Ltq orbitrap velos

Manufactured by Thermo Fisher Scientific

Sourced in United States, Germany, United Kingdom, Japan, Denmark

The LTQ Orbitrap Velos is a high-performance mass spectrometer that combines the high mass accuracy and resolution of the Orbitrap with the fast scan speed and sensitivity of the linear ion trap. It is designed for a wide range of applications, including proteomics, metabolomics, and small molecule analysis.

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448 protocols using ltq orbitrap velos

Quantitative Proteomics of Protein Modifications

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We used stable isotope labeling with amino acids in cell culture (SILAC; Ong et al, 2002), to measure changes in protein, lysine acetylation, and phosphorylation abundance. Peptide fractions were analyzed by online nanoflow LC‐MS/MS using a Proxeon easy nLC system (ThermoFisher Scientific) connected to an LTQ Orbitrap Velos (ThermoFisher Scientific) or Q‐Exactive (ThermoFisher Scientific) mass spectrometer. The LTQ Orbitrap Velos instrument was operated under Xcalibur 2.1 (ThermoFisher Scientific) with the LTQ Orbitrap Tune Plus Developers Kit version 2.6.0.1042 software in the data dependent mode to automatically switch between MS and MS/MS acquisition as described (Weinert et al, 2011). The Q‐Exactive was operated using Xcalibur 2.2 (ThermoFisher Scientific) in the data dependent mode to automatically switch between MS and MS/MS acquisition as described (Michalski et al, 2011; Kelstrup et al, 2012). All quantitative MS experiments performed in this study are summarized in Supplementary Table S1. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the PRIDE partner repository (Vizcaino et al, 2013) with the dataset identifier PXD000507.

Weinert B.T., Iesmantavicius V., Moustafa T., Schölz C., Wagner S.A., Magnes C., Zechner R, & Choudhary C. (2014). Acetylation dynamics and stoichiometry in Saccharomyces cerevisiae. Molecular Systems Biology, 10(1), 716.

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Peptide Separation and Identification

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Purified peptides were resuspend in buffer A* and separated on an EASY-nLC1000 system coupled to an LTQ-Orbitrap Velos (Thermo Scientific). Briefly, samples were loaded directly onto an in-house-packed 30-cm, 75-µm-inner-diameter, 360-µm-outer-diameter Reprosil-Pur C₁₈ AQ 3 µm column (Dr. Maisch, Ammerbuch-Entringen, Germany). Reverse-phase analytical separation was performed at 350 nl/min over a 180-min gradient by altering the buffer B concentration from 0 to 32% in 150 min, from 32 to 40% in the next 5 min, increasing it to 100% in 2.5 min, holding it at 100% for 2.5 min, and then dropping it to 0% for another 20 min. The LTQ-Orbitrap Velos was operated with Xcalibur v2.2 (Thermo Scientific) at a capillary temperature of 275°C with data-dependent acquisition and switching between collision-induced dissociation (CID) MS/MS (normalized collision energy [NCE], 35%; activation Q, 0.25; activation time, 10 ms; automated gain control [AGC] at 4 × 10⁴) and higher-energy collisional dissociation (HCD) MS/MS (resolution, 7,500; NCE, 45%; AGC at 2e5; maximum fill time, 200 ms).

Weber B.S., Hennon S.W., Wright M.S., Scott N.E., de Berardinis V., Foster L.J., Ayala J.A., Adams M.D, & Feldman M.F. (2016). Genetic Dissection of the Type VI Secretion System in Acinetobacter and Identification of a Novel Peptidoglycan Hydrolase, TagX, Required for Its Biogenesis. mBio, 7(5), e01253-16.

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Proteomic Analysis Using LC-MS/MS

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MS analysis was carried out using an LTQ-Orbitrap Velos (Thermo Scientific) interfaced with an UltiMate 3000 RSLCnano LC system (Dionex, Sunnyvale, CA, USA, now part of Thermo Scientific). Before loading, peptide mixtures were purified using ZipTip Pipette Tips (Millipore), according to the manufacturer’s recommendations. After loading, peptide mixtures (4 μg per run) were concentrated and desalted on a trapping pre-column (Acclaim PepMap C18, 75 μm × 2 cm nanoViper, 3 μm, 100 Å, Thermo Scientific), using 0.2% formic acid at a flow rate of 5 μl/min. The peptide separation was performed at 35°C using a C18 column (Acclaim PepMap RSLC C18, 75 μm × 15 cm nanoViper, 2 μm, 100 Å, Thermo Scientific) at a flow rate of 300 nL/min, using a 485 min gradient from 1 to 50% eluent B (0.2% formic acid in 95% ACN) in eluent A (0.2% formic acid in 5% ACN).
The mass spectrometer LTQ-Orbitrap Velos was set up in a data dependent MS/MS mode, as described previously [48 (link)]. Briefly, the lock mass option was enabled on a protonated polydimethylsiloxane background ion for internal recalibration, peptide ions were selected as the ten most intense peaks of the previous scan, and Higher Energy Collisional Dissociation (HCD) was chosen as the fragmentation method.

