To create a comprehensive neutrophil spectral library, selected healthy donor and patient peptide samples were eluted from StageTips using 80% acetonitrile (ACN) in 0.1% formic acid, dried in Eppendorf Concentrator plus 5305, reconstituted in SCX buffer A (5 mm K2HPO4 in 10% ACN) and pooled. 100 μg of pooled peptides were separated on a Shimadzu LC-20AD HPLC system using a PolyLC PolySULFOETHYL-A SCX column (100 × 2.1 mm, 3 μm beads, 300 Å pores) with a 12 min nonlinear gradient of SCX buffer B (1 m KCl, 5 mm K2HPO4 in 10% ACN) while collecting fractions every 15 s. The fractions were then concentrated, reconstituted in 0.1% TFA and desalted using C18-StageTips. Lower complexity fractions were pooled together prior to mass spectrometric analysis.
Concentrator plus 5305
Manufactured by Eppendorf
Sourced in Germany
The Concentrator plus 5305 is a benchtop centrifugal concentrator designed for evaporating solvents and concentrating samples in the laboratory. It features a temperature-controlled heating block and a vacuum pump to efficiently remove solvents from samples.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris, by Thermo Fisher Scientific (2 mentions)
Dithiothreitol (dtt), by Merck Group (2 mentions)
Iodoacetamide, by Merck Group (2 mentions)
Lc 20ad hplc system, by Shimadzu (1 mentions)
Scx column, by Shimadzu (1 mentions)
Strata x c18, by Phenomenex (1 mentions)
Tmt 6plex isobaric mass tag labeling kit, by Thermo Fisher Scientific (1 mentions)
Tissuelyser 2, by Qiagen (1 mentions)
U 13c pyruvate, by Cambridge Isotopes (1 mentions)
6 protocols using concentrator plus 5305
1
Comprehensive Neutrophil Spectral Library
2
Protein Quantification and TMT Labeling
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We use the SDT lysis method [29 (link)] to lyse the sample and the Bradford method for protein quantification. One hundred micrograms of sample protein meeting the experimental requirements was diluted with 8 M urea buffer. The protein disulfide bond was then reduced by DTT, the sulfhydryl group was protected by IAA alkylation, and the Trypsin buffer was used for enzymatic hydrolysis after TEBA dilution. The peptides after enzymolysis were desalted by a Strata X C18 (phenomenex) desalting column, dried on a benchtop refrigerated vacuum concentrator (Concentrator Plus 5305, Eppendorf), and then frozen and stored for further TMT labeling. One hundred micrograms of peptides from each sample were labeled according to the TMT 6 plex Isobaric Mass Tag Labeling kit (Thermo Fisher Scientific).
3
Mitochondrial Metabolite Extraction Protocol
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Fifty microliters isolated mitochondrial suspension was added into 450 uL modified KPBS (136 mM KCL, 10 mM KH2PO4, 10 mM HEPES, pH 7.25) containing 2 mM U-13C pyruvate (Cambridge Isotopes, CLM-2440-0.1) and incubated on ice for 5 min. After incubation, samples were centrifuged at 10,000 g × 30 s, and washed three times by adding 1 mL ice-cold KPBS. Subsequently, the supernatant was removed and 1 mL ice-cold LC/MS 80% methanol was added. To completely extract metabolites from the mitochondria, the sample was homogenized using TissueLyser II (Qiagen, 85300) for 5 min at 30 Hz, followed by centrifugation at 20,000 g for 15 min at 4 °C. The supernatant was kept on dry ice, and the pellet was resuspended with 500 uL 80% LC/MS-grade methanol, vortexed vigorously, and allowed to extract on ice. The samples were then centrifuged at 20,000 g for 10 min at 4 °C. The extraction was vacuum dried using a vacuum concentrator (Eppendorf, concentrator Plus 5305). Dried samples were solubilized in 50 µL LC/MS water. Metabolite analysis was conducted at the BIDMC metabolomics core. The data were normalized by protein concentration.
