The bacterial proteins for the LC/MS/MS analyses were extracted from 16-h cultures of WT-UTI89 and Δspr-UTI89. Three independent cultures for each strain were prepared from the analyses. The bacteria were collected from the cultures by centrifugation and resuspended in Tris-HCl buffer (50 mM, pH 7.5). The bacterial suspensions were subjected to French press at 8,000 lb/in2 followed by centrifugation at 6,000 g for 10 min to pellet and remove unbroken cells. The resulting supernatants were centrifuged at 100,000 g at 4°C for 1 h to pellet envelope fractions. The envelope fractions were resuspended in Tris-HCl buffer, and then subjected to 12.5% SDS-PAGE to separate the proteins in the samples. The gel lane of each sample was cut into two slices and was subjected to in-gel digestion with trypsin followed by protein identification with the Thermo LTQ-Orbitrap Velos system. The MS/MS spectra were searched against Escherichia coli SwissProt 2018_01 (556,568 sequences; 199,530,821 residues) using Proteome Discoverer 2.2 (Thermo Fisher, United Kingdom). The LC/MS/MS raw data are shown in Supplementary Table S2 . Subsequently, the protein identifications with two peptides in at least one of the samples were retained. The proteins exhibiting at least a 2-fold significance difference between WT-UTI89 and Δspr-UTI89 are shown in Table 2 .
Ltq orbitrap velos system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LTQ-Orbitrap Velos system is a high-performance mass spectrometer designed for advanced proteomics and metabolomics applications. It combines the sensitivity and resolution of the Orbitrap analyzer with the versatility of the linear ion trap. The system is capable of performing high-resolution, accurate-mass measurements and tandem mass spectrometry (MS/MS) analysis.
Automatically generated - may contain errors
Lab products found in correlation
Proteome discoverer 2, by Thermo Fisher Scientific (1 mentions)
Ultimate 3000 system, by Thermo Fisher Scientific (1 mentions)
Pro q emerald 300 glycoprotein gel stain kit, by Thermo Fisher Scientific (1 mentions)
Nupage bis tris precast gel, by Thermo Fisher Scientific (1 mentions)
Instantblue, by Abcam (1 mentions)
Mascot, by Matrix Science (1 mentions)
Easy nlc 2, by Thermo Fisher Scientific (1 mentions)
Sequencing grade modified trypsin, by Promega (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Nanoacquity system, by Waters Corporation (1 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (1 mentions)
Mascot algorithm, by Matrix Science (1 mentions)
Nanoacquity lc system, by Waters Corporation (1 mentions)
Kinetex evo c18 column, by Phenomenex (1 mentions)
Accela uplc, by Thermo Fisher Scientific (1 mentions)
Luna c18 column, by Phenomenex (1 mentions)
7 protocols using ltq orbitrap velos system
1
Profiling Bacterial Envelope Proteins
2
SFV1 Virion Proteomics Analysis
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The highly purified virions were analyzed by 4–12% gradient NuPAGE Bis-Tris precast gel (Thermo Fisher Scientific). Proteins were stained with Coomassie blue using InstantBlue (Expedeon), and the stained protein bands were excised from the gel. Proteins were in-gel tryptic digested, and the generated peptides were analyzed by nano-LC-MS/MS (Proteomics Platform, Institut Pasteur, France) using an Ultimate 3000 system (Dionex) coupled to an LTQ-Orbitrap Velos system (Thermo Fisher Scientific). Peptide masses were searched against annotated SFV1 proteins using Andromeda43 (link) with MaxQuant software, version 1.3.0.543 (link). Glycosylation of VPs was detected by using a Pro-Q Emerald 300 glycoprotein gel stain kit (Thermo Fisher Scientific).
3
Peptide Identification by Mass Spectrometry
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Peptide identification by MS was performed by the Mass Spectrometry Common Facility at the Institute of Biological Chemistry, Academia Sinica, using an LTQ-Orbitrap Velos system (Thermo Fisher Scientific, Waltham, MA, USA). Data interpretation and correlations between the spectra and amino acid sequences within a human EST database and customized FLAG-Trim28 sequence were analyzed using Mascot (Matrix Science software package).
4
Proteomic Profiling of Biological Samples
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Total proteins from three biological replicates were reduced with dithiothreitol (DTT), alkylated with iodoacetamide (Sigma Aldrich), and digested with trypsin (Promega Modified Trypsin Sequencing Grade). The resulting peptides were applied to a pump LC-MS nanoflow EASY-nLC II instrument coupled to a mass spectrometer LTQ Orbitrap-Velos system with nano-electrospray ionization (Thermo Fisher Scientific Co., San Jose, CA). To validate MS/MS-based peptide and protein identifications, algorithms, and tools were used as previously reported [41 (link)] and are described in the following sections.
