Unless otherwise stated, lyophilized samples were resuspended in 8 M Urea in 50 mM ammonium bicarbonate, reduced with 5 mM TCEP (Sigma-Aldrich) for 30 min at 37 °C on a thermomixer (Eppendorf) and subsequently alkylated with 10 mM IAA (Sigma) at room temperature in the dark. Urea was first diluted to a concentration of 5.5 M and Lys-C (Wako) at a 1:100 wt/wt ratio was added for 2.5–3 h at 37 °C. Subsequently, urea was diluted to 1 M and samples were further digested by addition of trypsin (Promega) at 1:50 wt/wt ratio overnight at 37 °C. Digested samples were acidified by addition of formic acid and purified using either SPE cartridges (Waters) or microspin columns (The Nest Group). Eluates were dried, resuspended in acetonitrile 2–5% and formic acid 0.1%, typically with addition of iRT peptides (Biognosys), sonicated and centrifuged for 5–10 min at 10,000 g before MS injection.
Microspin columns
Manufactured by Nest Group
Sourced in United States
Microspin columns are a type of laboratory equipment designed for the purification and separation of biomolecules, such as proteins, nucleic acids, and other small molecules. These columns are typically made of plastic or other inert materials and contain a porous medium that allows for the separation of target molecules based on their size or other physical properties. Microspin columns are commonly used in various applications, including sample preparation, DNA/RNA extraction, and protein purification.
Automatically generated - may contain errors
Lab products found in correlation
Lys c, by Fujifilm (3 mentions)
Spe cartridge, by Waters Corporation (3 mentions)
Irt peptide, by Biognosys (3 mentions)
Thermomixer, by Eppendorf (3 mentions)
Trypsin, by Promega (3 mentions)
Tmt 10 plex reagent, by Thermo Fisher Scientific (3 mentions)
High ph fractionation kit, by Thermo Fisher Scientific (3 mentions)
Pngase f, by New England Biolabs (3 mentions)
4 protocols using microspin columns
1
Protein Sample Preparation for MS
2
Protein Preparation and Labeling for TMT
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Frozen threads were thawed, and 33.3 μL 100-mM triethyl ammonium bicarbonate (TEAB), 2 μL 1% ProteaseMax detergent (Promega, Madison, WI, USA), and 1 μL 0.5 M dithiothreitol were added. Following vortexing, the samples were heated at 56°C for 20 minutes, then alkylated by addition of 2.7 μL 0.55 M iodoacetamide and incubated in the dark at room temperature for 15 minutes. Proteins were then digested by addition of 1 μL 1% ProteaseMax detergent and 10 μL 0.1 μg/μL trypsin (Pierce, MS Grade; Thermo Scientific, Waltham, MA, USA). After shaking at 37°C overnight, the solution was removed from the threads by transferring to another 0.5-mL centrifuge tube, and 5 μL 10% trifluoroacetic acid added. The samples were then stored at room temperature for 1 hour, spun at 16,000g for 5 minutes, and the supernatant removed. Peptides were then solid phase extracted using MicroSpin columns (The Nest Group, Southborough, MA, USA) and dried by vacuum centrifugation. Each digest was then dissolved by vigorous shaking for 10 minutes in 20 μL 100 mM TEAB, and 200 μg TMT 10-plex reagent (Thermo Scientific) dissolved in 12 μL anhydrous acetonitrile was added. Samples were then incubated for 1 hour at room temperature and 2 μL 5% hydroxylamine added followed by an additional 15 minutes incubation.
3
Affinity-Purified Protein Digestion
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Affinity-purified samples in Urea (8M) were reduced with 5 mM TCEP (30 min at 37 °C) and alkylated with (10 mM IAA at room temperature). Urea was diluted to a concentration of 5.5 M for Lys-C proteolysis (Wako) (0.4 µg, 3 h) and trypsin proteolysis (Promega) (1.2 µg, overnight). Digested samples were acidified by addition of formic acid, cleaned up with microspin columns (The Nest Group), and subjected to high pH fractionation, with a procedure based on the high pH fractionation kit by Thermo.
4
Deglycosylation of N-Linked Glycopeptides
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The eluted N-linked glycopeptides were deglycosylated by overnight incubation with 1000 Units of PNGase F (Glycerol-Free, New England Biolabs) as described previously [13 (link)]. Then the samples were desalted by using MicroSpin Columns, 5–200 µL loading volume (The Nest Group, Inc.; Southborough, MA, USA). Samples were centrifugally concentrated to dryness and resuspended in 40 µL 0.1 % FA.
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