Concentrated peptides (250 ng) were separated on a nanoHPLC system (nanoElute, Bruker Daltonics, Billerica, MA, USA). The samples were loaded onto an Acclaim PepMap100 C18 Trap Column (0.3 mm id × 5 mm, Dionex Corporation, Sunnyvale, CA, USA) at 4 µL/min consistent flow and peptides were eluted onto a PepMap C18 analytical nanocolumn (1.9 µm beads size, 75 µm × 25 cm, PepSep, Marslev, Denmark) heated at 50 °C. Peptides were eluted with solvent B (100% ACN and 0.1% FA) in a 5–37% linear gradient with a flowrate of 400 nL/min for ~2 h. The HPLC system was coupled to a TimsTOF Pro ion mobility mass spectrometer containing Captive Spray nano electrospray source (Bruker Daltonics). Data acquisition was undertaken using diaPASEF mode. For each individual Trapped Ion Mobility Spectrometry (TIMS) measurement in diaPASEF mode, a single mobility window consisting of 27 mass steps (with m/z ranging from 114 to 1414 and a mass width of 50 Da) was employed per cycle, with a 1.27 s duty cycle. This process involves scanning the diagonal line in the m/z-ion mobility plane for +2 and +3 charged peptides.
Captive spray nano electrospray source
Manufactured by Bruker
Sourced in United States
The Captive Spray nano electrospray source is a specialized lab equipment designed for the efficient ionization of liquid samples. It utilizes a controlled and stable electrospray process to generate charged droplets, enabling sensitive and accurate mass spectrometric analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nanoelute, by Bruker (4 mentions)
Acclaim pepmap 100 c18 column, by Thermo Fisher Scientific (3 mentions)
Timstof pro, by Bruker (2 mentions)
Acclaim pepmap 100 c18 trap column, by Thermo Fisher Scientific (1 mentions)
C18 spin column, by Thermo Fisher Scientific (1 mentions)
Impact 2 q tof spectrometer, by Bruker (1 mentions)
Trypsin, by Merck Group (1 mentions)
Orbitrap platform, by Thermo Fisher Scientific (1 mentions)
Solarix 15t ft icr ms, by Bruker (1 mentions)
Protein g magnetic beads, by GE Healthcare (1 mentions)
Trypsin, by Worthington (1 mentions)
Timstof pro mass spectrometer, by Bruker (1 mentions)
7 protocols using captive spray nano electrospray source
1
Peptide Profiling by nanoHPLC-TIMS-MS
2
Ubiquitination Reaction Protocol with Mass Spectrometry
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Ubiquitination reaction samples (3 µg protein) were diluted in denaturing buffer (3 M urea, 25 mM TEAB pH 8.5, 0.5 mM EDTA) and reduced using 2 mM TCEP for 10 min at 37°C, followed by alkylation with 50 mM chloroacetamide for 30 min at room temperature in the dark. Chloroacetamide was used to avoid iodoacetamide-induced artefacts that mimic ubiquitination [84 (link)]. Samples were diluted with 50 mM TEAB pH 8.5 to 1 M urea and digested with 0.5 µg trypsin (Sigma) for 3 h at 37°C. Digested peptides were purified using C18 Spin Columns (ThermoFisher) and resuspended in 0.1% formic acid. Peptides (0.5 µg) were captured and eluted from an Acclaim PepMap100 C18 column with a 2 h gradient of acetonitrile in 0.1% formic acid at 200 ml min−1. The eluted peptides were analysed with an Impact II Q-TOF spectrometer equipped with a Captive Spray nanoelectrospray source (Bruker). Data were acquired using data-dependent automatic tandem mass spectrometry (auto-MS/MS) and analysed with MaxQuant using a standard search procedure against a custom-made FASTA file including GST-Ub, Parkin, Ub and UbcH7. Methionine oxidation, Lys ubiquitination (diGly) and Ser/Thr phosphorylation were included as variable modifications. Cysteine carbamylation was included as fixed modification.
