Aliquots of the tryptic digests of STAT3 proteins treated with DMSO (control) and those treated with Stattic and TTI-101 were analyzed by LC-MS/MS on an Ultimate 3000 RSLC-Nano chromatograph interfaced to an Orbitrap Fusion high-resolution mass spectrometer (Thermo Scientific, Waltham MA). All MS/MS data were analyzed using Sequest-HT (Thermo Scientific). Proteins were identified by searching their fragment spectra against the Swiss-Prot protein database (EBI). The iodoacetamide derivative of cysteine, stattic adducts of cysteine, and the predicted TTI-101 adducts of cysteine were specified as variable modifications. To access for potential unknown modifications, the data was analyzed in MaxQuant using the dependent peptide search option [12 (link)]. An all-peptides output list was analyzed by comparing Stattic, TTI-101 and TTI-101ox with DMSO treated samples, as described in [13 ].
Ultimate 3000 rslc nano chromatograph
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ultimate 3000 RSLC-Nano chromatograph is a compact, high-performance liquid chromatography system designed for nano-scale separations. It features a robust design, advanced flow path, and high-precision pumps to deliver accurate and reproducible results in a wide range of applications.
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5 protocols using ultimate 3000 rslc nano chromatograph
1
Quantitative Proteomics of STAT3 Modifications
2
SILAC Protein Identification by Orbitrap
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SILAC samples were separated using UltiMate 3000 RSLCnano chromatograph (Thermo Fisher Scientific, Massachusetts, USA). Peptides were loaded on a pre-column (μ-precolumn, 30 mm i.d., 5 mm length, C18 PepMap 100, 5 μm particle size, 100 Å pore size) and further separated on an Acclaim PepMap RSLC column (75 mm i.d., length 500 mm, C18, particle size 2 mm, pore size 100 Å) with a 300 nl/min flow rate using a linear gradient: 2% B over 4 min, 2–40% B over 64 min, 40–98% B over 2 min, with A = 0.1% aq. formic acid and B = 80% AcN in 0.08% aq. formic acid. Peptides eluting from the column were introduced into an Orbitrap Elite (Thermo Fisher Scientific, Massachusetts, USA) operating in Top20 data dependent acquisition mode. A survey scan of 400–2000 m/z was performed in the Orbitrap at 120000 resolution with an AGC target of 1 × 106 and 200 ms injection time followed by twenty data-dependent MS2 scans performed in the LTQ linear ion trap with 1 microscan, 10 ms injection time and 10,000 AGC.
3
Quantitative Proteomics by Liquid Chromatography
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Tryptic peptides from isotopically labeled cells were separated using an UltiMate 3000 RSLCnano chromatograph (Thermo Fisher Scientific, Waltham, MA, USA). Tryptic peptides were loaded onto a precolumn (μ-precolumn, 30 µm i.d., 5 mm length, C18 PepMap 100, 5 µm particle size, 100 Å pore size) and separated using an Acclaim PepMap RSLC column (75 µm i.d., length 500 mm, C18, particle size 2 µm, pore size 100 Å). Tryptic peptides were separated by a linear gradient of mobile phase B (B = 80% (v/v) acetonitrile (ACN), 0.08% (v/v) formic acid (FA) in water) and A (A = 0.1% (v/v) FA in water) as follows: 2% B over 4 min, 2–40% B over 64 min, and 40–98% B over 2 min. The flow rate was 300 nL/min. Tryptic peptides eluting from the column were injected into an Orbitrap Elite (Thermo Fisher Scientific, Waltham, MA, USA) operating in Top20 data-dependent acquisition mode. Scanning was set to 400–2000 m/z, performed at a 120,000 resolution. The AGC target was 1 × 106 with a 200 ms injection time and twenty data-dependent MS2 scans (1 microscan, 10 ms injection time and 10,000 AGC).
4
Quantitative Proteomics of STAT3 Modifications
5
Glycopeptide Analysis by LC-MS/MS
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LC-MS/MS analysis was performed using an Ultimate 3000 RSLCnano chromatograph and Orbitrap Fusion Lumos Mass Spectrometer platform (Thermo) with a multi-nozzle emitter (NEWOMICS, Berkeley, CA, USA) used as the microflow sprayer. Glycopeptide separation was achieved in microflow mode using an Acclaim PepMap 100 capillary column 75 μm ID × 20 mm length, packed with C18 5 μm, 300 Å (Thermo). Glycopeptides were separated as follows: starting condition flow 5 µL, 2% ACN, 0.1% formic acid; 0–1 min flow 5 µL, 2% ACN, 0.1% formic acid; 1–2 min flow 1.5 µL, 2–5% ACN, 0.1% formic acid; 2–5 min flow 1.5 µL, 5–98% ACN, 0.1% formic acid; 7–9 min flow 1.5 µL, 98% ACN, 0.1% formic acid; followed by equilibration to starting conditions for an additional 4 min (Supplementary Figure S1 ).
We used a Parallel Reaction Monitoring (PRM) workflow with one MS1 full scan (400–1800 m/z, resolution 120 K, max IT 50 ms) and scheduled MS/MS fragmentation (Isolation window m/z 2.0, HCD fragmentation, resolution 30 K, scan range 200–1400, RF Lens 55%) for the analysis of the sialylated O-HPX glycopeptide TPLPPTSAHGNVAEGETKPDPVTER (Table 1 ).
We used a Parallel Reaction Monitoring (PRM) workflow with one MS
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