGC of 1e5, dynamic exclusion of 60 s and maximum injection time of 60 ms.

LC/MS-grade solvents were used for all the chromatographic steps. Each sample was loaded using splitless nano-ultraperformance liquid chromatography (nano-UPLC) (10,000-lb/in² nanoAcquity; Waters, Milford, MA). The mobile phases were H₂O plus 0.1% formic acid (mobile phase A) and acetonitrile plus 0.1% formic acid (mobile phase B). Desalting of the samples was performed online using a reversed-phase Symmetry C₁₈ trapping column (180-μm internal diameter, 20-mm length, and 5-μm particle size; Waters). The peptides were then separated on a T3 high-strength silica nanocolumn (75-μm internal diameter, 250-mm length, and 1.8-μm particle size; Waters) at 0.35 μl/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 25% buffer B in 155 min, 25% to 90% buffer B in 5 min, maintenance at 90% for 5 min, and then back to initial conditions.

ULC/MS‐grade solvents were used for all chromatographic steps. Each sample was loaded using split‐less nano‐Ultra Performance Liquid Chromatography (10 kpsi nanoAcquity; Waters, Milford, MA, USA). The mobile phase was: (A) H₂O + 0.1% formic acid, and (B) acetonitrile +0.1% formic acid. Desalting of the samples was performed online using a reversed‐phase Symmetry C18 trapping column (180 μm internal diameter, 20 mm length, 5 μm particle size; Waters). The peptides were then separated using a T3 HSS nano‐column (75 μm internal diameter, 250 mm length, 1.8 μm particle size; Waters) at 0.35 μl/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4–30% B over 55 min, 30–90% B over 5 min, maintained at 90% for 5 min and then back to the initial conditions. The nanoUPLC was coupled online through a nanoESI emitter (10 μm tip; New Objective, Woburn, MA, USA) to a quadrupole orbitrap mass spectrometer (Q Exactive HF; Thermo Scientific) using a FlexIon nanospray apparatus (Proxeon). Data were acquired in data‐dependent acquisition (DDA) mode, using a Top10 method. MS1 resolution was set to 120,000 (at 200 m/z), mass range of 375–1,650 m/z and AGC of 3e6, and maximum injection time was set to 60 ms. MS2 resolution was set to 15,000, quadrupole isolation 1.7 m/z, AGC of 1e5, dynamic exclusion of 20 s and maximum injection time of 60 ms.

Peptides were analysed by LC–MS/MS using a Waters nanoAcquity UPLC platform (Milford, MA, USA) coupled to a Thermo LTQ-Orbitrap XL mass spectrometer. The peptides (5 μl) were injected onto a 5 μm, 180 μm × 20 mm Symmetry C18 trapping column (Waters) followed by a 1.7 µm, 75 µm × 250 mm Ethylene Bridged Hybrid (BEH) C18 nanocolumn (Waters). A flow rate of 300 nl/minute was used with an ACN:water gradient with 0.1% formic acid (1% ACN for 1 min, followed by 0–62.5% ACN during 21 min, 62.5–85% ACN for 1.5 min, 85% ACN for 2 min and 100% ACN for 15 min). All analyses were performed in positive ion mode at a resolution of 30,000 over the mass to charge ratio (m/z) range 400–2000 using the lock mass setting. The top 5 precursor ions were automatically isolated and fragmented using collision induced dissociation (CID) energy of 35. Charge state screening was enabled, rejecting ions with unassigned or single positive charge states.

ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split-less nano-ultra performance liquid chromatography (10 kpsi nanoAcquity; Waters, Milford, MA, USA). The mobile phase was: A) H₂O + 0.1% formic acid and B) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed-phase Symmetry C18 trapping column (180 µm internal diameter, 20 mm length, 5 µm particle size; Waters). The peptides were then separated using a T3 HSS nano-column (75 µm internal diameter, 250 mm length, 1.8 µm particle size; Waters) at 0.35 µL/min. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 27%B in 105 min, 27% to 90%B in 5 min, maintained at 90% for 5 min and then back to initial conditions.

ULC/MS grade solvents were used for all chromatographic steps. Each sample was loaded using split‐less nano‐ultra performance liquid chromatography (10 kpsi MClass; Waters, Milford, MA, USA). The mobile phase was: a) H₂O + 0.1% formic acid and b) acetonitrile + 0.1% formic acid. Desalting of the samples was performed online using a reversed‐phase Symmetry C18 trapping column (180 µm internal diameter, 20 mm length, 5 µm particle size; Waters). The peptides were then separated using a T3 HSS nanocolumn (75 µm internal diameter, 250 mm length, 1.8 µm particle size; Waters) at 0.35 µL min⁻¹. Peptides were eluted from the column into the mass spectrometer using the following gradient: 4% to 30%B in 155 min, 30% to 90%B in 5 min, maintained at 90% for 5 min and then back to initial conditions.