The proteomics samples were measured on a Sciex TripleTOF 5600 instrument (Sciex, Concord, Canada). The peptides were separated by nano-flow liquid chromatography (NanoLC Ultra 2D, Eksigent) with a flow of 300 nl/min using a NanoSpray III source with a heated interface (Sciex, Concord, Canada). A fused silica PicoTip™ Emitter (inner diameter 75 μm) (New Objective, Woburn, USA) manually packed with 21 cm C18 beads (MAGIC, 3 μm, 200 Å, Michrom BioResources, Auburn, USA) was used to separate about 1 μg of peptides along a linear 120 min gradient from 2% to 35% Buffer B (98% acetonitrile and 0.1% formic acid in H2O) in Buffer A (2% acetonitrile and 0.1% formic acid in H2O). The TripleTOF 5600 was operated in positive ion, high sensitivity SWATH-mode using 64 variable windows between 400 and 1200 m/z (1 m/z overlap). The collision energy was calculated based on a formula for peptides with charge 2+ adding a spread of 15eV. An accumulation time of 250 ms for precursor ions and 50 ms for all fragment ion scans was used that resulted in a total cycle time of about 3.5 s. Samples from the same cell line and biological replicate were injected together in a block design, but randomized within each block. The injection order of the cell line blocks was also randomized.
Tripletof 5600 instrument
Manufactured by AB Sciex
Sourced in United States, Canada
The TripleTOF 5600+ instrument is a high-performance mass spectrometer designed for advanced analytical applications. It combines a triple quadrupole configuration with a time-of-flight (TOF) mass analyzer, providing accurate mass measurements and high-resolution data acquisition.
Automatically generated - may contain errors
Lab products found in correlation
Prominence nanolc system, by Shimadzu (4 mentions)
Eksigent expert nano lc 400 system, by AB Sciex (3 mentions)
Analyst tf v 1, by AB Sciex (3 mentions)
Proteinpilot 4.5 beta, by AB Sciex (3 mentions)
C18 ziptip, by Merck Group (2 mentions)
Nanospray 3, by AB Sciex (2 mentions)
Picotip emitter, by New Objective (1 mentions)
Nanolc ultra 2d, by AB Sciex (1 mentions)
Nanoacquity chromatographic system, by Waters Corporation (1 mentions)
Spectronaut software, by Biognosys (1 mentions)
Proteinpilot 5, by AB Sciex (1 mentions)
Autosampler 2 1d plus, by AB Sciex (1 mentions)
Monocap c18 high resolution 2000 column, by GL Sciences (1 mentions)
Nanolc ultra, by AB Sciex (1 mentions)
In gel tryptic digestion kit, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 rslc system, by Thermo Fisher Scientific (1 mentions)
Acclaim pepmap c18 column, by Thermo Fisher Scientific (1 mentions)
Vacuum concentrator, by Eppendorf (1 mentions)
Orbitrap elite, by Thermo Fisher Scientific (1 mentions)
Strata x 33 μm polymeric reversed phase column, by Phenomenex (1 mentions)
21 protocols using tripletof 5600 instrument
1
SWATH-MS Proteomics Protocol
2
Quantitative Yeast Proteome Profiling
3
Mass Spectrometric Analysis of Arabidopsis
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Mass spectrometric analysis was performed using a TripleTOF 5600 instrument (SCIEX) with an Autosampler-2 1D Plus and NanoLC Ultra (Eksigent). Each sample was isolated using a MonoCap C18 High-Resolution 2000 column (2000 mm × 100-μm inside diameter, 2-μm pore size; GL Science, Japan). Four microliters of the sample were concentrated through the analytical column at a flow rate of 500 nL/min for 30 min. The mobile phase comprised of 2% acetonitrile and 0.1% formic acid (A) and 80% acetonitrile and 0.1% formic acid (B). The following linear gradient was used in this analysis: A:B = 98:2 at 0 min to A:B = 60:40 over 300 min, A:B = 10:90 over 20 min and A:B = 98:2 over 40 min. The MS scan range was a mass/charge ratio (m/z) of 400 to 1250, and the top 20 precursor ions were selected for subsequent MS/MS scans in the high-sensitivity mode. The MS/MS data were analysed using ProteinPilot 5.0 software (SCIEX) and subsequently annotated using the A. thaliana TAIR10 protein database for peptide identification.
