BCN–EVCit–PABC–MMAF (20.7 μLof 3.7 mM stock solution in DMSO, 1.5 equivalent per azide group) was added to a solution of the mAb–linker conjugate in PBS (460 μL, 4.16 mg/mL), and the mixture was incubated at room temperature for 22 h. The reaction was monitored using either Agilent LC-MS system or Thermo LC-MS system equipped with a MabPac RP column (see above) and the crude products were purified by SEC to afford homogeneous ADC 1 (1.71 mg, 90% yield determined by BCA assay). Analysis and purification conditions were the same as described above. Homogeneity was confirmed by ESI-MS analysis. Homogeneous anti-EGFRvIII ADC 4 and anti-HER2 ADC 6 were prepared in the same manner.
Lc ms system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The LC-MS system is an analytical instrument that combines liquid chromatography (LC) and mass spectrometry (MS) technologies. The core function of the LC-MS system is to separate, identify, and quantify compounds in complex mixtures by coupling the high-performance liquid chromatography (HPLC) separation with the sensitive and selective mass spectrometric detection.
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Lab products found in correlation
Mabpac rp column, by Thermo Fisher Scientific (2 mentions)
Mini protean tgx precast protein gel, by Bio-Rad (1 mentions)
Autosampler, by Thermo Fisher Scientific (1 mentions)
Acquity hss t3 column, by Waters Corporation (1 mentions)
Antibiotics for cell culture, by Thermo Fisher Scientific (1 mentions)
Actin, by Santa Cruz Biotechnology (1 mentions)
Pgc 1α, by Santa Cruz Biotechnology (1 mentions)
P akt, by Santa Cruz Biotechnology (1 mentions)
Prep1, by Santa Cruz Biotechnology (1 mentions)
Protein electrophoresis and real time pcr reagents, by Bio-Rad (1 mentions)
Western blotting and ecl reagents, by Cytiva (1 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
Easy nlc 1200 lc system, by Thermo Fisher Scientific (1 mentions)
11 protocols using lc ms system
1
Antibody-Drug Conjugates Synthesis
2
Antibody-Drug Conjugates Synthesis
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BCN–EVCit–PABC–MMAF (20.7 μLof 3.7 mM stock solution in DMSO, 1.5 equivalent per azide group) was added to a solution of the mAb–linker conjugate in PBS (460 μL, 4.16 mg/mL), and the mixture was incubated at room temperature for 22 h. The reaction was monitored using either Agilent LC-MS system or Thermo LC-MS system equipped with a MabPac RP column (see above) and the crude products were purified by SEC to afford homogeneous ADC 1 (1.71 mg, 90% yield determined by BCA assay). Analysis and purification conditions were the same as described above. Homogeneity was confirmed by ESI-MS analysis. Homogeneous anti-EGFRvIII ADC 4 and anti-HER2 ADC 6 were prepared in the same manner.
3
Protein Coding Potential Assessment
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To assess the protein coding potential of Ntep potential, ORFs were calculated using the given sequence for Ntep from RefSeq (Supplementary Figure S6 ). We used two independent tools to enhance the quality of predictions; Sequence Manipulation Site: ORF Finder21 and Coding Potential Calculator.22 (link) To check 3T3 cells for potential peptides arising from the ORF, 3T3 cells stably overexpressing Ntep (3T3 pLV+Ntep) or harbouring a control vector (3T3 pLV+empty) were collected and the protein content isolated according to standard conditions. The protein lysate were alkylated using acrylamide (40%, 4K solution; Applichem, Darmstadt, Germany) for the purpose of mass spectrometry analysis. Thirty micrograms of each lysate were loaded onto 4–15% Mini-PROTEAN TGX Precast Protein Gels (Bio-Rad) and separated using SDS-PAGE. Proteins were stained using Coomassie Brillant Blue G250 (Thermo Fisher Scientific) according to standard procedure. The stained SDS-gel was further processed for mass spectrometry analysis at the MS Core Facility Proteomics at the Hannover Medical School. Therefore, the proteins samples in the designated range (Supplementary Figure S6 ) were in-gel digested and the harbouring peptides were analysed using the LC-MS System (Thermo Fisher Scientific, Waltham, MA, USA). Resulting data were searched for the predicted peptides against an in-house database.
4
Anti-EGFR mAb Conjugation and Characterization
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Aglycosylated anti-EGFR mAb (105 μL in PBS, 3.0 mg/mL, 315 μg antibody) was mixed with 1 M phosphate solution at pH 9 (10.5 μL) and MMAF-NHS (2.5 μL of 10mM stock solution in DMSO, 12 equiv.) and the mixture was incubated at room temperature for 3 h. The reaction was monitored using either Agilent LC-MS system or Thermo LC-MS system equipped with a MabPac RP column (see above). The crude products were purified by SEC to afford Lys conjugate 3 (197 μg, 63% yield determined by BCA assay, average DAR: 3.9). Analysis and purification conditions were the same as described above. The average DAR value was determined based on ion intensity of each DAR species in ESI-MS analysis. Heterogeneous anti-EGFRvIII ADC 5 and anti-HER2 ADC 7 were constructed in the same manner.
