Tryptic peptides were suspended in 3% (v/v) ACN and .1% (v/v) trifluoroacetic acid (TFA) and directly injected onto a reversed‐phase C18 1.7 μm, 130 Å, 75 × 250 mm M‐class column (Waters), using an Ultimate 3000 nanoUPLC (Thermos Scientific). Peptides were eluted at 300 nl/min with a gradient from 2% to 20% ACN in 100 min then to 32% ACN in 20 min then 1 min to 95% ACN and detected using a Q‐Exactive HF‐X mass spectrometer (Thermo Scientific). Precursor mass spectra (MS1) were acquired at a resolution of 120,000 from 380 to 1580 m/z with an automatic gain control (AGC) target of 3E6 and a maximum injection time of 45 ms. Precursor peptide ion isolation width for MS2 fragment scans was 1.4 m/z, and the top 12 most intense ions were sequenced. All MS2 spectra were acquired at a resolution of 15,000 with higher energy collision dissociation (HCD) at 27% normalized collision energy. An AGC target of 1E5 and 100‐ms maximum injection time was used. Dynamic exclusion was set for 25 s with a mass tolerance of ±10 ppm. Raw files were searched against the custom P. celeri database using MaxQuant v.2.0.3.0. Cysteine carbamidomethylation was considered a fixed modification, and methionine oxidation and protein N‐terminal acetylation were searched as variable modifications. All peptide and protein identifications were thresholded at a 1% false discovery rate (FDR).
Ultimate 3000 nanouplc
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ultimate 3000 nanoUPLC is a high-performance liquid chromatography (HPLC) system designed for the analysis of nanoscale samples. It features a compact design, precise flow control, and high-sensitivity detection capabilities to enable the separation and analysis of complex samples at the nanoliter scale.
Automatically generated - may contain errors
Lab products found in correlation
Q exactive hf x mass spectrometer, by Thermo Fisher Scientific (2 mentions)
Acclaim pepmap rslc c18 analytical column, by Thermo Fisher Scientific (2 mentions)
Acclaim pepmap 100 c18 trap column, by Thermo Fisher Scientific (2 mentions)
Q exactive hf hybrid quadrupole orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easy spray c18 column, by Thermo Fisher Scientific (1 mentions)
Speedbeads, by Cytiva (1 mentions)
Seramag, by Cytiva (1 mentions)
Hss t3 c18 1.8 μm, by Waters Corporation (1 mentions)
Orbitrap exploris 480, by Thermo Fisher Scientific (1 mentions)
Nanospray flex, by Thermo Fisher Scientific (1 mentions)
Q exactive hf mass spectrometry, by Thermo Fisher Scientific (1 mentions)
Trypsin solution, by Promega (1 mentions)
Acclaim pepmap rslc, by Thermo Fisher Scientific (1 mentions)
Proteasemax, by Promega (1 mentions)
Q exactive plus mass spectrometer, by Thermo Fisher Scientific (1 mentions)
7 protocols using ultimate 3000 nanouplc
1
Proteomics Workflow for P. celeri
2
Larval Peptide Characterization by LC-MS
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Larval peptides were prepared as previously described (46). Two micrograms of samples were injected onto an UltiMate 3000 nano UPLC equipped and chromatographic separation was achieved with a 50-cm-long C18 EASY-Spray column (Thermo Fisher Scientific) at 55°C. The following gradient was applied: 4–26% of solvent B (98% acetonitrile and 0.1% FA) in 90 min, 26–95% of solvent B in 5 min and 95% of solvent B for 5 min at a flow rate of 300 nl/min. The mass spectrometric acquisition on a Q Exactive HF Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, Bremen, Germany) comprised one survey full mass spectrum ranging from a mass/charge ratio (m/z) of 350–1600 at a resolution of R = 120 000 (at m/z 200) targeting 5 × 106 ions for a maximum injection time of 100 ms, followed by data-dependent higher-energy collision dissociation fragmentations of maximum 18 most intense precursor ions with a charge state 2+ to 7+, using 45-s dynamic exclusion. The tandem mass spectra were acquired with a resolution of R = 60 000, targeting 2 × 105 ions for a maximum injection time of 54 ms, setting isolation width to m/z 1.4 and normalized collision energy to 33% setting first mass at m/z 100.