4
Quantitative Metabolite Extraction from Biofluids
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Serum and cerebrospinal fluid (CSF) samples (200 ll) were spiked with 50 ll of the IS solution, vortexed (90 s), and added with 1,000 ll of acetonitrile/methanol (70/30; +1.0% formic acid). The samples were then sonicated (10 min, 4°C), centrifuged (27,627 g, 10 min, 10°C), and the supernatants were purified on Phree-SPE cartridges to remove endogenous phospholipids. Eluates were evaporated (Concentrator Plus #5305; Eppendorf AG, Hamburg, Germany) at 35°C and derivatized with 50 ll of Amplifex Keto Reagent for 1 h at room temperature. Subsequently, samples were added with 150 ll methanol/water (70/30), centrifuged (20,627 g, 10 min, 10°C), and transferred in autosampler vials for LC-MS/MS analysis. Injection volume: 10 ll.
5
Crosslinking and Mass Spectrometry Analysis of ubD2-I
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Purified ubD2–I (50 mM HEPES pH 7.5, 150 mM NaCl, 100 mM
imidazole and 1 mM TCEP) at a concentration of ~1 μM was
crosslinked with 2-fold molar ratio of disulfosuccinimidyl suberate (BS3) for 2
h on ice and the reaction was quenched with 50 mM NH4HCO3for 30 min at room temperature. Samples were then loaded on 4–12% NuPAGE
Bis-Tris (ThermoFisher Scientific). Gel bands of crosslinked ubD2–I
complex were excised and digested with trypsin, as previously
described56 (link). In brief,
proteins were reduced in 10 mM dithiothreitol (Sigma Aldrich, Germany) for 30
min at 37°C and alkylated in 55 mM iodoacetamide (Sigma Aldrich, Germany)
for 20 min at ambient temperature in the dark. Proteins were then digested
overnight at 37°C with 10 ng μl-1 trypsin (Pierce,
Germany) in 45 mM ABC and 10% v/v acetonitrile.
Following digestion, peptides were extracted from the gel and desalted
using C18 StageTips as described previously57 (link). Peptides were then eluted from the StageTip using 40
μl 80% v/v acetonitrile (ACN) in 0.1% v/v trifluoroacetic acid (TFA) into
a LowBind Eppendorf sample tube. Solvent in the eluate was removed using vacuum
centrifugation (Concentrator 5305 plus, Eppendorf, Germany). For LC-MS/MS
analysis, peptides were re-suspended with the sample loading buffer (0.1% v/v
formic acid (FA), 1.6% v/v ACN) to a concentration of ~ 0.3 μg
μl-1, and 3.5 μl was injected for LC-MS/MS
acquisition.
imidazole and 1 mM TCEP) at a concentration of ~1 μM was
crosslinked with 2-fold molar ratio of disulfosuccinimidyl suberate (BS3) for 2
h on ice and the reaction was quenched with 50 mM NH4HCO3for 30 min at room temperature. Samples were then loaded on 4–12% NuPAGE
Bis-Tris (ThermoFisher Scientific). Gel bands of crosslinked ubD2–I
complex were excised and digested with trypsin, as previously
described56 (link). In brief,
proteins were reduced in 10 mM dithiothreitol (Sigma Aldrich, Germany) for 30
min at 37°C and alkylated in 55 mM iodoacetamide (Sigma Aldrich, Germany)
for 20 min at ambient temperature in the dark. Proteins were then digested
overnight at 37°C with 10 ng μl-1 trypsin (Pierce,
Germany) in 45 mM ABC and 10% v/v acetonitrile.
Following digestion, peptides were extracted from the gel and desalted
using C18 StageTips as described previously57 (link). Peptides were then eluted from the StageTip using 40
μl 80% v/v acetonitrile (ACN) in 0.1% v/v trifluoroacetic acid (TFA) into
a LowBind Eppendorf sample tube. Solvent in the eluate was removed using vacuum
centrifugation (Concentrator 5305 plus, Eppendorf, Germany). For LC-MS/MS
analysis, peptides were re-suspended with the sample loading buffer (0.1% v/v
formic acid (FA), 1.6% v/v ACN) to a concentration of ~ 0.3 μg
μl-1, and 3.5 μl was injected for LC-MS/MS
acquisition.
6
Crosslinking and Mass Spectrometry Analysis of ubD2-I
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