Nanoacquity system

Manufactured by Waters Corporation

Sourced in United States, United Kingdom

The NanoAcquity system is a high-performance liquid chromatography (HPLC) instrument designed for nano-scale separations. It features a low-dispersion flow path and precise flow control capabilities, enabling the analysis of small sample volumes with high resolution and sensitivity.

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49 protocols using nanoacquity system

Nanoscale LC-MS/MS Proteomics Workflow

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LC-ESI-MS/MS was performed on a linear trap quadrupole (LTQ) Orbitrap Velos (Thermo Electron, San Jose, CA, USA) equipped with a NanoAcquity system (Waters). Peptides were trapped on a homemade 5 μm 200 Å Magic C18 AQ (Michrom) 0.1 × 20 mm precolumn and separated on a commercial 0.075 × 150 mm Nikkyo (Nikkyo Technology) analytical nanocolumn (C18, 5 μm, 100 Å). The analytical separation was run for 65 min using a gradient of H2O/FA 99.9%/0.1% (solvent A) and CH3CN/FA 99.9%/0.1% (solvent B). The gradient was initially per 0-1 min 95% A and 5% B and then to 65% A and 35% B for 55 min and 20% A and 80% B for 65 min at a flow rate of 220 nL/min. For MS survey scans, the orbitrap (OT) resolution was set to 60000 and the ion population was set to 5.0 × 10⁵ with an m/z window from 400 to 2000. For protein identification, up to eight precursor ions were selected for collision-induced dissociation (CID) in the LTQ. The ion population was set to 1.0 × 10⁴ (isolation width of 2 m/z) while, for MS/MS detection in the OT, it was set to 1.0 × 10⁵ with an isolation width of 2 m/z units. The normalized collision energies were set to 35% for CID.

Kramer M., Pierredon S., Ribaux P., Tille J.C., Petignat P, & Cohen M. (2015). Secretome Identifies Tenascin-X as a Potent Marker of Ovarian Cancer. BioMed Research International, 2015, 208017.

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Peptide Separation and Identification by Orbitrap MS

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Electrospray ionization (ESI) linear trap quadrupole (LTQ)-orbitrap (OT) mass spectrometry (MS) was performed on a LTQ Orbitrap Velos Pro from Thermo Electron (San Jose, CA) equipped with a NanoAcquity system from Waters (Waters Corporation, Manchester, UK). Peptides were trapped on a homemade 5 μm 200 Å Magic C18 AQ (Michrom, Auburn, CA) 0.1 × 20 mm precolumn and separated on a homemade 5 μm 100 Å Magic C18 AQ (Michrom) 0.75 × 150 mm column with a gravity-pulled emitter. The analytical separation was run for 65 min using a gradient of H₂O/FA 99.9%/0.1% (solvent A) and CH₃CN/FA 99.9%/0.1% (solvent B). The gradient was run at a flow rate of 220 nL/min as follows: 5% B for 1 min, from 5% to 35% B in 54 min, from 35% to 80% B in 10 min. For MS survey scans, the Orbitrap resolution was set to 60,000 and the ion population was set to 5 × 10⁵ with an m/z window from 400 to 2000. Eight precursor ions were selected for collision-induced dissociation (CID) in the LTQ. For this, the ion population was set to 7 × 10³ (isolation width of 2 m/z). The normalized collision energies were set to 35% for CID.

Jiménez E., Sánchez B., Farina A., Margolles A, & Rodríguez J.M. (2014). Characterization of the bile and gall bladder microbiota of healthy pigs. MicrobiologyOpen, 3(6), 937-949.

