Disulfide bond mapping of SLP_VHH22 and SLP_VHH50, each with four Cys residues, was performed essentially as described (Kim et al. 2012b (link); Hussack et al. 2011b (link)). Briefly, tryptic fragments for subsequent mass spectrometry (MS) analysis were prepared as described (Kim et al. 2012a (link)). Aliquots of VHH proteolytic digests were resuspended in 0.1 % (v/v) formic acid (aq) and analyzed by nanoflow reversed-phase HPLC MS (nanoRPLC-ESI-MS) with data-dependent analysis (DDA) using collision-induced dissociation (CID) on a nanoAcquity UPLC system coupled to a Q-TOF Ultima™ hybrid quadrupole/TOF mass spectrometer (Waters, Milford, MA, USA). The peptides were first loaded onto a 300 μm I.D. × 5 mm C18 PepMap100 μ-precolumn (Thermo Fisher) and then eluted into a 100 μm I.D. × 10 cm 1.7-μm BEH130C18 column (Waters) using a linear gradient from 0 to 36 % solvent B (acetonitrile + 0.1 % formic acid) over 36 min followed by 36–90 % solvent B for 2 min. Solvent A was 0.1 % formic acid in water. The peptide MS2 spectra were compared with VHH protein sequences using the Mascot™ database searching algorithm (Matrix Science, London, UK). The MS2 spectra of the disulfide-linked peptides were de-convoluted using the MaxEnt 3 program (Waters) for de novo sequencing to confirm and/or determine the exact disulfide linkage positions.
C18 pepmap100 μ precolumn
Manufactured by Thermo Fisher Scientific
Sourced in Belgium
The C18 PepMap100 μ-precolumn is a chromatographic column used in liquid chromatography-mass spectrometry (LC-MS) analysis. It is designed to concentrate and desalt peptide samples prior to separation on the analytical column.
Automatically generated - may contain errors
Lab products found in correlation
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
Nanoacquity uplc system, by Waters Corporation (1 mentions)
Maxent3 program, by Waters Corporation (1 mentions)
Beh130 c18 column, by Waters Corporation (1 mentions)
Variable wavelength uv detector, by Thermo Fisher Scientific (1 mentions)
Orbitrap fusion tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 nanoflow lc system, by Thermo Fisher Scientific (1 mentions)
Q exactive hf mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easy spray pepmap rslc c18, by Thermo Fisher Scientific (1 mentions)
4 protocols using c18 pepmap100 μ precolumn
1
Disulfide Mapping of VHH Antibodies
2
Capillary LC-UV Analysis of Analytes
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Chromatographic analysis was performed on an UltiMate 3000 series RSLC nano system, equipped with a binary capillary pump, degasser, autosampler, thermostatted column compartment and a variable wavelength UV detector (Dionex, Amsterdam, the Netherlands). Data acquisition and peak processing were performed using Chromeleon version 6.8 chromatography data software (Dionex).
An Acclaim Pepmap RSLC C18 capillary column (150 mm x 300 μm i.d., 2 μm, 100 Å) equipped with a C18 Pepmap100 μ-precolumn (5 mm x 300 μm i.d., 5 μm, 100 Å), both from Thermo Fisher Scientific (Erembodegem, Belgium) was used for analysis. The column was thermostatted at 40°C and samples were kept at 5°C in the autosampler. A volume of 20 μL of MEPS extract was injected into the capillary LC system. Mobile phases consisted of 10 mM ammonium acetate with 0.05% triethylamine, adjusted to pH 3.00 ± 0.01 using acetic acid (A), and acetonitrile (B). The separation was performed in gradient mode using the following time program: 10% B (min 0); 10% B (min 3); 16% B (min 12); 31% B (min 12.1); 31% B (min 23); 35% B (min 38); 35% B (min 40); 90% B (min 40.1); 90% B (min 45). A flow rate of 4 μL.min -1 was applied and UV detection was performed at 254 nm.
