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Protein deconvolution 2

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Protein Deconvolution 2.0 is a software tool designed for the analysis and interpretation of mass spectrometry data. It provides advanced algorithms for deconvoluting and resolving complex protein spectra, allowing for the identification and characterization of individual protein components within a sample.

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Lab products found in correlation

Protein peptide editor module of biolynx, by Waters Corporation (2 mentions) Masslynx v4, by Waters Corporation (2 mentions) C18 ziptip, by Merck Group (2 mentions) Thermo orbitrap elite, by Thermo Fisher Scientific (1 mentions) Acquity uplc 1 class system, by Waters Corporation (1 mentions) Acquity uplc protein beh c4 column, by Waters Corporation (1 mentions) Molecular biology grade water, by Quality Biological (1 mentions) Fusion tribrid mass spectrometer, by Thermo Fisher Scientific (1 mentions) Rituxan, by Genentech (1 mentions) Reditux, by Roche (1 mentions) Ristova, by Roche (1 mentions) Remicade, by Johnson & Johnson (1 mentions) Q exactive orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions) Q exactive, by Thermo Fisher Scientific (1 mentions)

4 protocols using protein deconvolution 2

1

Intact Protein Mass Analysis by UPLC-MS

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The intact masses of the proteins were analyzed using a Waters ACQUITY I class UPLC system (Milford, MA) with an ACQUITY UPLC Protein BEH C4 column (2.1 mm × 100 mm, 1.7 μm particle size; Waters). The mobile phases were 0.1% formic acid in water (eluent A) and 0.1% formic acid in acetonitrile (eluent B). The gradient applied was: 0–3 min, 5% eluent B; 3–13 min, linear increase to 50% eluent B at 0.2 ml/min. The eluent was injected into a Thermo Orbitrap Elite (Thermo Fisher Scientific, Waltham, MA) and ionized with an electrospray source. MS spectra were acquired in the mass range of 400–2,000 m/z and 120,000 resolution at m/z 200. The deconvoluted mass spectra were generated using Protein Deconvolution 2.0 (Thermo Fisher Scientific, Waltham, MA).
Lee B.S., Choi W.J., Lee S.W., Ko B.J, & Yoo T.H. (2021). Towards Engineering an Orthogonal Protein Translation Initiation System. Frontiers in Chemistry, 9, 772648.
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2

Characterization of Monoclonal Antibody Drugs

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The formulated rituximab drug product vial, Rituxan® (Genentech), contains a protein concentration of 10 mg/mL in a 10 mL injection vial. The formulated infliximab drug product vial, Remicade ® (Janssen), contains 100 mg of lyophilized powder for reconstitution at 10 mg/mL; in our case by adding 10 mL molecular biology grade water (Quality Biological, Gaithersburg, MD). Roughly 20 mg protein per lot was used to obtain one complete dataset from the SEC-FPLC, SEC-HPLC-MALS, 1D 1H NMR and CD assays. The amount of protein needed can be lowered significantly if analytical SEC-FPLC and an NMR cryogenic probe are used instead of the instruments applied here. In addition, the two non-US FDA approved Indian-sourced rituximab drug product vials, Reditux® (Dr. Reddy’s) and Ristova® (Roche), containing mAb concentrations of 10 mg/mL in a 10 mL injection vial were used for NMR data comparison only.
For intact mass spectrometry (MS), around 0.3 mg of the mAbs were buffer exchanged from formulation into an ElectroSpray Ionization (ESI) compatible buffer (40% acetonitrile and 1% formic acid in water). Intact MS data were collected using a Fusion Tribrid Mass Spectrometer (Thermo Scientific, Bremen, Germany). The molecular weight was determined using the ReSpect algorithm within Protein Deconvolution 2.0 (Thermo Scientific). The detail experimental procedure is in the supporting information.
Chen K., Long D.S., Lute S.C., Levy M.J., Brorson K.A, & Keire D.A. (2016). Simple NMR methods for evaluating higher order structures of monoclonal antibody therapeutics with quinary structure. Journal of pharmaceutical and biomedical analysis, 128, 398-407.
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3

Legumain and Kgp Protease Interactions

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Legumain was incubated with Mcp1a or Clt2 in a 1:1 molar ratio in a buffer composed of 20 mM citric acid pH 4 and 50 mM NaCl for 30 min at 37 °C. Subsequently, samples were analyzed by SDS-PAGE and mass spectrometry, utilizing an ESI-Orbitrap setup. Similarly, Kgp was incubated with Mcp1a-N74K or Mcp1a-I72A-N74K in a 1:10 molar ratio in Kgp assay buffer at 37 °C for 3 h and analyzed by SDS-PAGE and mass spectrometry. For mass spectrometric analysis, samples were desalted with C18 ZipTips (Merck Millipore), eluted from the tips with 50% acetonitrile in 0.1% formic acid and directly infused into the mass spectrometer (Q Exactive Orbitrap mass spectrometer, Thermo Fisher Scientific) at a flow rate of 1 μl/min. Capillary voltage at the nanospray head was 2 kV. Raw data were processed with Protein Deconvolution 2.0 (Thermo Fisher Scientific). Masses were assigned to the protein sequence with the Protein/Peptide Editor module of BioLynx (part of MassLynx V4.1, Waters).
Elamin T., Santos N.P., Briza P., Brandstetter H, & Dall E. (2022). Structural and functional studies of legumain–mycocypin complexes revealed a competitive, exosite-regulated mode of interaction. The Journal of Biological Chemistry, 298(10), 102502.
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4

Mass Spectrometry Analysis of AtCYT6-AtLEGβ Interaction

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AtCYT6‐derived constructs were incubated with AtLEGβ in a 1:5 molar ratio in a buffer composed of 100 mm citric acid pH 4.0 or 5.5, 100 mm NaCl, and 2 mm DTT at 21°C for 30 min. For mass spectrometric analysis, samples were desalted with C18 ZipTips (Merck, Darmstadt, Germany), eluted from the tips with 50% acetonitrile in 0.1% formic acid, and directly infused into the mass spectrometer (Q‐Exactive; Thermo Fisher Scientific, Waltham, Massachusetts) at a flow rate of 1 μl min−1. Capillary voltage at the nanospray head was 2 kV. Raw data were processed with Protein Deconvolution 2.0 (Thermo Fisher Scientific, Waltham, Massachusetts). Masses were assigned to the protein sequence with the Protein/Peptide Editor module of BioLynx (part of MassLynx V4.1; Waters, Eschborn, Germany).
Santos N.P., Soh W.T., Demir F., Tenhaken R., Briza P., Huesgen P.F., Brandstetter H, & Dall E. (2023). Phytocystatin 6 is a context‐dependent, tight‐binding inhibitor of Arabidopsis thaliana legumain isoform β. The Plant Journal, 116(6), 1681-1695.
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