Tanca A., Abbondio M., Pisanu S., Pagnozzi D., Uzzau S, & Addis M.F. (2014). Critical comparison of sample preparation strategies for shotgun proteomic analysis of formalin-fixed, paraffin-embedded samples: insights from liver tissue. Clinical Proteomics, 11(1), 28.

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Quantitative Proteomics Analysis via LC-MS/MS

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Labeled peptides were analyzed via LC-MS/MS with an LTQ-Orbitrap Velos (Thermo-Fisher Scientific) interfaced with an Eksigent nanoLC ultra 2D plus system (AB Sciex, Switzerland). Peptides were injected onto a Pepmap 100 trap column (300 μm × 5 mm, 5 μm, 100 Å) at a flow rate of 400 nL/min. For analytical separation, the trap was switched inline to an Acclaim Pepmap C18 column (75 μm × 15 cm, 3 μm, 100 Å) using a 240 min linear gradient from 5 to 30% acetonitrile in 0.1% formic acid at a flow rate of 400 nL/min. MS/MS analyses were performed using an LTQ Orbitrap Velos (Thermo Fisher Scientific) with a nanoelectrospray ion source. The LTQ-Orbitrap Velos settings included one 30,000 resolution scan for precursor ions followed by MS2 scans of the 20 most intense precursor ions in positive ion mode. MS/MS data acquisition was completed using Xcalibur 2.1 (Thermo Fisher Scientific). The fragmentation method used for identification of TMT labeled peptides was based on higher energy collisional dissociation (HCD) with 40% fixed collision energy (CE).

Castillo J., Bogle O.A., Jodar M., Torabi F., Delgado-Dueñas D., Estanyol J.M., Ballescà J.L., Miller D, & Oliva R. (2019). Proteomic Changes in Human Sperm During Sequential in vitro Capacitation and Acrosome Reaction. Frontiers in Cell and Developmental Biology, 7, 295.

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Tryptic Peptide Analysis by LTQ-Orbitrap

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The tryptic peptides were analysed using an LTQ-Orbitrap Velos mass spectrometer coupled with EASY nLC 1000 (Thermo Finnigan, San Jose, CA). Tryptic peptides were separated by an analytic column (75 μm × 12 cm) packed with C18 resin. A linear 60 min gradient was used from 100% solvent A (0.1% formic acid in 2% acetonitrile) to 60% solvent B (0.1% formic acid in 98% acetonitrile) at a flow rate of 0.3 μL/min. The separated peptides were electrosprayed with nanoESI. The electrospray voltage was 2.6 kV using 35% normalized collision energy for MS/MS. All MS/MS spectra were acquired in data-dependent scans for the fragmentation of the 10 most abundant spectrums from full MS scan. The repeat count for dynamic exclusion was set to 1, the repeat duration was 30 s, the dynamic exclusion duration was set at 180 s, the exclusion mass width was 1.5 Da and the list of dynamic exclusion was 50.

Choi D., Go G., Kim D.K., Lee J., Park S.M., Di Vizio D, & Gho Y.S. (2020). Quantitative proteomic analysis of trypsin-treated extracellular vesicles to identify the real-vesicular proteins. Journal of Extracellular Vesicles, 9(1), 1757209.

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Quantitative Proteomic Analysis of S. aureus

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Two µg of endogenous peptides of S. aureus were analyzed using an LTQ Orbitrap Velos (Thermo Finnigan, San Jose, CA). First, the peptides were loaded into the precolumn (75 µm × 2 cm, Acclaim Pep Map 100 C18 column, 3 µm, 100 A; Thermo Fisher Scientific) at a flow rate of 10 µL/min and then were transferred to an analytical column (75 µm × 25 cm, Acclaim Pep Map RSLC C18 column, 2 m, 100 A; Thermo Fisher Scientific) at a flow rate of 300 nL/min. The peptides were reverse-phase separated with buffer A (0.51% acetic acid) and buffer B (100% ACN and 0.1% acetic acid) under a 95-min gradient (3–5% buffer B for 5 s, 5–15% buffer B for 40 min, 15–28% buffer B for 34 min and 50 s, 28–38% buffer B for 12 min, 38–100% buffer B for 5 s, and 100% buffer B for 8 min). MS survey scans were performed for mass-to-charge ratios ranging from 350–1,800, and the 20 most intense ions from the survey scans were analyzed by MS/MS spectra in the LTQ, which were determined using Xcalibur mass spectrometer software in real-time. With siloxane (m/z 445.120025) as a lock mass, dynamic mass exclusion windows of 60 s were used similar to previous studies (Wang et al., 2015 (link); Zhou et al., 2015 (link)).