An Acclaim Pepmap RSLC C18 capillary column (150 mm x 300 μm i.d., 2 μm, 100 Å) equipped with a C18 Pepmap100 μ-precolumn (5 mm x 300 μm i.d., 5 μm, 100 Å), both from Thermo Fisher Scientific (Erembodegem, Belgium) was used for analysis. The column was thermostatted at 40°C and samples were kept at 5°C in the autosampler. A volume of 20 μL of MEPS extract was injected into the capillary LC system. Mobile phases consisted of 10 mM ammonium acetate with 0.05% triethylamine, adjusted to pH 3.00 ± 0.01 using acetic acid (A), and acetonitrile (B). The separation was performed in gradient mode using the following time program: 10% B (min 0); 10% B (min 3); 16% B (min 12); 31% B (min 12.1); 31% B (min 23); 35% B (min 38); 35% B (min 40); 90% B (min 40.1); 90% B (min 45). A flow rate of 4 μL.min -1 was applied and UV detection was performed at 254 nm.
3
nLC-ESI-MS/MS for Proteomics
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NanoLiquid Chromatography-ElectroSpray Ionization-tandem mass spectrometry (nLC-ESI–MS/MS) experiments were carried out using an UltiMate3000 RSLCnano System coupled to a Orbitrap Fusion Tribrid mass spectrometer through a nanoESI source (EASY-Spray NG) (Thermo Fisher Scientific) as already published42 (link). An EASY-Spray PepMap RSLC C18 column (2 μm particle size, 100 Å pore size, 75 μm i.d. × 50 cm length, Thermo Fisher Scientific) was employed to separate the digested samples hyphenated with a μ-Precolumn C18 PepMap100 (5 µm particle size, 100 Å pore size, 300 µm i.d. x 5 mm length, Thermo Fisher Scientific) operating at 10 μL/min, 3 min, to concentrate and desalt 1.95 μg of peptides. Reverse-phase chromatography was performed in 143 min, and total LC-run of 181 min at a flow rate of 250 nL/min and temperature of 35 °C. MS analysis was done with a full MS scan from 375 to 1,500 m/z in Orbitrap at a resolving power of 120 K and positive ionization mode, followed by data-dependent MS/MS scan recorded by Ion Trap at rapid scan rate, in top speed mode with a 3 s cycle-time, with dynamic exclusion enabled for 1 min. Two technical replicates were acquired for each sample.
4
Peptide Analysis by Q-Exactive HF MS
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Samples were analyzed using a Q-Exactive HF mass spectrometer (Thermo Scientific, Bremen, Germany) coupled with an UltiMate 3000 Nanoflow LC system (Thermo Scientific, Germering, Germany). Peptides were concentrated at the trap column (μ-Precolumn C18 PepMap100, Thermo Scientific, 5 μm, 300 μm i.d. 5 mm, 100 Å) and eluted from an analytical column (EASY-Spray PepMap RSLC C18, Thermo Scientific, 2 μm, 75 μm i.d. 500 mm, 100 Å) with a gradient of acetonitrile.
Mass spectrometry measurements were performed using data-dependent acquisition mode (Top 12). MS1 spectra were acquired from 300 to 1400 Th, with a resolving power of 120 000 at m/z 200. Precursor ions were isolated with the m/z window of 1.4 Th followed by their fragmentation using higher-energy collision dissociation (HCD). Fragment ions were measured in an Orbitrap mass-analyzer with a resolving power of 15 000 at m/z 200. Each sample was analyzed in triplicate. The data were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE34 (link) partner repository with the dataset identifiers PXD007648 and 10.6019/PXD007648.
Mass spectrometry measurements were performed using data-dependent acquisition mode (Top 12). MS1 spectra were acquired from 300 to 1400 Th, with a resolving power of 120 000 at m/z 200. Precursor ions were isolated with the m/z window of 1.4 Th followed by their fragmentation using higher-energy collision dissociation (HCD). Fragment ions were measured in an Orbitrap mass-analyzer with a resolving power of 15 000 at m/z 200. Each sample was analyzed in triplicate. The data were deposited in the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE34 (link) partner repository with the dataset identifiers PXD007648 and 10.6019/PXD007648.
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