Iodomethane, sodium hydroxide beads, acetic acid, and ammonium borane complex were purchased from Sigma Aldrich (St. Louis, MO, USA). Isolute® C18 (EC) cartridges were purchased from Biotage (Charlotte, NC, USA) and 10k Amicon Ultra-0.5 mL Centrifugal Filters were purchased from Millipore Sigma (Burlington, MA, USA). Microspin columns were purchased from Harvard Apparatus (Hollison, MA, USA). N-glycosidase F enzyme (PNGase F) was acquired from New England Biolabs (Ipswich, MA, USA). Solvents, including high-performance liquid chromatography (HPLC)-grade water, acetonitrile, methanol, and dimethyl sulfoxide were purchased from Fisher Scientific (Pittsburgh, PA, USA). Pooled CSF was acquired from Golden West Biologicals, Inc. (Temecula, CA, USA).
Microspin columns
Manufactured by Harvard Apparatus
Sourced in United States
Microspin columns are a type of laboratory equipment used for the purification and separation of biomolecules, such as proteins, nucleic acids, and other macromolecules. These columns are designed to facilitate the efficient and rapid processing of small sample volumes, making them a versatile tool for various applications in biochemistry, molecular biology, and analytical chemistry.
Automatically generated - may contain errors
Lab products found in correlation
Iodomethane, by Merck Group (5 mentions)
Sodium hydroxide beads, by Merck Group (4 mentions)
Borane ammonia complex, by Merck Group (3 mentions)
Pngase f, by New England Biolabs (3 mentions)
Acetonitrile, by Thermo Fisher Scientific (2 mentions)
Methanol, by Thermo Fisher Scientific (2 mentions)
Isolute c18 ec cartridges, by Biotage (2 mentions)
Acetic acid, by Merck Group (2 mentions)
Carbon nanoparticles, by Merck Group (2 mentions)
Human alpha 1 acid glycoprotein agp, by Merck Group (2 mentions)
Pngase f with 10 g7 reaction buffer, by New England Biolabs (2 mentions)
Macrospin columns, by Harvard Apparatus (2 mentions)
3100 offgel low res kit, by Agilent Technologies (2 mentions)
Isopropanol, by Thermo Fisher Scientific (2 mentions)
Acetic acid, by Thermo Fisher Scientific (2 mentions)
Ms grade formic acid, by Merck Group (2 mentions)
Chloroform, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Trifluoroacetic acid, by Merck Group (2 mentions)
Hplc grade water, by Mallinckrodt (2 mentions)
14 protocols using microspin columns
1
Glycoprotein Analysis in Cerebrospinal Fluid
2
Enrichment and Identification of Cross-Linked Peptides
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Amounts of 150–175 μg of
constructs A and B at a final protein
concentration of 1 μg/μL or less were incubated with 2
mM SDA (100 mM stock in DMSO) for 30 min at room temperature. The
cross-linking reactions were quenched with 50 mM Tris-HCl. Proteins
were digested by trypsin, and peptides were acidified with trifluoroacetic
acid (TFA) to a final concentration of 0.5% (v/v), desalted on MicroSpin
Columns (Harvard Apparatus) following manufacturer’s instructions,
and vacuum-dried. To enrich cross-linked peptide species by peptide
size exclusion chromatography, we subjected fractions that eluted
first and contained the cross-linked peptide pairs to LC-MS/MS analysis.
Cross-linked peptides were measured in technical duplicates on an
Orbitrap Fusion Tribrid mass spectrometer or on a Q Exactive HF-X
coupled to a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific)
equipped with an in house-packed C18 column (ReproSil-Pur
120 C18-AQ, 1.9 μm pore size, 75 μm inner diameter,
30 cm length, Dr. Maisch GmbH). ProteomeDiscoverer 1.4 (Thermo Fisher
Scientific) was used for converting raw files into.mgf format (signal-to-noise
ratio 1.5, 1000–10000 Da precursor mass). The generated.mgf
files were subjected to pLink v. 1.23 (pFind group)45 (link) to identify cross-linked peptides. All spectra were evaluated
manually. For more information, see theSupporting
Information .
constructs A and B at a final protein
concentration of 1 μg/μL or less were incubated with 2
mM SDA (100 mM stock in DMSO) for 30 min at room temperature. The
cross-linking reactions were quenched with 50 mM Tris-HCl. Proteins
were digested by trypsin, and peptides were acidified with trifluoroacetic
acid (TFA) to a final concentration of 0.5% (v/v), desalted on MicroSpin
Columns (Harvard Apparatus) following manufacturer’s instructions,
and vacuum-dried. To enrich cross-linked peptide species by peptide
size exclusion chromatography, we subjected fractions that eluted
first and contained the cross-linked peptide pairs to LC-MS/MS analysis.
