Mitochondrial HspB2 was immunopurified from mice harboring either the wild type HSPB2 (HSPB2wt), transgenic cardiac HSPB2 (HSPB2cTg), or an HSPB2 cardiac knockout (HSPB2cKO, [9 (link)]). For each group, mitochondria from four mouse hearts were combined, lysed in 0.1% NP-40 homogenization buffer, and 2 mg was incubated with anti-HspB2 antibodies [9 (link)]. The IP eluates were then fractionated on SDS-PAGE, excised and analyzed by LC-MS/MS. Samples had one biological replicate and two technical replicates. Technical replicates showed greater than 90% overlap. The raw data were analyzed by BioWorks (ThermoFisher Scientific, version 3.3.1 SP1) and proteins were identified using SEQUEST (ThermoFisher Scientific, version 3.3.1) and Scaffold (Proteome Software, version 3.3.3). At least two peptides and 99.0% protein confidence were required for protein algorithms; the global false discovery rate was approximately 0.1%.
Bioworks
Manufactured by Thermo Fisher Scientific
BioWorks is a comprehensive suite of software solutions designed to streamline laboratory operations. It provides tools for data management, instrument control, and workflow automation, enabling efficient and reliable execution of scientific experiments.
Automatically generated - may contain errors
Lab products found in correlation
Lxq linear ion trap mass spectrometer, by Thermo Fisher Scientific (4 mentions)
Accela lc system, by Thermo Fisher Scientific (4 mentions)
C18 omix tip, by Agilent Technologies (1 mentions)
Orbitrap xl, by Thermo Fisher Scientific (1 mentions)
Nanolc 2d, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Ltq orbitrap hybrid mass spectrometer, by Thermo Fisher Scientific (1 mentions)
Picofrit c18 reverse phase column, by New Objective (1 mentions)
Sil 20ac autosampler, by Shimadzu (1 mentions)
7 protocols using bioworks
1
Proteomic Analysis of HspB2 Mitochondria
2
Endoglycosidase-Mediated Antibody Modification
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Reactions were set up using 5 nM EndoS or EndoS mutants, or 100 nM EndoBT and 5 µM Rituximab or high-mannose-type IgG1 in PBS pH 7.4 at room temperature. Rituximab, a chimeric anti-human CD20 monoclonal antibody approved for treatment of B-cell lymphoma in adults, is produced in mammalian cell (Chinese Hamster Ovary) culture with the most abundant glycoforms being G0F, G1F, and G2F (antibody purchased from Premium Health Services, Inc.)57 (link). At various time intervals, 10 µl aliquots of the reaction were taken in duplicate and quenched with 1.1 µl of 1% trifluoroacetic acid. The quenched reaction was then mixed with 50 mM TCEP, and analyzed by LC-MS using an Accela LC System attached to a LXQ linear ion trap mass spectrometer (Thermo Scientific, Waltham, MA). Relative amount of the substrate and the hydrolysis products were quantified after deconvolution of the raw data and identification of the corresponding MS peaks using BioWorks (Thermo Scientific, Waltham, MA). The data were plotted in GraphPad Prism, and fit with a one-phase exponential decay curve.
3
Alanine Scan Mutants: Enzymatic Kinetics
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Reactions for the alanine scan mutants were set up using 100 nM EndoBT-3987WT for reactions with RNAseB and 500 nM for reactions with high-mannose IgG1. The enzymes were mixed with 5 μM substrate in PBS pH 7.4 at room temperature. For the alanine scan, 10 μl aliquots of the reaction were taken in triplicate and allowed to progress for 30 and 45 min for RNAseB and high-mannose IgG1 substrates, respectively. All reactions were quenched using 1.1 μL of 1% trifluoroacetic acid. The quenched reactions were then mixed with 50 mM TCEP and analyzed by LC-MS using an Accela LC System attached to a LXQ linear ion trap mass spectrometer (Thermo Scientific, Waltham, MA). Relative amounts of the substrate and hydrolysis products were quantified after deconvolution of the raw data and identification of the corresponding peaks using BioWorks (Thermo Scientific, Waltham, MA). The data were plotted and statistical significance was determined using a multiple comparisons test (Tukey method) in GraphPad (GraphPad Software, LaJolla, CA).
