Peptide samples were loaded onto a first dimension trap column (Waters Xbridge, C18, 10 um particle size, 100 A pore size, 4 cm packing length 150 μm column inner diameter). Online peptide separation coupled to MS/MS was performed with a 2D-nanoLC system (nanoAcquity UPLC system, Waters) and a Velos-Pro/Orbitrap-Elite hybrid mass spectrometer (ThermoFisher Scientific). Six discrete elutions were performed at 1.5 μL/min with 5 mM ammonium formate, pH 10, using increasing concentrations of ACN (1%, 3%, 6%, 15%, 25% and 44%) and diluted with 6 μL/min 0.1% formic acid (FA) prior to loading onto a second dimension trap column (Dr. Maisch ReproSil-Pur, C18, 5 um particle size, 120 A pore size, 4 cm packing length 150 μM column inner diameter) connected to an analytical column (Orochem Reliasil, C18, 3 μM particle size, 90 A pore size, 20–25 cm packing length 50 μM column inner diameter) with an incorporated electrospray emitter. Peptide separation was achieved using a gradient from 3 to 80% (V/V) of ACN in 0.1% FA over 115 minutes at a flow rate of 200 nL/min. The mass spectrometer was operated in data-dependent mode using a Top 10 method. Full MS scans (m/z 300–2000) were acquired in the Orbitrap analyzer (resolution = 120,000), followed by high energy collision induced dissociation (HCD) MS/MS (fm/z 100–2000, resolution = 15,000) at a normalized collision energy of 35%.
Velos pro orbitrap elite hybrid mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Velos Pro/Orbitrap Elite hybrid mass spectrometer is a high-performance analytical instrument designed for advanced proteomics and metabolomics research. It combines the high-resolution and accurate mass capabilities of the Orbitrap mass analyzer with the fast scanning and high sensitivity of the dual-pressure linear ion trap. The instrument is capable of performing high-resolution accurate mass (HRAM) MS and MS/MS analysis to enable comprehensive characterization of complex biological samples.
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3 protocols using velos pro orbitrap elite hybrid mass spectrometer
1
Multi-Dimension Peptide Separation and LC-MS/MS
2
Identifying S-layer Proteins in L. amylovorus
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The presence of S-layer proteins on the surface of the L. amylovorus intestinal isolates GRL 1112 – GRL 1118 has previously been described [28 (link)]. The putative slp encoding genes were identified in silico in the draft genomes of the L. amylovorus strains based on homology with the publicly available L. acidophilus slp gene sequences. The identification of the expressed slp genes was based on the observed molecular weights of the proteins, obtained by analyzing overnight cultures of the strains by standard SDS-PAGE in 12% gels, and on the amino-terminal and/or internal amino acid sequences of the Slp:s. The amino-terminal sequences were obtained by an Edman-degradation-based Procise 494 HT sequencer (Life Technologies, Carlsbad, CA), and internal peptide sequences through a peptide mapping analysis: the proteins were digested in-gel by trypsin followed by analysis with liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) carried out with an EASY-nLC liquid chromatograph (Thermo Fisher Scientific, Germany) connected to a Velos Pro-Orbitrap Elite hybrid mass spectrometer (Thermo Fisher Scientific, Germany) with a nano-electrospray ion source (Thermo Fisher Scientific, Germany). Both amino-terminal sequencing and peptide mapping were performed in the Institute of Biotechnology (University of Helsinki, Finland).
3
Multi-Dimension Peptide Separation and LC-MS/MS
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Peptide samples were loaded onto a first dimension trap column (Waters Xbridge, C18, 10 um particle size, 100 A pore size, 4 cm packing length 150 μm column inner diameter). Online peptide separation coupled to MS/MS was performed with a 2D-nanoLC system (nanoAcquity UPLC system, Waters) and a Velos-Pro/Orbitrap-Elite hybrid mass spectrometer (ThermoFisher Scientific). Six discrete elutions were performed at 1.5 μL/min with 5 mM ammonium formate, pH 10, using increasing concentrations of ACN (1%, 3%, 6%, 15%, 25% and 44%) and diluted with 6 μL/min 0.1% formic acid (FA) prior to loading onto a second dimension trap column (Dr. Maisch ReproSil-Pur, C18, 5 um particle size, 120 A pore size, 4 cm packing length 150 μM column inner diameter) connected to an analytical column (Orochem Reliasil, C18, 3 μM particle size, 90 A pore size, 20–25 cm packing length 50 μM column inner diameter) with an incorporated electrospray emitter. Peptide separation was achieved using a gradient from 3 to 80% (V/V) of ACN in 0.1% FA over 115 minutes at a flow rate of 200 nL/min. The mass spectrometer was operated in data-dependent mode using a Top 10 method. Full MS scans (m/z 300–2000) were acquired in the Orbitrap analyzer (resolution = 120,000), followed by high energy collision induced dissociation (HCD) MS/MS (fm/z 100–2000, resolution = 15,000) at a normalized collision energy of 35%.
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