3
Mass Spectrometry Analysis of Protein Complexes
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Co-IP lysates were prepared as above using 40 μL α-FLAG-conjugated magnetic beads. Following Co-IP, samples were resuspended in Co-IP buffer with reduced Triton X-100 (0.1%). To prepare samples for mass spectrometry, FLAG-tagged affinity purifications were eluted, reduced, and alkylated using 5% (w/v) sodium dodecyl-sulfate, 10 mM tris(2-carboxyethylphosphine), 40 mM 2-chloroacetamide, 50 mM Tris-HCl, pH 8.5, boiled for 10 min, and incubated shaking at 1000 rpm at 37°C for 30 min. Affinity-purified proteins were digested using the SP3 method with Sera-Mag™ carboxylate-functionalized SpeedBeads (Cytiva).54 (link) Cleaned-up peptides were then dried in a SpeedVac vacuum concentrator and stored at −20°C until analysis.
Tryptic peptides were suspended in 3% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and directly injected onto a reversed-phase C18 1.7 μm, 130 Å, 75 mm × 250 mm M-class column (Waters), using an Ultimate 3000 nanoUPLC (ThermoFisher). Peptides were eluted with an acetonitrile gradient and detected using a Q-Exactive HF-X mass spectrometer (ThermoFisher). Raw files were searched against the Uniprot Human database UP000005640 using MaxQuant v.1.6.14.0. All peptide and protein identifications were thresholded at a 1% false discovery rate. Statistical analysis was performed on log2-transformed iBAQ intensities using limma.
Tryptic peptides were suspended in 3% (v/v) acetonitrile, 0.1% (v/v) trifluoroacetic acid and directly injected onto a reversed-phase C18 1.7 μm, 130 Å, 75 mm × 250 mm M-class column (Waters), using an Ultimate 3000 nanoUPLC (ThermoFisher). Peptides were eluted with an acetonitrile gradient and detected using a Q-Exactive HF-X mass spectrometer (ThermoFisher). Raw files were searched against the Uniprot Human database UP000005640 using MaxQuant v.1.6.14.0. All peptide and protein identifications were thresholded at a 1% false discovery rate. Statistical analysis was performed on log2-transformed iBAQ intensities using limma.
4
Peptide Separation and Identification by Nano-UPLC-MS/MS
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A 2 µL of the reconstituted peptides were separated on an Ultimate 3000 nanoUPLC (Thermo Scientific) with 300 nL/min by a reversed-phase C18 column (HSS-T3 C18 1.8 μm, 75 μm × 250 mm, Waters Corporation) at 55 °C using a 90 min linear gradient from 5% Eluent A (0.1% TFA/3% DMSO/Water) to 35% Eluent B (0.1% TFA/3% DMSO/ACN) followed by ionization using a Nanospray Flex electrospray ionization source (Thermo Scientific). Mass-to-charge analysis of the eluting peptides was performed using an Orbitrap Exploris 480 (Thermo Scientific) in data-dependent acquisition (DDA) mode. Full scan MS1 spectra were collected over a range of 350–1600 m/z with a mass resolution of 60,000 @ 200 m/z using an automatic gain control (AGC) target of 300%, maximum injection time was set to “Auto” and RF lens to 40%. The Top20 most intense peaks above the signal threshold of 2 × 104, harboring a charge of 2–6, were selected within an isolation window of 1.4 Da as precursors for fragmentation using higher-energy collisional dissociation (HCD) with a normalized collision energy of 30. The resulting fragment ion m/z ratios were measured as MS2 spectra over an automatically selected m/z range with a mass resolution of 15,000@ 200 m/z, AGC target was set to “Standard” and maximum injection time to “Auto”.
5
Nano-LC-MS/MS Peptide Analysis
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The sample was loaded to the column equilibrated with buffers A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile). The Acclaim PepMap 100 C18 trap column (75 μm × 2 cm, Dionex) was equilibrated with liquid A by Ultimate 3000 nanoUPLC (Dionex), and the sample was eluted onto an Acclaim PepMap RSLC C18 analytical column (75 μm × 25 cm, Dionex) at a flow rate of 300 nl/min. The liquid-phase gradient was given as follows: 0–6 min, where the linear gradient of buffer B was from 2 to 10%; 7–51 min, where the linear gradient of buffer B was from 10 to 20%; 51–53 min, where the linear gradient of buffer B was from 20 to 80%; 53–57 min, where the linear gradient of buffer B solution was held at 80%.