5
Synthesis and Characterization of PS5 Peptide
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Both PS5 and negative control (NC) peptides were synthesized on a 50 μmol scale following standard Fmoc procedures of SPPS, as already reported [45 (link)]. The molecular weight of the peptides and the purity of fractions were evaluated by LC-MS analysis. To perform cellular assays, both peptides were linked to the fragment 48–60 of the HIV Tat protein at the N-terminus of the peptides and labelled with carboxytetramethylrhodamine (TAMRA) fluorophore. Purified peptides were lyophilized, then dissolved in a water solution at 1 mM stock concentration, filter-sterilized and stored aliquoted at −20 °C under N2 until further use. Reagents for peptide synthesis were from Iris Biotech (Germany); reversed phase columns for peptide analysis and the LC-MS system were from ThermoFisher (Waltham, MA); solvents for peptide synthesis and HPLC were from Romil (Dublin, Ireland).
6
Anti-EGFR mAb Conjugation and Characterization
7
Plasma Metabolite Extraction Protocol
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Plasma deproteinization was performed by adding methanol containing IS (as internal standards) to plasma. Then, samples were mixed vigorously and centrifuged. The collected supernatant was transferred to an autosampler (San Jose, CA, USA) and injected into a LC/MS system (Thermo Scientific, Milford, CT, USA).
8
Amyloid Peptide Synthesis and Preparation
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Amyloid peptides analyzed in this study were synthesized as already reported [35 (link),43 (link)]. Their sequences are reported in Table 1 . Reagents for peptide synthesis were from Iris Biotech (Marktredwitz, Germany), solvents for peptide synthesis and HPLC analyses were from Romil (Dublin, Ireland); reversed phase columns for peptide analysis and the LC-MS system were from ThermoFisher (Waltham, MA, USA). Peptides’ purity and identity were confirmed by LC-MS. Purified peptides were lyophilized and stored at −20 °C until use. Prior to be analyzed they were all treated for 30 min with HFIP to ensure a monomeric state (at 50% (v/v) in water), and then the organic solvent was removed by evaporation.
9
Metabolic Kinetics of TA Esters
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The metabolic kinetics of ester bonds of TA6, TA8, and TA9, in normal saline (NS), phosphate-buffer solution (PBS, pH7.4), and rat plasma were assessed. TA6, TA8, or TA9 (0.1 mM) were dissolved in 1 mL NS or PBS and then incubated at 37 °C, respectively. Then, 50 μL of the incubated solutions were collected at 4 h, 12 h, and 24 h for further analysis of TA6, TA8, and TA9. For the metabolic study in rat plasma, 0.1 mL of the TA6, TA8, or TA9 solutions (1 mM/L) were incubated with 1.9 mL of fresh rat plasma at 37 °C. Afterward, 20 μL of the mixtures were collected and added into 40 μL glacial acetonitrile to terminate reactions at the specified time. The resulting solutions were centrifuged at 15,000 g for 10 min at 4 °C, and the supernatants were analyzed.
The collected samples from NS, PBS, and rat plasma metabolic incubation mixtures were analyzed using an LC-MS system with a heated electrospray ionization (HESI) source (Thermo Fisher, Germany). The samples were separated with an Acquity HSS T3 column (100 mm × 2.1 mm, 1.8 mm, Waters, Ireland), which was maintained at 45 °C. The mobile phase was 0.2% formic acid aqueous solution and acetonitrile, and the flow rate was set as 0.4 mL/min.
The collected samples from NS, PBS, and rat plasma metabolic incubation mixtures were analyzed using an LC-MS system with a heated electrospray ionization (HESI) source (Thermo Fisher, Germany). The samples were separated with an Acquity HSS T3 column (100 mm × 2.1 mm, 1.8 mm, Waters, Ireland), which was maintained at 45 °C. The mobile phase was 0.2% formic acid aqueous solution and acetonitrile, and the flow rate was set as 0.4 mL/min.
10
Peptide Synthesis Protocol Optimization
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Media, sera, antibiotics for cell culture and the Lipofectamine reagent were all from Invitrogen (Grand Island, NY, USA). Ceramide D-erythro-Sphingosine, N-Acetyl- (C2 Ceramide), pY and IRS1 antibodies were from Merck Millipore (Darmstadt, Germany). Cycloheximide (CHX), palmitate and Biotin Reagents were obtained from Sigma-Aldrich (Darmstadt, Germany). Prep1, PGC-1α, pAkt, Akt and actin antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). IR antibody was from Cell Signaling Technology (Beverly, MA, USA). p160 antibody was purchased from Zymed Laboratories (San Francisco, CA, USA). Glucose Transporter 4 (GLUT4) was from Abcam In. (Cambridge, MA). Protein electrophoresis and real-time PCR reagents were from Bio-Rad (Richmond, VA, USA). Western blotting and ECL reagents were purchased from Amersham Biosciences (Arlington Heights, IL, USA). Reagents for peptide synthesis including Fmoc-protected amino acids and resins, activation and deprotection reagents, were purchased from Calbiochem-Novabiochem (Laufelfingen, Switzerland) and Sigma-Aldrich (Darmstadt, Germany). Solvents for peptide synthesis and HPLC analyses were purchased from Delchimica (Naples, Italy); C18 Biobasic columns for peptide analysis and the LC-MS system were from ThermoFisher (Milan, Italy).
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