Peptides were eluted from the StageTips using 80% acetonitrile/0.1% formic acid and concentrated prior to loading either on an uHPLC nLC-1000 system coupled to a Q Exactive Plus mass spectrometer (Thermo) or an uHPLC nLC-1200 system coupled to an Exploris 480 mass spectrometer (Thermo). The peptides were loaded on a 20 cm (Q Exactive Plus) or 50 cm (Exploris 480) column (75 μm inner diameter) in-house packed with Reprosil C18 (Dr. Maisch GmbH) and eluted with a 73- or 88-min optimized gradient increasing from 2% to 40% mixture of 80% acetonitrile/0.1% formic acid at a flow rate of 225 nl/min or 250 nl/min. The Q Exactive Plus was operated in positive ion mode with a data-dependent acquisition strategy of one MS full scan (scan range 300 - 1,650 m/z; 70,000 resolution; AGC target 3e6; max IT 20 ms) and up to ten MS/MS scans (17,500 resolution; AGC target 1e5, max IT 120 ms; isolation window 1.8 m/z) with peptide match preferred using HCD fragmentation. The Exploris 480 was operated in positive ion mode with a data-dependent acquisition strategy of one MS full scan (scan range 300 - 1,650 m/z; 60,000 resolution; normalized AGC target 300%; max IT 28 ms) and up to twenty MS/MS scans (15,000 resolution; AGC target 100%, max IT 40 ms; isolation window 1.4 m/z) with peptide match preferred using HCD fragmentation.
Q exactive plus
Manufactured by Dr. Maisch
The Q Exactive Plus is a high-resolution, accurate-mass hybrid quadrupole-Orbitrap mass spectrometer. It provides high-resolution, accurate-mass full-scan MS data and targeted fragmentation data in a single run.
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Lab products found in correlation
Exploris 480 mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Q exactive plus mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200 system, by Thermo Fisher Scientific (1 mentions)
Q exactive hf x, by Thermo Fisher Scientific (1 mentions)
Q exactive plus, by Thermo Fisher Scientific (1 mentions)
2 protocols using q exactive plus
1
High-resolution Mass Spectrometry Peptide Analysis
2
Mass Spectrometry-based Proteomics Protocol
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Data acquisition for LFQ comparison of ADC and SCC, as well as all TMT experiments, was performed on a Q Exactive HF-X instrument (Thermo Scientific, Xcalibur software version 4.1.31.9) coupled to an easy nLC 1200 system (Thermo Scientific). For the cooking time comparison, a Q Exactive Plus instrument (Thermo Scientific, Xcalibur software version 3.0.63) with an easy nLC 1200 system was used. The peptides were separated over a 110 min gradient with a flow rate of 250 nL with increasing concentration of buffer B (up to 60%) on a 20 cm reversed-phase column packed in-house with 1.9 µm beads (ReproSil Pur, Dr. Maisch GmbH).
The Q Exactive Plus was operated in data-dependent mode with 70 K MS1 resolution, AGC target of 3 × 106 ions, and a maximum injection time of 50 ms, choosing the top 20 ions for MS2 scans with 17.5 K resolution, AGC target of 5 × 104 ions and maximum injection time of 250 ms.
The Q Exactive HF-X was set up for 60 K MS1 resolution with an AGC target of 3 × 106 ions and maximum injection time of 10 ms, followed by 20 MS2 scans with 45 K resolution, AGC target of 1 × 105 ions, and maximum injection time of 86 ms.
The Q Exactive Plus was operated in data-dependent mode with 70 K MS1 resolution, AGC target of 3 × 106 ions, and a maximum injection time of 50 ms, choosing the top 20 ions for MS2 scans with 17.5 K resolution, AGC target of 5 × 104 ions and maximum injection time of 250 ms.
The Q Exactive HF-X was set up for 60 K MS1 resolution with an AGC target of 3 × 106 ions and maximum injection time of 10 ms, followed by 20 MS2 scans with 45 K resolution, AGC target of 1 × 105 ions, and maximum injection time of 86 ms.
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