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Label-free quantitative proteomics analysis

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For
label-free, relative, quantitative analysis, six replicates of each
sample were analyzed by nano-LC–MS/MS. For each run, 1 μg
of the digest was injected on a 100 μm × 100 mm, reverse-phase
C18 BEH column with 1.7 um particles and a 300 Å pore size (Waters,
Milford, MA) using a Waters nanoAcquity system. Chromatography solvents
were water (A) and acetonitrile (B), both with 0.1% formic acid. Peptides
were eluted from the column with the following gradient 3 to 35% B
(130 min). At 140 min, the gradient increased to 95% B and was held
there for 10 min. At 160 min, the gradient returned to 3% to re-equilibrate
the column for the next injection. A short 50 min linear gradient
blank was run between samples to prevent sample carryover. Peptides
eluting from the column were analyzed by data-dependent MS/MS on a
Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, MA).
A top-15 method was used to acquire data. In brief, the instrument
settings were as follows: resolution was set to 70 000 for
MS scans and 17 500 for the data-dependent MS/MS scans to increase
speed. The MS AGC target was set to 10⁶ counts, while MS/MS
AGC target was set to 10⁵. The MS scan range was from 300
to 2000 m/z. MS scans were recorded
in profile mode, while the MS/MS was recorded in centroid mode, to
reduce data file size. Dynamic exclusion was set to a repeat count
of 1 with a 25 s duration.

Cholewa B.D., Pellitteri-Hahn M.C., Scarlett C.O, & Ahmad N. (2014). Large-Scale Label-Free Comparative Proteomics Analysis of Polo-Like Kinase 1 Inhibition via the Small-Molecule Inhibitor BI 6727 (Volasertib) in BRAFV600E Mutant Melanoma Cells. Journal of Proteome Research, 13(11), 5041-5050.

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Tandem Mass Spectrometry of Biotherapeutic Proteins

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To obtain the tandem mass spectra for the unique peptides in the biotherapeutics (m/z 806.89 and 624.32), the scFv and anti-PD-1 mAb proteins were separated by SDS/PAGE, stained with Simply Blue (Life Technologies Scientific), enzymatically digested in situ with trypsin ⁴³, and desalted ⁴⁴. The purified peptides were diluted to 0.1% formic acid and analyzed separately by high resolution LC-MS/MS in data dependent mode. We used a Waters NanoAcquity system (with a 100-μm inner diameter × 10-cm length C18 column (1.7 μm BEH130; Waters) configured with a 180-μm × 2-cm trap column coupled to a Thermo Q-Exactive Plus orbitrap mass spectrometer. Trapping was performed at 15 μL/min buffer A for 1 minute and elution with a 50% linear acetonitrile gradient over 120 minutes. MS data were collected in data dependent acquisition mode. Full scan MS1 spectra were acquired over 380–1600 m/z at a resolution of 70,000 (m/z 400) with automatic gain control (AGC) at 3 × 10⁶ ions. The top 15 most intense precursor ions were selected for HCD fragmentation performed at normalized collision energy (NCE) 25% with target ion accumulation value of 5 X 10(4). MS/MS spectra were collected with resolution of 17,500. See Fig. S3 for MS/MS spectra.

Rafiq S., Yeku O.O., Jackson H.J., Purdon T.J., van Leeuwen D.G., Drakes D.J., Song M., Miele M.M., Li Z., Wang P., Yan S., Xiang J., Ma X., Seshan V.E., Henderickson R.C., Liu C, & Brentjens R.J. (2018). Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo. Nature biotechnology, 36(9), 847-856.

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Mitochondrial Proteome Analysis by Mass Spectrometry

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Mitochondria treated with ONOO⁻ were lysed and the proteins were resolved by SDS-PAGE. Protein bands were stained with Coomassie blue, excised and cut into small pieces and then subjected to ingel digestion by trypsin as described previously (Yang et al., 2012 (link)). Extracted tryptic fragments were purified for mass spectrometry analysis with a Thermo Orbitrap Velos instrument. The digests were dissolved in 2 µl of 2% acetonitrile 0.1% formic acid; there were two LC-MS runs of 1.5 µl sample injection each. They were run for 120 min on a water/acetonitrile 0.1% formic acid gradient on a Waters NanoAcquity system with a 10 cm x 75 µm column packed with 3 µm Magic C18AQ stationary phase. The MS/MS data were processed with the Sorcerer Sequest software for searching against a UniProt guinea pig database to which common, ubiquitous laboratory contaminants were added. The results were exported into Scaffold files. The search criteria were set to: minimum peptide count of 2; minimum peptide probability of 0.95; peptide False Discovery Rate <5%; minimum protein probability of 0.95 and protein False Discovery Rate <1%. Those proteins that matched the experimental data and met the search criteria were considered to have a statistically significant probability of being present in the sample. The database searches were conducted with tyrosine nitration as a variable modification.

