Five μL of each fractionated peptide sample was separated on a 2-column custom-built capillary LC system similar as reported previously(16 (link)). Reversed-phase separations were carried out on 35cm × 75 μm i.d. fused silica columns packed in house using 3μ Jupiter C18 particles (Phenomenex). Mobile phases consisted of 0.1% formic acid in water (A) and 0.1% formic acid in 100% acetonitrile (B) with a 100 min gradient. MS analyses were performed using an Orbitrap Velos Pro Hybrid mass spectrometer (ThermoScientific) outfitted with a custom electrospray ionization (ESI) interface, which was operated in data dependent MS/MS mode, with one high resolution (Resolution of 60,000 at 400 m/z) MS scan followed by HCD MS/MS scan events (resolution of 15,000 at 400 m/z) at normalized collision energy of 32.
Orbitrap velos pro hybrid mass spectrometer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Orbitrap Velos Pro Hybrid mass spectrometer is a high-performance analytical instrument designed for advanced mass spectrometry applications. It combines the Orbitrap mass analyzer with a linear ion trap, providing high mass accuracy, resolution, and sensitivity. The core function of this system is to enable precise and reliable identification and quantification of a wide range of molecules, including proteins, peptides, and small molecules.
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Lab products found in correlation
Ultimate 3000 rslcnano system, by Thermo Fisher Scientific (2 mentions)
Proteome discoverer 1, by Thermo Fisher Scientific (2 mentions)
Proteome discoverer ver 1, by Thermo Fisher Scientific (1 mentions)
Maxquant 1, by Thermo Fisher Scientific (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
5 protocols using orbitrap velos pro hybrid mass spectrometer
1
Capillary LC-MS/MS Proteomic Analysis
2
Quantitative Proteomics by Mass Spectrometry
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Tryptic digested peptides were purified and subjected to ESI-LC-MS/MS analysis at a concentration of 1,000 femtomole per injection performed in duplicate. The purified peptides were analyzed using an Orbitrap Velos Pro Hybrid mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) in positive ion mode electrospray ionization using an EASY-spray column (PepMap RSLC, C18, 2 μm, 100 Å, 75 μm×50 cm or 15 cm), as described earlier. Proteome Discoverer ver. 1.4 (Thermo Fisher Scientific) was used to identify proteins from raw data generated by tandem mass spectrometric analysis (.raw/.msf). By searching against the UniProt human database, the in-built SequestHT and Mascot search algorithms were used to match tandem mass spectra to peptide sequences. As reported previously [7 (link)], proteins with high peptide confidence and rank one were considered for identification after an effective screening of missed cleavages. The identified proteins were further checked for complementary DNA fragments, isoforms, uncharacterized entries, and normalized using the normalized spectral abundance factor method [8 (link)].
3
High-Resolution Mass Spectrometry for Lipid Profiling
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The quantitative evaluation of cholesterol (chol) and the phospholipids phosphatidylcholine (PC), phosphatidylserine (PS), sphingomyelin (SM), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and lysophosphatidylcholine (LPC) was conducted using high-resolution mass spectrometry, as recently described by our group [10 (link)]. Lipids were extracted from cell pellets (107 cells) with an established MTBE protocol [18 (link)]. Data acquisition was performed using Orbitrap-MS (Orbitrap Velos Pro hybrid mass spectrometer, Thermo Fisher Scientific, Waltham, MA, USA) [19 (link)]. Complete scan profile spectra from m/z 320 to 1050 for positive ion mode and from m/z 500 to 1000 for negative ion mode were obtained in the Orbitrap mass analyzer at a resolution of 100k at m/z 400 and <2 ppm mass accuracy. All samples were measured once in positive polarity and once in negative polarity. For MS/MS experiments, the 10 most abundant ions of the entire scan spectrum were sequentially fragmented in the ion trap using He as collision gas (normalized collision energy: 50; isolation width: 1.5; activation Q: 0.2; and activation time: 10), and centroided product spectra at a normal scan rate (33 kDa/s) were obtained. Data analysis was accomplished by Lipid Data Analyzer, a custom-developed software tool [19 (link),20 (link),21 (link)].
4
Mass Spectrometry-based Proteomics Workflow
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Trypsin digestion of proteins was performed as published by Shevchenko and collaborators using Sequencing Grade Modified Trypsin (Promega) [144 (link)]. Following this procedure peptides were purified using the StageTip purification method [145 (link),146 (link)]. Purified peptides were separated by reversed-phase liquid chromatography employing an RSLCnano Ultimate 3000 system (Thermo Scientific) followed by mass analysis with an Orbitrap Velos ProHybrid mass spectrometer (Thermo Scientific) as described [98 (link),143 (link),147 (link),148 (link)]. For further details see [149 (link)].
MS/MS2 data processing for peptide analysis and protein identification was performed either with the MaxQuant 1.5.1.0 and Perseus 1.5.3 or the Proteome Discoverer 1.4 software (Thermo Scientific) and the Mascot and SequestHT search algorithms. Phosphosite probabilities were calculated with the phosphoRS search algorithm [150 (link),151 (link)].
Three unique peptides [152 (link)] and three MS/MS counts were demanded for positive protein identification. Furthermore, only proteins identified from at least two out of three biological repetitions were considered further. Proteins also identified from the control strain (AGB596) were regarded as false-positives and excluded from further consideration.
MS/MS2 data processing for peptide analysis and protein identification was performed either with the MaxQuant 1.5.1.0 and Perseus 1.5.3 or the Proteome Discoverer 1.4 software (Thermo Scientific) and the Mascot and SequestHT search algorithms. Phosphosite probabilities were calculated with the phosphoRS search algorithm [150 (link),151 (link)].
Three unique peptides [152 (link)] and three MS/MS counts were demanded for positive protein identification. Furthermore, only proteins identified from at least two out of three biological repetitions were considered further. Proteins also identified from the control strain (AGB596) were regarded as false-positives and excluded from further consideration.
5
Proteomic Analysis of Protein Samples
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Trypsin digestion of proteins was performed as described earlier (Shevchenko et al., 1996 (link)). Briefly, the Stage TipStageTip method was used to purify peptides (Rappsilber et al., 2007 (link)), which were subsequently separated by reversed-phase liquid chromatography. For analysis an RSLCnano Ultimate 3000 system (Thermo Scientific) followed by mass analysis with an Orbitrap Velos ProHybrid mass spectrometer (Thermo Scientific) was applied as described in more detail elsewhere (Lin et al., 2015 (link); Schmitt et al., 2017 (link)). MS/MS2 data processing for peptide analysis and protein identification was performed with the Proteome Discoverer 1.4 software (Thermo Scientific) and the Mascot and SequestHT search algorithms. Proteins identified in empty vector control (pGP172 with human serum) were regarded as unspecific binding and excluded.
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