Peptide Isolation and Nano-LC-MS/MS Analysis

Peptides were eluted into 96-well microtitre plates with 20 μl 40% MeCN, 0.5% AcOH followed by 20 μl 50% MeCN, 0.5% AcOH. Organic solvents were removed by vacuum centrifugation in a speed-vac and dried to ∼2 μl. The peptides were reconstituted with 10 μl of 2% MeCN, 0.5% AcOH, 0.1% TFA. Five microlitres of this eluate was analysed by online reversed-phase C18 nanoscale LC-MS/MS on an LTQ-Orbitrap Velos mass spectrometer (Thermo Electron) using a top10 higher-energy collisional dissociation (HCD) fragmentation method as described previously34 (link). The LC-MS analysis was performed with a nanoflow Easy –nLC system (Proxeon Biosystems) connected through a nano-electrospray ion source to the MS. Peptides were separated by a linear MeCN gradient for 220 min in a 15-cm fused-silica emitter in house packed with reversed-phase ReproSil-Pur C18-AQ 3 μm resin (Dr Maisch GmbH). Full-scan MS spectra were acquired from 350 to 1,750 m/z at a target value of 1e6 and a resolution of 30,000, and the HCD-MS/MS spectra were recorded at a target value of 5e4 and with resolution of 7,500 using a normalized collision energy of 40%.

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Secher A., Kelstrup C.D., Conde-Frieboes K.W., Pyke C., Raun K., Wulff B.S, & Olsen J.V. (2016). Analytic framework for peptidomics applied to large-scale neuropeptide identification. Nature Communications, 7, 11436.

Publication 2016

LTQ-Orbitrap Velos mass spectrometer ReproSil-Pur C18-AQ 3 μm resin

Centrifugation Electron Ms ms Peptides Reprosil Resin Scan Silica Solvents Vacuum

Corresponding Organization :

Other organizations : Novo Nordisk Foundation, University of Copenhagen, Novo Nordisk (Denmark)

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Variable analysis

independent variables

Organic solvents used for elution (40% MeCN, 0.5% AcOH followed by 50% MeCN, 0.5% AcOH)

dependent variables

Peptides eluted into 96-well microtitre plates
Organic solvents removed by vacuum centrifugation
Peptides reconstituted with 10 μl of 2% MeCN, 0.5% AcOH, 0.1% TFA
Peptides analyzed by online reversed-phase C18 nanoscale LC-MS/MS on an LTQ-Orbitrap Velos mass spectrometer using a top10 higher-energy collisional dissociation (HCD) fragmentation method

control variables

Volume of organic solvents used for elution (20 μl each)
Concentration of organic solvents used for elution (40% and 50% MeCN, 0.5% AcOH)
Volume of reconstitution solution (10 μl of 2% MeCN, 0.5% AcOH, 0.1% TFA)
Mass spectrometry parameters (target value of 1e6 and resolution of 30,000 for full-scan MS spectra, target value of 5e4 and resolution of 7,500 for HCD-MS/MS spectra, normalized collision energy of 40%)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

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