Reagents for tissue culture, including DMEM, fetal bovine serum (FBS), penicillin/streptomycin, and phosphate-buffered saline (PBS) were obtained from Gibco. Mass spectrometry-grade trypsin and Lys-C protease were purchased from ThermoFisher Scientific and Wako, respectively. Isobaric TMT reagents and the BCA protein concentration assay kit were from ThermoFisher Scientific. Empore-C18 material for in-house StageTips was acquired from 3 M and Sep-Pak cartridges were purchased from Waters. Sera-Mag Speed Beads (cat. nos. 45152105050350 and 65152105050350) were from GE Life Sciences (Marlborough, MA). All solvents used for liquid chromatography were purchased from VWR. Unless otherwise noted, all other chemicals were purchased from ThermoFisher Scientific.
Lys c protease
Manufactured by Thermo Fisher Scientific
Sourced in United States
Lys-C protease is a laboratory reagent used for protein analysis. It is an enzyme that specifically cleaves peptide bonds on the C-terminal side of lysine residues in proteins. This targeted cleavage can be used to facilitate protein identification and characterization through techniques such as mass spectrometry.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein concentration assay kit, by Thermo Fisher Scientific (3 mentions)
Isobaric tmt reagents, by Thermo Fisher Scientific (3 mentions)
Sep pak cartridges, by Waters Corporation (2 mentions)
Mass spectrometry grade trypsin, by Thermo Fisher Scientific (2 mentions)
Ms grade trypsin, by Thermo Fisher Scientific (2 mentions)
Acquity uplc, by Thermo Fisher Scientific (2 mentions)
Ltq ot, by Thermo Fisher Scientific (2 mentions)
N glycanase, by New England Biolabs (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Phosphate buffered saline (pbs), by Thermo Fisher Scientific (1 mentions)
Sep pak cartridges 100 mg, by Waters Corporation (1 mentions)
Complete protease inhibitor, by Merck Group (1 mentions)
Ammonium formate, by Thermo Fisher Scientific (1 mentions)
Formic acid, by Thermo Fisher Scientific (1 mentions)
High performance liquid chromatography grade acetonitrile, by Thermo Fisher Scientific (1 mentions)
Glu c, by Thermo Fisher Scientific (1 mentions)
α cyano 4 hydroxycinnamic acid, by Merck Group (1 mentions)
Triton x 100, by Merck Group (1 mentions)
9 protocols using lys c protease
1
Mass Spectrometry-Based Proteomic Workflow
2
TMT Labeling and BCA Assay
3
Comprehensive Proteomic Sample Preparation
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Isobaric TMT reagents and the BCA protein concentration assay kit were from ThermoFisher Scientific (Rockford, IL, USA). Empore-C18 material for in-house made Stage Tips was from 3 M (Saint Paul, MN, USA). Sep-Pak cartridges (100 mg size) were purchased from Waters (Milford, MA, USA). All solvents used for Liquid chromatography (LC) were purchased from J.T. Baker (Central Valley, PA, USA). Mass spectrometry (MS)-grade trypsin and Lys-C protease were purchased from ThermoFisher Scientific and Wako (Boston, MA, USA), respectively. Complete protease inhibitors were from Millipore Sigma (Saint Louis, MO, USA). Unless otherwise noted, all other chemicals were purchased from ThermoFisher Scientific.
4
Mass Spectrometry Sample Preparation
5
On-Beads Digestion for Mass Spectrometry
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We performed on-beads digestion based on previous reported protocol [20] . After overnight incubation with striatal homogenates, NeutrAvidin beads were rinsed for five times in one ml lysis buffer (6 M Guanidine, 50 mM HEPES, pH8.5), then added one ml lysis buffer. Dithiothreitol (DTT, DOT Scientific Inc, Cat# DSD11000) was applied to a final concentration of 5 mM. After incubation at RT for 20 min, iodoacetamide (IAA, Sigma-Aldrich, Cat# I1149) was added to a final concentration of 15 mM and incubated for 20 min at RT in the dark. Excess IAA was quenched with DTT for 15 min. Samples were diluted with buffer (100 mM HEPES, pH 8.5, 1.5 M Guanidine), and digested for three hrs with Lys-C protease (1:100, ThermoFisher Scientific, Cat# 90307_3668048707) at 37 °C. Trypsin (1:100, Promega, Cat# V5280) was then added for overnight incubation at 37 °C with intensive agitation (1000 rpm). The next day, reaction was quenched by adding 1% trifluoroacetic acid (TFA, Fisher Scientific, O4902-100). The samples were desalted using HyperSep C18 Cartridges (Thermo Fisher Scientific, Cat# 60108-301) and vacuum centrifuged to dry.