Mass analysis was performed by nano-spray ionization-mass spectrometry (NSI-MS) and Q-Exactive HF mass spectrometry (Thermo Scientific). The intact peptide was detected by the Orbitrap, the scanning range was set to 250–1,500 m/z, the automatic gain control (AGC) target was 3E6, the resolution was 70,000, the max injection time (IT) was 250 ms, and the dynamic exclusion time was 15 s. The peptide was selected and fragmented for MS/MS using 28% NCE; ion fragments were detected in the Orbitrap, the resolution was 17,500, AGC was 1E5 or 5E4, and the maximum IT was 100 or 200 ms. LC-MS was performed by Micrometer Biotech (Hangzhou, China).
Mass analysis was performed by nano-spray ionization-mass spectrometry (NSI-MS) and Q-Exactive HF mass spectrometry (Thermo Scientific). The intact peptide was detected by the Orbitrap, the scanning range was set to 250–1,500 m/z, the automatic gain control (AGC) target was 3E6, the resolution was 70,000, the max injection time (IT) was 250 ms, and the dynamic exclusion time was 15 s. The peptide was selected and fragmented for MS/MS using 28% NCE; ion fragments were detected in the Orbitrap, the resolution was 17,500, AGC was 1E5 or 5E4, and the maximum IT was 100 or 200 ms. LC-MS was performed by Micrometer Biotech (Hangzhou, China).
6
Peptide Characterization by Nanoflow LC-MS/MS
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The lyophilized peptide was resuspended in buffer A (2% ACN, 0.1% FA), loaded onto an Acclaim PepMap 100 C18 trap column (75 μm × 2 cm, Dionex, Waltham, MA, USA) with Ultimate 3000 nanoUPLC (Dionex) and eluted onto an Acclaim PepMap RSLC C18 analytical column (Dionex, 75 μm × 25 cm). Buffer A linear gradient was run at 300 nL min−1 for 45 min, starting from 11% to 20% buffer B, followed by 2 min gradient to 80% buffer B, and maintained at 80% buffer B for 3 min. The peptide was subjected to NSI source in mass spectrometry Q Exactive plus (Waltham, MA, USA) coupled online to the UPLC. MS1 spectra were acquired at the resolution of 70 000 (at 200 m/z) with an AGC (automatic gain control) of 3 000 000 and a max IT of 250 ms. MS/MS data were acquired at the resolution of 17 500 through isolation windows of 2 Da, and the AGC and max IT were set to 100 000 and 100 ms, the NCE (normalized collision energy) was set to 28%. And the loop count was set to 15 which means 15 MS/MS scans would be acquired between each full scan.
7
Tryptic Peptide Identification by Mass Spectrometry
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The bands of interest were first excised from gels. Classical steps of washes (100 mM ammonium bicarbonate/acetonitrile, 50:50 v/v) were followed by reduction (10 mM dithiothreitol for 1 h. at 56 °C), alkylation (55 mM iodoacetamide for 30 min at room temperature) and digestion by a trypsin solution (10 ng/µL, Promega) containing ProteaseMAX 0.025% (w/v) (Promega) in 50 mM ammonium bicarbonate overnight at 37 °C. Tryptic peptides were extracted by 0.1% TFA in water/acetonitrile (50:50 v/v) and dried into a speed vacuum. Mass spectrometry was performed on a Q Exactive Plus mass spectrometer (ThermoFisher Scientific, Bremen, Germany) equipped with a nanospray ion source and coupled to an Ultimate 3000 nano UPLC (Dionex, ThermoScientific, Sunnyvale, CA, USA). Dried tryptic peptides were dissolved in 2% acetonitrile/0.05% TFA in water and desalted on a C18 µ-precolumn (PepMap100, 300 µm × 5 mm, 5 µm, 100 Å, Dionex) before elution onto a C18 column (Acclaim PepMap, RSLC, 75 µm × 150 mm, 2 µm, 100 Å, Dionex). Peptides were eluted with a linear gradient from 6 to 40% of mobile phase B (20% water, 80% acetonitrile/0.1% formic acid) in A (0.1% formic acid in water) for 52 min. Peptides were detected with a workflow combining full MS (350- 1900 m/z range at 70000 resolution)/data dependent MS/MS Top 10 (high collision dissociation, 150 –2250 m/z range).
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