Tu H., Xu F., Cheng Y., Pan Q., Cai X., Wang S., Ge S., Cao M., Su D, & Li Y. (2021). Proteomic profiling of the endogenous peptides of MRSA and MSSA. PeerJ, 9, e12508.

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Tat Protein Peptide Mapping

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The N-terminal Cy5-labeled Tat was separated by SDS-PAGE, identified by staining with Coomassie Blue, and in-gel digested. The LC-MS analysis was performed with an LTQ OrbitrapVelos mass spectrometer (ThermoFinnigan, USA). Data were acquired in data-dependent mode to simultaneous recording of full-scan mass and collision-induced dissociation (CID) spectra with multistage activation. For Tat protein peptide mapping, the CID spectra was compared to the sequence of HIV-1 Tat using Sequest (Bioworks; Thermo Electron, USA).

Kim J.M, & Seong B.L. (2017). Highly chromophoric Cy5-methionine for N-terminal fluorescent tagging of proteins in eukaryotic translation systems. Scientific Reports, 7, 11642.

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Peptide Analysis via LC-MS/MS

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Peptide samples
from in-gel digestion
and bRPLC fractionation were analyzed on LTQ-Orbitrap Velos and LTQ-Orbitrap
Elite mass spectrometers (Thermo Electron, Bremen, Germany) interfaced
with an Easy-nLC II nanoflow liquid chromatography system (Thermo
Scientific, Odense, Southern Denmark). The peptide digests were reconstituted
in 0.1% formic acid and loaded onto a trap column (75 μm ×
2 cm) packed in-house with Magic C₁₈ AQ (Michrom Bioresources,
Inc., Auburn, CA) at a flow rate of 5 μL/min with solvent A.
Peptides were resolved on an analytical column (75 μm ×
10 cm) at a flow rate of 350 nL/min using a linear gradient of 7–30%
solvent B (0.1% formic acid in 95% acetonitrile) over 60 min. Data-dependent
acquisition with full scans in the 350–1800 m/z range were carried out using an Orbitrap mass
analyzer at a mass resolution of 60 000 in Velos and 120 000
in Elite at 400 m/z, respectively.
Twenty of the most intense precursor ions from a survey scan were
selected for MS/MS and were fragmented using higher-energy collision dissociation
(HCD) with 35% normalized collision energy and detected at a mass
resolution of 15 000 and 30 000 at 400 m/z in Velos and Elite, respectively. Dynamic exclusion
was set for 30 s with a 10 ppm mass window.

Pinto S.M., Manda S.S., Kim M.S., Taylor K., Selvan L.D., Balakrishnan L., Subbannayya T., Yan F., Prasad T.S., Gowda H., Lee C., Hancock W.S, & Pandey A. (2014). Functional Annotation of Proteome Encoded by Human Chromosome 22. Journal of Proteome Research, 13(6), 2749-2760.

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Proteome Analysis by LC-MS/MS

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Protein extracts were loaded on NuPAGE 4–12% bis–Tris acrylamide gels (Life Technologies) to stack proteins in a single band that was stained with Imperial Blue (Pierce, Rockford. IL), cut from the gel, and digested with high-sequencing-grade trypsin (Promega, Madison, WI). Samples (injected in quadruplicate) were analyzed by liquid chromatography (LC)–tandem mass spectrometry (MS/MS) in an LTQ-Orbitrap-Velos (Thermo Electron, Bremen, Germany) online with a nanoLC Ultimate 3000 chromatography system (Dionex, Sunnyvale, CA). Peptides were separated on a Dionex Acclaim PepMap RSLC C18 column. Samples were measured in a data-dependent acquisition mode. The peptide masses were measured in a survey full scan. In parallel to the high-resolution full scan, the data-dependent collision-induced dissociation scans of the 10 most intense precursor ions were fragmented and measured in the linear ion trap to have maximum sensitivity and maximum amount of MS/MS data.

Daou P., Hasan S., Breitsprecher D., Baudelet E., Camoin L., Audebert S., Goode B.L, & Badache A. (2014). Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration. Molecular Biology of the Cell, 25(5), 658-668.

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Mass Spectrometry Analysis of mRNPs

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Purified mRNPs were precipitated in cold acetone (Thermo Scientific), subjected to trypsin digestion, and analyzed on a LTQ Orbitrap Velos mass spectrometer (Thermo Electron), as previously described (Ge et al. 2016 (link)). MASCOT software (Matrix Science) was used to determine peptide and protein identities, and Scaffold (Proteome Software) was used for further analysis. Proteins identified by five or more spectral counts were considered putative mRNP components.

Fritz S.E., Haque N, & Hogg J.R. (2018). Highly efficient in vitro translation of authentic affinity-purified messenger ribonucleoprotein complexes. RNA, 24(7), 982-989.

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