Cross-linked peptides were measured in technical duplicates on an
Orbitrap Fusion Tribrid mass spectrometer or on a Q Exactive HF-X
coupled to a Dionex UltiMate 3000 UHPLC system (Thermo Fisher Scientific)
equipped with an in house-packed C18 column (ReproSil-Pur
120 C18-AQ, 1.9 μm pore size, 75 μm inner diameter,
30 cm length, Dr. Maisch GmbH). ProteomeDiscoverer 1.4 (Thermo Fisher
Scientific) was used for converting raw files into.mgf format (signal-to-noise
ratio 1.5, 1000–10000 Da precursor mass). The generated.mgf
files were subjected to pLink v. 1.23 (pFind group)45 (link) to identify cross-linked peptides. All spectra were evaluated
manually. For more information, see the
Information
3
Glycoprotein Characterization Protocol
4
Off-Gel Electrophoresis Sample Preparation
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Before OGE, samples were dried under speed-vacuum then purified by using Macrospin columns (Harvard Apparatus, Holliston, US-MA) according to manufacturer's recommendations. Tubes were dried under speed-vacuum and A 3100 OFFGEL Fractionator (Agilent technologies, Santa Clara, US-CA) was performed over night to separate the sample. Guidelines available in Agilent datasheet were followed, using a 13cm IPG strip (Immobiline DryStrip pH 3-10, 13cm GE Healthcare, Little Chalfont, UK) and 12 OGE wells (20, 21) . After fractionation, microspin columns (Harvard Apparatus, Holliston, US-MA) were used according to the manufacturer's recommendations and the 12 fractions of each experiment were dried under speed-vacuum. Peptide concentration of the fractions was theoretically approximated, considering that 1/12 of the pooled sample was found in each fraction after OGE.
5
Peptide Fractionation by pI Separation
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Previously dried samples were resuspended in 5% CAN and 0.1%FA and purified under Macrospin columns (Harvard Apparatus, Holliston, MA). A 3100 OFFGEL Fractionator (Agilent Technologies, Les Ulis, France) was then used to separate peptides according to their pI, as reported previously16 (link) with a 13 cm IPG strip (immobiline Dry strip pH 3–10, 13 cm; GE Healthcare, Little Chalfont, UK) and 12 OGE wells. The focusing parameters were 20 Kvh, 800v, 50 uA, 200 mW, and 100 s. The hold parameters were 500 V, 20 uA, and 50 mW. After overnight fractionation, microspin columns (Harvard Apparatus) were performed according to manufacturer's recommendations and the 12 fractions were dried under speed vacuum.
6
Mass Spectrometry of Phage Particles
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For mass spectrometry, 5 μL of concentrated phage lysate (6 × 1010 PFU/mL; protein concentration determined as 2.5 mg/mL using a Pierce BCA kit) and purified phage particles (2 × 1012 PFU/mL; protein concentration of 2.8 mg/mL) were used for trypsin digestion. The samples were prepared for mass spectrometry essentially as described in [45 (link)]. Phage samples were mixed with 8 M urea–100 mM ammonium bicarbonate to a final volume of 50 μL, and the cysteine bonds were reduced with 5 mM tris(2-carboxyethyl)phosphine (TCEP) (37 °C for 60 min) with subsequent alkylation using 10 mM iodoacetamide (22 °C for 30 min). Ammonium bicarbonate, at 100 mM, was used to dilute the urea concentration of the samples to 1.5 M. Proteins were digested for 18 h at 37 °C with sequencing grade trypsin (Promega). Formic acid (10%) was used to lower the pH of the samples to 3.0, and the peptides were subsequently purified with C18 reverse-phase spin columns according to the manufacturer’s instructions (Microspin Columns, Harvard Apparatus). The dried peptides were reconstituted in 2% acetonitrile and 0.2% formic acid prior to mass spectrometric analyses.
7
Peptide Separation by Isoelectric Point
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The peptides were separated by their isoelectric point using OFFGEL fractionator with a 12-well setup (3100 OFFGEL Low Res Kit, pH 3–10; Agilent Technologies, Santa Clara, CA, USA). The protocol was followed as per the manufacturer's instructions. Micro Spin columns (Harvard Apparatus, Holliston, MA, USA) were used for chemical reagent clearance.
8
Glycoprotein Analysis by MALDI-TOF MS
9
Glycoprotein Analysis via Mass Spectrometry
10
PNGase F Glycan Modification Protocol
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PNGase F was obtained from New England Biolabs (Ipswich, MA, USA). Borane-ammonia complex, sodium hydroxide beads, acetic acid, and iodomethane were acquired from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was bought from Mallinckrodt Chemicals (Phillipsburg, NJ, USA). HPLC grade acetonitrile, water, and isopropanol were purchased from Fisher Scientific (Fair Lawn, NJ, USA). Isolute® C18 (EC) cartridges were purchased from Biotage (Charlotte, NC, USA), and microspin columns were purchased from Harvard Apparatus (Holliston, MA, USA).
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