3
Peptide Analysis using TimsTOF Pro
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All mass spectrometry experiments were performed at the Mass spectrometry and Proteomics platform at Université de Sherbrooke. From each fraction, 250 ng of peptides were injected into an HPLC (nanoElute, Bruker Daltonics) and loaded onto a trap column (constant flow of 4 µl/min; Acclaim PepMap100 C18 column, 0.3 mm internal diameter×5 mm, Dionex Corporation). An analytical C18 column (1.9 µm beads size, 75 µm×25 cm, PepSep) was used for elution for over a 2 h gradient of acetonitrile (5-37%) in 0.1% FA at 400 nl/min while being injected into a TimsTOF Pro trapped ion mobility mass spectrometer (TIMS) equipped with a Captive Spray nano electrospray source (Bruker Daltonics). Data were acquired using data-dependent auto-MS/MS with a 100-1700 m/z mass range, with parallel accumulation-serial fragmentation (PASEF) enabled and PASEF scans set at 10 (1.17 s duty cycle) and a dynamic exclusion of 0.4 min, m/z-dependent isolation window and collision energy of 42.0 eV. The intensity threshold was set at 2500, and the target intensity was set at 20,000.
4
Offline and Online LC-MS/MS Protocol
5
Targeted Protein Quantification in Tissues
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Protein extraction, digestion, immuno-affinity enrichment, and analysis by liquid chromatography–multiple reaction monitoring were conducted as previously described (12 (link), 13 (link)) with the following modifications. Frozen tissue lysates were cleared by centrifugation at 20,000×g for 10 min at 4°C, and protein concentration was determined using a Micro BCA Protein Assay Kit (Pierce, Rockford, IL, USA, #23235) prior to storage in liquid nitrogen until digestion. Digestion of plasma was performed with Lys-C for 2 h at 37°C [1:50 (w/w) enzyme:substrate ratio] followed by trypsin (Worthington, #LS003740) for overnight digestion at 37°C [1:50 (w/w) enzyme:substrate ratio]. Enrichments were performed using a mixture of 45 antibodies (four capture more than one peptide) coupled to protein G magnetic beads (GE Sepharose, #28-9513-79) at a 5-μg antibody:1-μl bead slurry ratio. For liquid chromatography, the trap and analytical columns were heated at 45°C and interfaced to the mass spectrometer using a CaptiveSpray nano electrospray source (Bruker, Billerica, MA, USA).
6
Peptide Separation and Identification Using TIMS-MS
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250 ng of peptides from each fraction were injected into an HPLC (nanoElute, Bruker Daltonics) and loaded onto a trap column with a constant flow of 4 µl/min (Acclaim PepMap100 C18 column, 0.3 mm id×5 mm, Dionex Corporation) then eluted onto an analytical C18 Column (1.9 µm beads size, 75 µm×25 cm, PepSep). Peptides were eluted over a 2-h gradient of ACN (5-37%) in 0.1% FA at 400 nl/min, while being injected into a TimsTOF Pro ion mobility mass spectrometer equipped with a Captive Spray nano electrospray source (Bruker Daltonics). Data was acquired using data-dependent auto-MS/MS with a 100-1700 m/z mass range, with PASEF enabled with a number of PASEF scans set at 10 (1.17 s duty cycle) and a dynamic exclusion of 0.4 min, m/z dependent isolation window and collision energy of 42.0 eV. The target intensity was set to 20,000, with an intensity threshold of 2500.
7
HPLC-TIMS-TOF Mass Spectrometry of Peptides
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Peptides were injected into an HPLC (nanoElute, Bruker Daltonics) and loaded onto a trap column with a constant flow of 4 µL/min (Acclaim PepMap100 C18 column, 0.3 mm id × 5 mm, Dionex Corporation) and eluted onto an analytical C18 Column (1.9 µm beads size, 75 µm × 25 cm, PepSep). Peptides were eluted over a 2-h gradient of acetonitrile (5–37%) in 0.1% formic acid at 400 nL/min while being injected into a TimsTOF Pro Mass Spectrometer equipped with a Captive Spray nano-electrospray source (Bruker Daltonics). Data were acquired using data-dependent auto-MS/MS with a 100–1700 m/z mass range, with PASEF enabled with a number of PASEF scans set at 10 (1.27 s duty cycle) and a dynamic exclusion of 0.4 min, m/z dependent isolation window and collision energy of 42.0 eV. The target intensity was set to 20,000, with an intensity threshold of 2500.
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