Yang M., Xu Y., Heisner J.S., Sun J., Stowe D.F., Kwok W.M, & Camara A.K. (2018). Peroxynitrite Nitrates Adenine Nucleotide Translocase and Voltage-Dependent Anion Channel 1 and Alters their Interactions and Association with Hexokinase II in Mitochondria. Mitochondrion, 46, 380-392.

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Proteomic Analysis by NanoLC-nanoESI-MS/MS

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NanoLC-nanoESI-MS/MS analysis was performed on a nanoAcquity system (Waters, MA, USA) connected to the Orbitrap Elite hybrid mass spectrometer (Thermo Electron, Bremen, Germany) equipped with a PicoView nanospray interface (New Objective, MA, USA). Peptide mixtures were loaded onto a 75 μm inner diameter, 25 cm length C18 BEH column (Waters) packed with 1.7 μm particles with a pore width of 130 Å and separated using a segmented gradient in 180 min from 5% to 40% solvent B (acetonitrile with 0.1% formic acid) at a flow rate of 300 nl/min and a column temperature of 35 °C. Solvent A was 0.1% formic acid in water. The mass spectrometer was operated in the data-dependent mode. Briefly, survey full scan MS spectra were acquired in the Orbitrap (m/z 350–1600) with the resolution set to 120K at m/z 400 and automatic gain control target at 10⁶. The 15 most intense ions were sequentially isolated for higher-energy collisional dissociation (HCD) MS/MS fragmentation and detection in the Orbitrap with previously selected ions dynamically excluded for 60 s. For MS/MS, we used a resolution of 15,000, an isolation window of 2 m/z, and a target value of 50,000 ions, with maximum accumulation times of 200 ms. Fragmentation was performed with normalized collision energy of 35% and an activation time of 0.1 ms. Ions with singly and unrecognized charge state were also excluded.

Quantitative Proteomics Analysis Reveals the Min System of Escherichia coli Modulates Reversible Protein Association with the Inner Membrane. (2016). Molecular & Cellular Proteomics : MCP, 15(5), 1572-1583.

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GAPDH Interactome Identification by NI-NTA Pull-Down

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Nickel-nitrilotriacetic acid resin pull down assay is an affinity chromatography procedure that uses a bait protein, P. lutzii GAPDH in our case, immobilized and incubated with a protein source containing putative protein preys. The assay was performed in native conditions and the results included direct and indirect associations between bait and prey proteins. Firstly, recombinant GAPDH was immobilized onto Ni-NTA resin following the purification assay without the elution step. Then, 300 μg of P. lutzii cell lysate containing the protein extract was incubated for 3 h on ice and under gentle agitation. Next, the column containing bait and prey proteins was washed with native wash buffer 5 times to reduce unspecific interactions or contaminants and then, the complex bait-preys was eluted with native elution buffer. The eluted sample underwent tryptic digestion and the digested peptides were separated further via NanoUPLC-MS^E and analyzed using a nanoACQUITY system (Waters Corporation, Milford, Manchester, United Kingdom) in order to identify the proteins that possibly interacted with GAPDH.
A control sample was prepared similarly but 300 μg of P. lutzii protein extract was incubated with Ni-NTA without the immobilized GAPDH and the proteins identified in both experiments were excluded from the results.

e Silva K.S., Lima R.M., Baeza L.C., Lima P.D., Cordeiro T.D., Charneau S., da Silva R.A., Soares C.M, & Pereira M. (2019). Interactome of Glyceraldehyde-3-Phosphate Dehydrogenase Points to the Existence of Metabolons in Paracoccidioides lutzii. Frontiers in Microbiology, 10, 1537.