4
Shotgun Proteomics Protocol for Identification
5
LC-MS/MS Proteomic Workflow for Protein Identification
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LC-MS/MS experiments were performed on LTQ-Orbitrap hybrid mass spectrometer (Thermo Fisher Scientific, Inc.) coupled to a Shimadzu LC-20AD LC system (Shimadzu Corporation) and SIL-20AC autosampler (Shimadzu Corporation). Tryptic peptides were separated by a PICOFRIT C18 reverse-phase column (0.075×100 mm, New Objective Inc.) at a flow rate of 300 nl/min with a 110 min-gradient. Each peptide mixture was loaded in solvent A [95% H2O, 5% acetonitrile (ACN), 0.1% formic acid (FA)] and eluted by 5% solvent B (5% H2O, 95% ACN, 0.1% FA) for 5 min followed by a linear gradient to 45% solvent B in the next 90 min, then the column was re-equilibrated at initial condition for 15 min. The entire eluant was sprayed into the LTQ-Orbitrap mass spectrometer via a dynamic nanospray probe (Thermo Fisher Scientific, Inc.) and analyzed in positive mode. The 3 most abundant precursor ions detected in the full MS survey scan (m/z range of 400–2000, R=60,000) by Orbitrap were isolated for further MS/MS analyzing in the linear ion trap. The automatic gain control target was set to 1,000,000 and 10,000 for MS scans and MS/MS scans, respectively. Tandem mass spectra were extracted by Bioworks (version 3.3.1 SP1, Thermo Fisher Scientific, Inc.) and submitted to Human UniProtKB/Swiss-Prot database (Proteome ID: UP000005640, 20199 entries) using TurboSequest v.28 (36 (link)) search engine.
6
Rituximab Glycosylation Activity Assay
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Activity on fully glycosylated Rituximab was measured using 5 µM Rituximab with 1 µM AlfCWT or AlfCE274A in PBS for 1 month at 25 °C. Activity on partially deglycosylated Rituximab was measured using 5 µM Rituximab with 50 nM EndoS2 and 1 µM AlfCWT in PBS overnight at 25 °C. EndoS2 rapidly hydrolyzes antibody glycans between the first and second N-acetylglucosamine residues, leaving one N-acetylglucosamine residue and fucose attached to each heavy chain of the antibody. After the designated incubation, 10 μl aliquots of each reactions were quenched by the addition of 1.1 μl trifluoroacetic acid. The quenched reactions were then mixed with 50 mM TCEP, and analyzed by LC–MS using an Accela LC System attached to an LXQ linear ion trap mass spectrometer (ThermoScientific, Waltham, MA), as previously described39 (link). Relative amounts of substrate and hydrolysis products were quantified after deconvolution of the raw data using BioWorks (ThermoScientific, Waltham, MA). Relative intensities by mass were then exported to GraphPad Prism and replotted, overlayed, and annotated.
7
Crosslinking and Mass Spectrometry of FliD
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FliD78–405 crystals were crosslinked using 2% formaldehyde, harvested and washed in mother liquor, dissolved in water and the crosslinking reversed by heating the samples to 95°C for 20 min. The samples were analyzed by liquid chromatography (LC)-electrospray ionization (ESI)-mass spectrometry (MS) using a gradient of mobile phase A (0.1% formic acid in water) and mobile phase B (0.1% formic acid in acetonitrile) increasing from 0% B to 90% B in 20 min. The Accela LC System was attached to a LXQ linear ion trap mass spectrometer (Thermo Scientific). Raw MS data were analyzed using Xcalibus Qual Browser (Thermo Scientific) and deconvoluted using BioWorks (Thermo Scientific, Waltham, MA).
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