Nanoflex source

All experiments were performed on
a Thermo Orbitrap Fusion Lumos instrument. Proteins were introduced
into the instrument via nanospray using a nano flex source from Thermo
Scientific that was modified with a platinum wire to allow the use
of tips pulled from borosilicate glass (Harvard Apparatus GC100T-10).
Nanospray tips were ∼1–15 μm in diameter and had
a taper length of ∼1 mm. Proteins were isolated using the quadrupole
and subjected to CID, HCD, or ETD prior to analysis in an Orbitrap
mass analyzer. In subthreshold CID experiments, CID energy was incrementally
increased until just below the observation of fragment ions, and the
protein ion was then reisolated and subjected to MS³ fragmentation
by CID, HCD, or ETD. For proton transfer charge reduction experiments
(PTCR), nitrogen-adducted fluoranthene (m/z 216 Da) was used as a proton-scavenging anion.^{43 (link)} Following proton transfer during MS², the desired charge state was isolated and subjected to MS³ fragmentation. All mass spectra were acquired in the Orbitrap mass
analyzer using a resolution of 120,000, and 200 scans were averaged.
Replicate spectra for each condition were collected immediately after
the initial run.

The collected fractions were examined by nano-RPLC-MS/MS using a Dionex's Ultimate 3000 RSLCnano system (Dionex, Sunnyvale, CA) coupled to a Thermo Scientific Q Exactive mass spectrometer equipped with a NanoFlex source (Thermo Fisher Scientific). A capillary RP-LC column (75 um i.d. x 150mm, filled with Acclaim PepMap RSLC C18, 100Å, 2 um, nanoViper Dionex, Sunnyvale, CA) was used for separation of peptides by LC. First, samples were desalted from the autosampler at 5 μL/min by loading onto a trap column (Acclaim PepMap 100 C18, 100 Å, 3 μm, 75 μm×2 cm, Dionex, Sunnyvale, CA). Thereafter, desalted samples were washed for 12 min with 0.1% FA in HPLC grade water and the system changed into line with the analytical RP capillary column. The tryptic digest was analyzed within 65 min 3-step gradient (80% ACN in 0.1% methanol from 4% to 50% over 45 min, 50% to 90% over 5 min and kept at 95% for 15 min) at a flow rate of 300 nL/min. Key parameter settings for the Thermo Scientific Q Exactive mass spectrometer were as follows:

Samples were analyzed on a LC/MS system comprised of an Easy nLC 1000 (Thermo) coupled to a Q Exactive hybrid quadrupole-Orbitrap mass spectrometer with a NanoFlex source (Thermo Scientific). Peptides were trapped on an Acclaim PepMap 100 (75 µm × 20 mm, Thermo Scientific) and desalted. Chromatography was performed on an analytical column (75 µm × 150 mm, Thermo Scientific) packed with C18, 2 µm particles using a 115-min water/acetonitrile reversed-phase gradient method.
The Q Exactive was operated in data-dependent acquisition (DDA) mode. Full scan MS spectra (m/z 400–1800) were acquired in the Orbitrap analyzer with a resolving power of 70,000 (at m/z 200). The fifteen most intense multiply charged ions (z ≥ 2) were sequentially isolated with a 1.0 Da isolation width and fragmented in the collision cell by higher-energy collisional dissociation (HCD). Fragment ions were mass analyzed to a 35,000 resolving power (at m/z 200).