Peptides were separated by nano-LC-MS/MS on an Acclaim® PepMap100 15 cm × 75 μm packed with C18 material (3 μm; 100 Å, Thermo Fisher) using an Easy-LC nano-HPLC (Thermo Fisher). The HPLC gradient was 0–35% solvent B (A = 0.1% formic acid; B = Acetonitrile, 0.1% formic acid) in a total of 70 min run at a flow of 250 nL/min. Mass spectrometric analysis was performed using an LTQ OrbitrapVelosETD (Thermo Scientific, Bremen, Germany). An MS scan (400–2000m/z) was recorded in the Orbitrap mass analyser at a resolution of 60,000 at 400m/z for a target of 106 ions followed by data-dependent collision-induced dissociation (CID) MS/MS analysis of the top twenty most intense ions with charge state ≥ 2.The following parameters: activation time = 10 ms, normalized energy = 35, Q-activation = 0.25, dynamic exclusion = enabled with repeat count 1, exclusion duration = 30 s and, intensity threshold = 10,000, target ions = 104 (link).
Easylc nano hplc
Manufactured by Thermo Fisher Scientific
The EasyLC nano-HPLC is a high-performance liquid chromatography (HPLC) system designed for nano-scale separations. It is capable of handling nano-flow rates and providing high-resolution chromatographic separations.
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3 protocols using easylc nano hplc
1
Nano-LC-MS/MS Proteomics Workflow
2
Comprehensive Protein Identification by Mass Spectrometry
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Eluted proteins were purified by SDS-PAGE and Coomassie-stained gel pieces were digested in gel with trypsin as described previously (Borchert et al., 2010 (link)). After desalting using C18 Stage tips, extracted peptides were separated on an EasyLC nano-HPLC (ThermoFisher) coupled to an LTQ Orbitrap Elite (ThermoFisher) as described elsewhere (Franz-Wachtel et al., 2012 (link)) with slight modifications: the peptide mixtures were separated using a 127-min segmented gradient from to 5–33% and 50–90% of HPLC solvent B (80% acetonitrile in 0.1% formic acid) in HPLC solvent A (0.1% formic acid) at a flow rate of 200 nL/min. The 15 most intense precursor ions were sequentially fragmented in each scan cycle using collision-induced dissociation (CID). The target values for MS/MS fragmentation were 5,000 charges and 106 charges for the MS scan. Acquired MS spectra were processed with MaxQuant software with integrated Andromeda search engine (Cox et al., 2011 (link)). Database search was performed against a target decoy Saccharomyces cerevisiae database obtained from Uniprot, the sequences of the constructs DHFR-3HA and Porin-3HA, and 285 commonly observed contaminants. Initial maximum allowed mass tolerance was set to 4.5 ppm (for the survey scan) and 0.5 D for CID fragment ions. A false discovery rate of 1% was applied at the peptide and protein level.
3
Nano-HPLC-MS/MS Proteomics Protocol
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Coomassie-stained gel pieces were digested in gel with trypsin as described previously (Borchert et al., 2010) . After desalting using C18 Stage tips (Rappsilber et al., 2007) , extracted peptides were separated on an EasyLC nano-HPLC coupled to a Q Exactive HF mass spectrometer (both Thermo Fisher Scientific) as described elsewhere (Kelstrup et al., 2014) with slight modifications: the peptide mixtures were injected onto the column in HPLC solvent A (0.1% formic acid) at a flow rate of 500 nl/min and subsequently eluted with a 57minute segmented gradient of 10-33-50-90% of HPLC solvent B (80% acetonitrile in 0.1% formic acid) at a flow rate of 200 nl/min. Full scan was acquired in the mass range from m/z 300 to 1650 at a resolution of 120,000 followed by HCD fragmentation of the 7 most intense precursor ions. High-resolution HCD MS/MS spectra were acquired with a resolution of 60,000. The target values for the MS scan and MS/MS fragmentation were 3x 10 6 and 10 5 charges, respectively. Precursor ions were excluded from sequencing for 30 s after MS/MS.
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