Total IgG1 antibodies were captured from 2 μl of serum using Protein G Sepharose 4 Fast Flow beads (GE Healthcare, Uppsala, Sweden) in a 96-well filter plate (Millipore Multiscreen, Amsterdam, the Netherlands) as previously described (12 (link)) or by using Protein G cartridges on the AssayMAP Bravo (Agilent Technologies, Santa Clara, USA). Briefly, 1 μl of serum diluted in PBS was applied to the cartridges, followed by washes with PBS and LC-MS pure water. IgG antibodies were then eluted with 1% formic acid.
Assaymap bravo
Manufactured by Agilent Technologies
Sourced in United States
The AssayMAP Bravo is a high-throughput, automated liquid handling platform designed for sample preparation and assay workflows. It features a modular design and flexible configuration options to accommodate a variety of laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Peptide desalted columns, by Thermo Fisher Scientific (2 mentions)
S trap, by Protifi (2 mentions)
Bca protein assay, by Thermo Fisher Scientific (2 mentions)
Protein g sepharose 4 fast flow beads, by GE Healthcare (1 mentions)
Easylc 1200, by Thermo Fisher Scientific (1 mentions)
Q exactive hf x, by Thermo Fisher Scientific (1 mentions)
Zeba spin desalting column, by Thermo Fisher Scientific (1 mentions)
Nanodrop uv spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Mass spec grade trypsin lys c, by Promega (1 mentions)
Trypsin lys c mix, by Promega (1 mentions)
Dtag 13 degrader compound, by Bio-Techne (1 mentions)
General plasticware, by Corning (1 mentions)
Assaymap plasticware, by Agilent Technologies (1 mentions)
Ethylene glycol, by Merck Group (1 mentions)
Assaymap c18 cartridges, by Agilent Technologies (1 mentions)
12 protocols using assaymap bravo
1
Capturing Total IgG1 Antibodies
2
Phosphoproteomic Profiling of Protein
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Pelleted cells were solubilized in 10% SDS; 100mM TEAB, probe sonicated, vortexed, and centrifuged to remove insoluble material. Total protein was quantified by Micro BCA (Pierce). 1.5 mg protein was digested with trypsin on Straps (Protifi), and desalted on Peptide Desalted Columns (ThermoFisher Scientific). Phosphopeptides were enriched with Fe cartridges on an AssayMAP Bravo (Agilent). Phosphopeptide enrichments were loaded onto an EASY C18, 1.7μm 2.1 × 50cm column at 300 nL/min with an UltiMate™ 3000 RSLCnano HPLC system, eluted over a 120-min gradient, and analyzed on Orbitrap Eclipse™. The instrument was operated in MS2. MS1 spectra were acquired at a resolving power of 120,000. MS2 spectra acquired in the Orbitrap with CID normalized collision energy = 38. Dynamic exclusion was enabled to minimize the redundant selection of peptides previously selected for MS/MS. Phosphopeptides were identified (FDR 0.05) and quantified in Proteome Discoverer (2.5).
3
Phosphopeptide Enrichment and MS Analysis
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Mass spectrometric measurements were performed on a Q Exactive HFX mass spectrometer coupled to an EasyLC 1200 (Thermo Fisher Scientific). Before analysis, phosphopeptides were enriched on Automated Liquid Handling Platform (AssayMap Bravo; Agilent) using Fe(III)-NTA cartridges (5 μl) essentially as described previously (Post et al, 2017 (link)). The MS raw data files were analyzed by MaxQuant software (Cox & Mann, 2008 (link)), version 1.4.1.2, using a UniProt Saccharomyces cerevisiae database from March 2016 containing common contaminants such as keratins and enzymes used for in-gel digestion.
4
Phosphoproteomic Profiling of Protein
5
Protein Extraction and Digestion
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The protocols for protein extraction and digestion have been previously described [59 (link)]. Protein extraction was performed on sectioned, fresh-frozen human metastatic tissue (10 µm) using the Bioruptor plus, model UCD-300 (Dieagenode). Protein digestion and peptide desalting were performed on the AssayMAP Bravo (Agilent Technologies, Lexington, MA, USA) platform with the urea solution digest and peptide cleanup v2.0 protocols.
6
Automated Monoclonal Antibody Purification
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Purified monoclonal antibody was obtained through use of the Agilent AssayMAP Bravo automated liquid handling platform. Briefly, approximately 100 µg of protein was loaded onto a 5 µL bed of Protein A resin (Agilent, Cat. G5496-60000) pre-equilibrated with 75 mM phosphate, 100 mM sodium chloride, pH 7.4. The resin was washed sequentially with 50 mM Bis-Tris, 1 M sodium chloride, pH 7.4, and 25 mM Bis-Tris, pH 5.9. Finally, the monoclonal antibody was eluted in a 20 µL volume with 75 mM acetate, pH 3.3, immediately neutralized with 2.4 M Tris, pH 8.0, and buffer exchanged into 100 mM Tris-HCl, pH 7.5, using a 7 kDa MWCO Zeba spin desalting column (Thermo Fisher Scientific, Cat. 89882).
7
Phosphopeptide Enrichment by IMAC
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Each fraction was reconstituted in 100 μl of 80% acetonitrile in 0.1% TFA and transferred to a 96-well plate. IMAC-based phosphopeptide enrichment was performed using Fe(III)-NTA resin (G5496-60082) on an AssayMAP Bravo (Agilent Technologies) platform following the manufacturer's instructions.
8
Peptide Fractionation and LC-MS Analysis
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Peptide concentration was determined using a NanoDrop™ UV spectrophotometer (Thermo Scientific™, Waltham, MA, USA). A total of 100 μg of peptides was fractionated by basic pH reversed-phase material (RPS cartridge tips; 5 μL PS-DVB resin, Agilent, Santa Clara, CA, USA) into six fractions using the Agilent AssayMAP Bravo pipetting system. The RPS cartridges were primed, washed and equilibrated according to the manufacturer’s protocol. Peptides were reconstituted in 100 μL of 25 mM ammonium formate (pH 10) and loaded onto the cartridges. Peptides were fractionated by increasing acetonitrile concentrations (5%, 10%, 15%, 20%, 25%, 30%, 80%). The seven elution steps were combined into 6 fractions, combining the 5% and 80% fractions. All fractions were acidified with formic acid to a final concentration of 1%. The fractionated peptides were dried down in the speed-vac and stored at −20 °C until MS measurement. Before analysis by LC-MS, peptides were dissolved in 0.1% formic acid and were spiked with retention time standard peptides PROCAL [33 (link)] at 100 fmol per injection.
9
CSF Protein Digestion for Mass Spec
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In solution digest of CSF was performed on an AssayMap Bravo liquid handling instrument (Agilent) using a custom designed protocol. 100uL CSF, to which 5 μL Pierce digest indicator (Thermo 84841) is added to a final concentration of 0.15 μg. DTT is added to a concentration of 6.4 mM and heated at 60°C for 30 min. The samples are cooled for an additional 30 min followed by the addition of 12 mM iodoacetamide at room temperature for 30 min. 2 μg Mass Spec grade Trypsin/LysC (Promega) is added to each sample and incubated at 37°C overnight. The digestion is halted by acidifying the samples to 12.5% formic acid.
10
Mass Spectrometry-based Proteomics of Mouse Splenocytes
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