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Peptide Separation and Identification

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Each sample was analyzed
on a Waters NanoAcquity system (Waters Corporation, Milford, MA).
The peptides were initially loaded onto a Symmetry C18 180 μm
× 20 mm 5 μm trap column to desalt and chromatographically
focus the peptides prior to elution onto a HSS T3 C18 75 μm
× 150 mm, 1.7 μm analytical column. Solvent A, HPLC grade
water with 0.1% formic acid; solvent B, acetonitrile with 0.1% formic
acid was used. The flow rate was set at 0.3 μL/min. The gradient
began following a 3 min (5 μL/min) trapping stage on the trap
column. At time zero, solvent A was 99% while solvent B was 1%. Solvent
B increased linearly to 40% at 90 min and to 85% at 92 min. The gradient
was held at 85% solvent B at 93 min and returned to starting conditions
at 95 min to equilibrate.

Daly C.E., Ng L.L., Hakimi A., Willingale R, & Jones D.J. (2014). Qualitative and Quantitative Characterization of Plasma Proteins When Incorporating Traveling Wave Ion Mobility into a Liquid Chromatography–Mass Spectrometry Workflow for Biomarker Discovery: Use of Product Ion Quantitation As an Alternative Data Analysis Tool for Label Free Quantitation. Analytical Chemistry, 86(4), 1972-1979.

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Trypsin Digestion and LC-MS/MS Analysis

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Two μl of the same sample used in the crystallization experiments was diluted in 50 μl of 50 mM ammonium bicarbonate pH 7.5. The sample was subsequently digested with trypsin (Porcine, sequencing grade, Promega, Madison, WI) O/N at 37 °C using an enzyme to protein ratio 1:20. Formic acid was added to a final v/v concentration of 0.1% before the liquid chromatography mass spectrometry analysis. The digest was separated using an ultra performance Nano Acquity system coupled to a Synapt G1 mass spectrometer (Waters, Milford, MA). The peptides were separated on an HSS T3 (75 μm × 250 μm, 1.8 μm particles) column (Waters) with a gradient of 3–40% of buffer A (0.1% formic acid) and buffer B (100% acetonitrile with 0.1% formic acid). The spectra were acquired in Liquid-chromatography mass spectrometry in elevated energy mode (LCMS^E) mode (alternating low and high collision energy) with mass range from 125 to 2,000 m/z using a collision energy ramp (10–40 V)61 (link). The data were analysed using the Proteinlynx Global Server V2.5 platform (Waters) against UniProt database using C-mannosylation as a variable modification.

Desmet J., Verstraete K., Bloch Y., Lorent E., Wen Y., Devreese B., Vandenbroucke K., Loverix S., Hettmann T., Deroo S., Somers K., Henderikx P., Lasters I, & Savvides S.N. (2014). Structural basis of IL-23 antagonism by an Alphabody protein scaffold. Nature Communications, 5, 5237.

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Proteomic Identification of Differentially Expressed Proteins

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The spots differentially expressed between 2 and 4 hag. were excised from gels and placed in microtubes. Then, they were bleached and subjected to trypsin digestion [23 (link)]. Next, the samples containing recovered peptides were vacuum concentrated until they reached a volume of 10–15 μL.
The resulting peptides from tryptic digestion were subjected to liquid chromatography with tandem mass spectrometry (LC-MS/MS) on a nanoAcquity system (Waters, Milford, MA) coupled with a Q-ToF micro mass spectrometer (Waters), according to methods described elsewhere [23 (link)]. The raw data were processed, and the resulting spectra were analyzed in the ProteinLynx Global Server 4.2 software (Waters) and compared with the SwissProt database (http://www.uniprot.org/downloads, October 2011). For comparison with the NCBI database, the MASCOT tool MS/MS IonSearch (www.matrixscience.com) was used with the following settings: tryptic digestion, with 1 cleavage site lost, cysteines modified by carbamidomethylation and methionine oxidation, error tolerance for the peptide of 30 ppm, and fragment mass error of MS/MS equal to 0.1 Da. According to MASCOT analysis probability, only the significant “hits” (p < 0.05) were accepted. After protein identification, their ontology and biological processes were classified in Blast2Go.

Mares J.H., Gramacho K.P., Santos E.C., da Silva Santiago A., Santana J.O., de Sousa A.O., Alvim F.C, & Pirovani C.P. (2017). Proteomic analysis during of spore germination of Moniliophthora perniciosa, the causal agent of witches’ broom disease in cacao. BMC Microbiology, 17, 176.

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