5
Isolation and Analysis of AGO2 Complexes
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Nuclear extracts from 11 g of 0–24 h OR embryos were prepared as previously described [26 (link)] and lysed in 2.5 mL HBSMT-0.3% + 1 M KCl (50 mM HEPES, 150 m MNaCl, 1 M KCl, 3 mM MgCl2, 0.3% Triton X-100 [v/v] at pH 7) including 1 mM PMSF, and Complete protease inhibitor cocktail (Roche). For AGO2 immunoaffinity purification, 6 mL of 9D6 tissue culture supernatant was covalently crosslinked to rProtA sepharose beads using 20 mM dimethylpimelimidate in 0.2 M sodium borate, pH 9.0 quenched with 0.2 M ethanolamine, pH 7.9. For control sample, 2.4 μg normal mouse IgG (Santa Cruz) was used. Crosslinked beads were incubated with 5.5 mg nuclear lysate overnight at 4°C and washed as described previously [1 (link)]). Samples were eluted in formic acid, trypsin digested, subjected to ultraperformance liquid chromatography on a NanoAcquity system (Waters), and analyzed on an LTQ-Orbitrap-Velos system (Thermo) at the NIDDK Mass Spectrometry Facility. Results were analyzed by Mascot algorithm (Matrix Science).
6
Catabolic Profiling of Polatuzumab Vedotin
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To characterize the catabolic profile of polatuzumab vedotin conjugated with [3H]-MMAE, bile and urine samples from the study with [3H]-MMAE polatuzumab vedotin were pooled across time points that covered >90% of the radioactivity excreted in that route and were investigated using a nanoACQUITY LC system (Waters, MA, USA) coupled with an LTQ-Orbitrap Velos system (Thermo Scientific, San Jose, CA) and an online β-RAM 5C radiodetector (Lab Logics, Brandon, FL, USA) for radioprofiling.
Chromatographic separation was performed on a Kinetex EVO C18 column (100 × 2 mm, 1.7 µm particle size, Phenomenex, Torrance, CA, USA) with mobile phases A (0.1% formic acid in water with 10 mM ammonium acetate) and B (0.1% formic acid in 90% acetonitrile in water with 10 mM ammonium acetate) at a constant flow rate of 0.1 mL/min. The gradient was as follows: initial holding at 10% B for 2 min, increased to 22% B at 4 min, 58% B at 45 min, 95% B at 50 min, holding at 95% B until 54 min, decreasing to 10% B at 54.5 min, and then column re-equilibration until 60 min. The flow was split 4:1 post-column for the radiomeasurements and mass spectrometry, respectively.
Chromatographic separation was performed on a Kinetex EVO C18 column (100 × 2 mm, 1.7 µm particle size, Phenomenex, Torrance, CA, USA) with mobile phases A (0.1% formic acid in water with 10 mM ammonium acetate) and B (0.1% formic acid in 90% acetonitrile in water with 10 mM ammonium acetate) at a constant flow rate of 0.1 mL/min. The gradient was as follows: initial holding at 10% B for 2 min, increased to 22% B at 4 min, 58% B at 45 min, 95% B at 50 min, holding at 95% B until 54 min, decreasing to 10% B at 54.5 min, and then column re-equilibration until 60 min. The flow was split 4:1 post-column for the radiomeasurements and mass spectrometry, respectively.
7
Metabolic Profiling of [3H]-MMAE
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To characterize the metabolic profile of [3H]-MMAE, the bile samples were analyzed across time points that covered the majority of the radioactivity (up to 6 h with 75% of the total radioactivity injected) excreted. Due to the low radioactivity recovered in the urine, the urine samples were analyzed for up to 8 h. Both the bile and urine samples were investigated using Accela UPLC coupled with a Linear Trap Quadrupole (LTQ)-Orbitrap Velos system (Thermo Scientific, San Jose, CA, USA) and an online β-RAM 5C radiodetector (Lab Logics, Tampa, FL, USA) for radioprofiling.
Chromatographic separation was performed on a Luna C18 column (150 × 4.6 mm, 3 μm particle size, Phenomenex, Torrance, CA, USA) with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at a constant flow rate of 1 mL/min. The gradient was as follows: initial holding at 5% B for 2 min, increased to 15% B at 4 min, 42% B at 44 min, 75% B at 49 min, 95% B at 50 min, holding at 95% B until 55 min, decreasing to 5% B at 55.1 min, and then column re-equilibration until 60 min. The flow was split 3:1 post-column for radiomeasurements and mass spectrometry, respectively. As the majority of the metabolite was the intact [3H]-MMAE, only the intact [3H]-MMAE was quantified.
Chromatographic separation was performed on a Luna C18 column (150 × 4.6 mm, 3 μm particle size, Phenomenex, Torrance, CA, USA) with mobile phases A (0.1% formic acid in water) and B (0.1% formic acid in acetonitrile) at a constant flow rate of 1 mL/min. The gradient was as follows: initial holding at 5% B for 2 min, increased to 15% B at 4 min, 42% B at 44 min, 75% B at 49 min, 95% B at 50 min, holding at 95% B until 55 min, decreasing to 5% B at 55.1 min, and then column re-equilibration until 60 min. The flow was split 3:1 post-column for radiomeasurements and mass spectrometry, respectively. As the majority of the metabolite was the intact [3H]-MMAE, only the intact [3H]-MMAE was quantified.
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