4
Protein Identification by Mass Spectrometry
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IgE‐reactive protein bands were excised from Coomassie Blue stained SDS‐PAGE gels. After washing and destaining, proteins fixed in the gel were reduced with dithiothreitol and alkylated with iodoacetamide.[58
] In‐gel digestion was performed either using the In‐Gel Tryptic Digestion kit (Thermo Fisher Scientific), or with trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, Madison, WI, USA) using a final trypsin concentration of 20 ng µL−1 in 50 mM aqueous ammonium bicarbonate and 5 mM CaCl2 for 8 h at 37 °C.[59 (link)
] Afterwards, peptides were extracted with 5% trifluoroacetic acid (TFA) in 50% aqueous acetonitrile supported by ultrasonication. Extracted peptides were dried down in a vacuum concentrator (Eppendorf, Hamburg, Germany) and then resuspended in 0.1% TFA.
The extracted peptides were then injected into an Ultimate 3000 RSLC system (Dionex), using a 25 cm Acclaim PepMap C18 column (Thermo Fisher Scientific) for separation, which was coupled to a TripleTOF 5600 instrument (Sciex).[60 (link)
] A UniProt database with the species Bos taurus (txid 9913) in combination with the cRAP contaminant‐database was used for identification of the proteins.
] In‐gel digestion was performed either using the In‐Gel Tryptic Digestion kit (Thermo Fisher Scientific), or with trypsin (Trypsin Gold, Mass Spectrometry Grade, Promega, Madison, WI, USA) using a final trypsin concentration of 20 ng µL−1 in 50 mM aqueous ammonium bicarbonate and 5 mM CaCl2 for 8 h at 37 °C.[59 (link)
] Afterwards, peptides were extracted with 5% trifluoroacetic acid (TFA) in 50% aqueous acetonitrile supported by ultrasonication. Extracted peptides were dried down in a vacuum concentrator (Eppendorf, Hamburg, Germany) and then resuspended in 0.1% TFA.
The extracted peptides were then injected into an Ultimate 3000 RSLC system (Dionex), using a 25 cm Acclaim PepMap C18 column (Thermo Fisher Scientific) for separation, which was coupled to a TripleTOF 5600 instrument (Sciex).[60 (link)
] A UniProt database with the species Bos taurus (txid 9913) in combination with the cRAP contaminant‐database was used for identification of the proteins.
5
Intact Protein and Tryptic Peptide Analysis
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For intact protein analysis, 0.5 µg of protein was analysed using a Dionex Ultimate 3000 nanoLC system and Orbitrap Elite (Thermo) mass spectrometer. Proteins were desalted on a C4 PepMap300 pre-column (300 µm x 5mm, 5 µm 300Å) using buffer A (0.1% formic acid) at 30 µL/min for 5 min, a gradient of 10-98% buffer B (80% acetonitrile, 0.1% formic acid) over 5 min, and 98% buffer B for 9 min. The MS was operated in positive ion mode with the Orbitrap analyser set at 120,000 resolution. Source Deconvoluted data is reported as uncharged, monoisotopic mass. For tryptic peptide analysis, proteins were denatured, reduced/alkylated, and digested to peptides as previously described [23] . Digested peptides were desalted with C18 ZipTips (Millipore) and measured by LC-ESI-MS/MS using a Prominence nanoLC system (Shimadzu) and a TripleTof 5600 instrument (SCIEX) with a Nanospray III interface as described [24] .
6
Peptide Purification and Mass Spectrometry Analysis
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Peptides were desalted using C18 ZipTips (Millipore). LC-ESI-MS/MS analysis was performed using a Prominence nanoLC system (Shimadzu) and TripleTof 5600 instrument (SCIEX) as described previously (59) . Approximately 2 or 0.4 µg peptides were injected for data dependent acquisition (DDA) or data independent acquisition (DIA; Sequential Window Acquisition of All THeoretical Mass Spectra, SWATH-MS) (60) , respectively. Peptides were separated on a VYDAC EVEREST reversed-phase C18 HPLC column (300 Å pore size, 5 µm particle size, 150 µm i.d. x 150 mm) at a flow rate of 1 µl/min with a gradient of 10-60% buffer B over 45 min, with buffer A (1% acetonitrile and 0.1% formic acid) and buffer B (80% acetonitrile with 0.1% formic acid). LC parameters were identical for DDA and DIA, and DDA and DIA MS parameters were set as previously described (61, 62) .
