Ultimate 3000 nano lc system

Generated peptides were analyzed on a Dionex Ultimate 3000 nano LC system coupled to a Q-Exactive HF mass spectrometer (Thermo Scientific, Germany). Peptides were delivered to a trap column (75 μm × 2 cm, self-packed with Reprosil-Pur C18 ODS-3 5 μm resin, Dr. Maisch, Ammerbuch) at a flow rate of 5 μL min^-1 in solvent A0 (0.1% formic acid in water). Peptides were separated on an analytical column (75 μm × 40 cm, self-packed with Reprosil-Gold C18, 3 μm resin, Dr. Maisch, Ammerbuch) using a 120 min linear gradient from 4–32% solvent B (0.1% formic acid, 5% DMSO in acetonitrile) and solvent A1 (0.1% formic acid, 5% DMSO in water) at a flow rate of 300 nL min^-1. The mass spectrometer was operated in data-dependent acquisition (DDA) mode, automatically switching between MS and twenty MS2 spectra.
MS1 spectra were acquired over a mass-to-charge (m/z) range of m/z 360–1 300 at a resolution of 60 000 (at m/z 200) using a maximum injection time of 10 ms and an AGC target value of 3e6. Up to 20 peptide precursors were isolated (isolation window m/z 1.7, maximum injection time 50 ms, AGC value 2e5), fragmented by HCD using 25% NCE and analyzed at a resolution of 30 000 with a scan range from m/z 200 to 2 000. Precursor ions that were singly-charged, unassigned or with charge states >6+ were excluded. The dynamic exclusion duration of fragmented precursor ions was 35 s.

Protein estimation for each sample was performed using the Pierce BCA protein assay kit to ensure that each sample contained at least 300 μg of protein. Platelet lysate was prepared by three repeated freeze-thaw cycles and the frozen samples were sent on dry ice to The Australian Proteome Analysis Facility (APAF), which performed the proteomics analysis. For this, the samples were digested using commercially procured S-Traps (Protifi) and equal total peptide quantities were prepared for mass spectrometry using Tandem Mass Tag (TMT; Thermo Scientific) reagent labelling. The TMT-labelled peptides were fractionated using Offline Basic pH reversed-phase fractionation, dried by vacuum centrifugation, and reconstituted in 0.1% formic acid for liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis, which was performed using the UltiMate 3000 nanoLC system (Thermo Fisher Scientific) and a Q-Exactive HF-X mass spectrometer (Thermo Fisher Scientific). It is of note that while this traditional approach identifies a large number of differentially expressed proteins, information about their structural changes is not detected. Raw data files were processed using the Proteome Discoverer software (Thermo Scientific, v2.1.0.81). The data were searched using the search engines SequestHT and Mascot against a sequence database for Mus musculus.

The separation was achieved using a
Dionex UltiMate 3000 Nano LC system (NCS-3200RS, Thermo Scientific,
Germering, Germany) fitted with a micro-LC flow selector to deliver
a mobile phase. Channel A contained 0.1% trifluoroacetic acid (TFA,
Sigma Aldrich) in Optima LC-MS grade water (Fisher Chemical), and
channel B contained 0.1% TFA in LC-MS grade acetonitrile (Fisher Chemical).
Isocratic elution at 20% channel B was used at a constant flow rate
of 2 μL/min for the entirety of the experiment. The pump was
connected to an externally mounted 6-port two-position Cheminert injection
valve (C72x-669D, VICI Valco, Houston, TX) using a 750 mm × 0.100
mm nanoViper capillary. A 140 mm × 0.100 mm nanoViper capillary
was used as a 1.1 μL sample loop. A 350 mm × 0.025 mm i.d.
× 0.360 mm o.d. fused silica capillary was used to connect the
outlet of the column to a Waters Acquity TUV detector fitted with
a 10 nL nano flow cell (Waters Corporation, Milford, MA) set to 214
nm. An Atlas analog-to-digital converter and Chromeleon version 6.8
software (Thermo) were used to acquire data at 100 Hz.
The yaGfl
standards were injected in triplicate (21 data). The regression results
from data (peak area vs injected concentration) indicated an intercept
indistinguishable from 0 (95% CI of −0.0026 to +0.0049) and
a slope of 0.00575 (95% CI of 0.00566 to 0.00583)

Tryptic peptides were analyzed using a Dionex Ultimate 3000 nano-LC system (Sunnyvale, CA, USA) connected to an Orbitrap Fusion Tribrid Mass Spectrometer (Thermo Scientific, Bremen, Germany) equipped with a nano-electrospray ion source according to the procedure previously described [50 (link)].

One µg total of sample was analysed by a Dionex Ultimate 3000 nano-LC system (Sunnyvale, CA, USA) combined with an Orbitrap Fusion™ Tribrid™ Mass Spectrometer (Thermo Scientific, Bremen, Germany) equipped with a nano electrospray ion source. Peptide mixtures were pre-concentrated onto an Acclaim PepMap 100—100 μm × 2 cm C18 (Thermo Fisher Scientific, Waltham, MA, USA) and separated on EASY-Spray column, 25 cm × 75 μm ID packed with Thermo Scientific Acclaim PepMap RSLC C18, 3 μm, 100 Å, at 35 °C and flow rate 300 nL/min. Mobile phases were the following: 0.1% formic acid (FA) in water (Buffer A); 0.1% FA in water/acetonitrile with 2/8 ratio (Buffer B). The elution gradient was from 96% Buffer A to 95% Buffer B for 110 min. MS spectra were collected over an m/z range of 375–1500 Da at 120,000 resolutions, operating in the data dependent mode, cycle time 3 s between master scans. Higher-energy collision dissociation was carried out with collision energy set at 35 eV and a positive polarity. The experiment was repeated three times and each sample was run in three technical replicates.