6
Mosaic Nanoparticle Composition Analysis
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Label-free quantitation was performed by peptide mass spectrometry to determine the relative abundance of each HA present in the mosaic nanoparticle samples. Each mosaic nanoparticle, either before or after SEC purification, along with a standard mixture of each purified HA-I53_dn5B fusion protein at equimolar concentrations (1:1:1:1), was denatured and reduced using guanidine hydrochloride and DTT. Samples were then alkylated with iodoacetamide, deglycosylated with N-glycanase (New England Biolabs), and digested overnight with LysC protease (ThermoFisher scientific). LC-MS was performed using a Waters Acquity UPLC coupled to a Thermo LTQ-OT using data-dependent acquisition. Peptides were resolved over a Waters CSH C18 1.7 μm, 2.1 × 100 mm column with a linear gradient from 3% to 40% B over 30 minutes (A: 0.1% formic acid; B: acetonitrile with 0.1% formic acid). Peptides were identified from MS/MS data using Protein Prospector using a score cutoff of 15 (http://prospector.ucsf.edu/ ). Due to the high sequence identity between the HA constructs, only four peptides unique to each specific HA were observed that could be used for label-free quantitation. The integrated peak areas for these peptides relative to the areas from an equimolar mixture of each HA were used to estimate the total abundance of each HA within the mosaic nanoparticle samples (Supplementary Table 3 ).
7
Mosaic Nanoparticle Composition Analysis
8
Blue Native Western Blot for Metabolic Complexes
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Antibodies used were total OXPHOS Blue Native (BN) WB antibody cocktail (mouse monoclonal, 1:1,000; ab110412; Abcam; RRID:AB_2847807), anti-VDAC1 (rabbit polyclonal, 1:500, ab34726; Abcam; RRID:AB_778788), anti-VCP (rabbit polyclonal, 1:1,000; 111740; Abcam; RRID:AB_10861709), goat anti-rabbit IgG HRP (1:10,000; 32260; Thermo Fisher Scientific; RRID:AB_1965959), and goat anti-mouse IgG HRP (1:10,000; 32230; Thermo Fisher Scientific; RRID:AB_1965958). BN-PAGE reagents were prepared using an Invitrogen NativePAGE Sample Prep Kit (BN2008), run on 3–12% Bis-Tris NativePAGE gels (BN1001; Invitrogen); running buffers were prepared using NativePAGE running buffer kit (BN2007; Invitrogen); and gels were transferred using NuPAGE transfer buffer (NP00061; Invitrogen) onto polyvinylidene difluoride membranes (1620264; Bio-Rad). Other reagents were Halt Phosphatase Inhibitor cocktail (1:100; Thermo Fisher Scientific; 78420), DSSO (A33545; Thermo Fisher Scientific), DMSO (D2650; Sigma-Aldrich), sequencing-grade modified trypsin (V5280; Promega), ProteaseMAX (V2072; Promega), Tris(2-carboxyethyl)phosphine (TCEP; C4706; Sigma-Aldrich), iodoacetamide (IAA; I1149; Sigma-Aldrich), Lys-C Protease (PI90307; Thermo Fisher Scientific), and DTT (DSD11000; DOT Scientific).
9
Protein Purification and Fractionation
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Eluates from affinity purification were mixed with four times ice-cold Acetone (Sigma Aldrich) and NaCl for precipitation of proteins. The mixture was stored at -20°C for 1 h and later centrifuged the supernatant was removed. Dried protein pellet was solubilized in 8 M urea / 2 M thiourea solution, reduced using 10 mM DTT for 30 min at RT and derivatized with 30 mM IAA over 20 min at RT and in the dark. Digestion with Lys-C protease (Thermo Scientific) in a ratio 1:100 (w/w) was carried out for 2 h at 25 °C. The sample was diluted 5-fold in 50 mM ABC and trypsin protease was then added at a ratio of ~1:50 (m/m). After 16 h at 25 °C the digestion was quenched by acidification with TFA. Peptides were cleaned up using C18 StageTips. Eluted peptides were fractionated using a Superdex Peptide 3.2/300 column (GE Healthcare) at a flow rate of 10 µl min-1 using 30% (v/v) ACN and 0.1 % (v/v) TFA as mobile phase. For each experiment and replica peptide-containing fractions were pooled and absorbance at 215 nm was quantified.
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