7
Tandem Mass Spectrometry for Protein Identification
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Tandem mass spectrometric analysis was carried out using AB SCIEX TripleTOF 5600+ instrument (AB SCIEX, Redwood City, CA, USA) coupled to an Eksigent expert nano-LC 400 system (AB SCIEX). MS and MS/MS data was acquired using Analyst® TF v.1.6 (AB SCIEX).
Mass spectrometry data was analyzed by using ProteinPilot 4.5 Beta (AB SCIEX) for the peptide identifications. The detailed protocol is described inSupplementary File 6 .
Mass spectrometry data was analyzed by using ProteinPilot 4.5 Beta (AB SCIEX) for the peptide identifications. The detailed protocol is described in
8
Tandem Mass Spectrometry Proteomic Analysis
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Tandem mass spectrometric analysis was carried out using AB SCIEX TripleTOF 5600+ instrument (AB SCIEX, Redwood City, CA, USA) coupled to Eksigent expert nano-LC 400 system (AB SCIEX). MS and MS/MS data was acquired using Analyst® TF v.1.6 (AB SCIEX).
Mass spectrometry data was analyzed by using ProteinPilot 4.5 Beta (AB SCIEX) for the peptide identifications. Detailed protocol inSupplementary File S5 . The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium [33 ] via the PRIDE partner repository with the dataset identifier PXD003018.
Mass spectrometry data was analyzed by using ProteinPilot 4.5 Beta (AB SCIEX) for the peptide identifications. Detailed protocol in
9
Tandem Mass Spectrometry Proteomics Analysis
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Tandem mass spectrometric analysis was carried out using an AB SCIEX TripleTOF 5600+ instrument (AB SCIEX, Redwood City, CA, USA) coupled to an Eksigent expert nano-LC 400 system (AB SCIEX). MS and MS/MS data were acquired using Analyst® TF v.1.6 (AB SCIEX).
Mass spectrometry data was analyzed using ProteinPilot 4.5 Beta (AB SCIEX) for peptide identification.
Mass spectrometry data was analyzed using ProteinPilot 4.5 Beta (AB SCIEX) for peptide identification.
10
SCX Fractionation and Nano-HPLC-MS/MS Analysis
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Labeled samples were pooled and subjected to the SCX fractionation column connected with an HPLC system (LC-20ab, Shimadzu, Kyoto, Japan). Peptides were eluted using buffer-1 (25 mM NaH2PO4 in 25% ACN, pH 2.7) and a gradient of buffer-2 (25 mM NaH2PO4, 1 M KCl in 25% ACN, pH 2.7). The fractionating procedure was as follows: 100% buffer A for 10 min, 5%–35% buffer B for 20 min, 35%–80% buffer-2 for 1 min. Flow rate was kept at 1 mL/min. Fractions were desalted using a Strata X 33-μm Polymeric Reversed Phase column (Phenomenex, Torrance, CA, USA) and vacuum-dried.
Peptide fractions were analyzed using Nano HPLC (LC-20AD Shimadzu, Kyoto, Japan) and a 10-cm eluting C18 column (Shimadzu, Kyoto, Japan). A Triple TOF 5600 instrument (AB SCIEX, Concord, ON, Canada), fitted with Nanospray III (AB SCIEX) and a pulled quartz-tip emitter (New Objectives, Woburn, MA, USA), was used for mass spectrometry [49 (link)]. This procedure was carried out by Guangzhou Gene denovo Biotechnology Co., Ltd. (Guangzhou, China).
Peptide fractions were analyzed using Nano HPLC (LC-20AD Shimadzu, Kyoto, Japan) and a 10-cm eluting C18 column (Shimadzu, Kyoto, Japan). A Triple TOF 5600 instrument (AB SCIEX, Concord, ON, Canada), fitted with Nanospray III (AB SCIEX) and a pulled quartz-tip emitter (New Objectives, Woburn, MA, USA), was used for mass spectrometry [49 (link)]. This procedure was carried out by Guangzhou Gene denovo Biotechnology Co., Ltd. (Guangzhou, China).
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