Crosslinked samples were processed essentially as described28 (link). In short, samples were dried (Eppendorf, Concentrator plus), resuspended in 8 M urea, reduced, alkylated and digested with trypsin (Promega). Digested peptides were separated from the solution and retained by a solid-phase extraction system (SepPak, Waters). Crosslinked peptides were enriched by size exclusion chromatography (SEC) using an ÄKTAmicro chromatography system (GE Healthcare) equipped with a Superdex Peptide 3.2/30 column (column volume, 2.4 ml). For each crosslinked sample four fractions were measured in technical duplicates. Therefore, the elution fractions 0.9–1.0 and 1.0–1.1 ml were pooled and the three elution fractions 1.1–1.2, 1.2–1.3 and 1.3–1.4 ml were analyzed separately by liquid chromatography with tandem MS (LC–MS/MS). Absorption levels at 215 nm of each fraction were used to normalize peptide amounts prior to LC–MS/MS analysis.
Superdex peptide 3.2 30 column
Manufactured by GE Healthcare
The Superdex™ Peptide 3.2/30 column is a size exclusion chromatography column used for the separation and purification of peptides. The column has a bed volume of 2.4 mL and a separation range of 100 to 7,000 Da. It is designed for use with various liquid chromatography systems.
Automatically generated - may contain errors
3 protocols using superdex peptide 3.2 30 column
1
Crosslinking Peptide Enrichment Protocol
2
Enrichment and Analysis of Crosslinked Peptides
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Crosslinked samples were dried (Eppendorf, Concentrator plus), resuspended in 100 μl 8M Urea, reduced, alkylated, and digested with trypsin (Promega). Digested peptides were separated from the solution and retained by a solid phase extraction system (SepPak, Waters). Crosslinked peptides were enriched by size exclusion chromatography using an ÄKTAmicro chromatography system (GE Healthcare) equipped with a Superdex™ Peptide 3.2/30 column (column volume = 2.4 ml). Fractions were collected in 100 μl units and analyzed by LC-MS/MS. For each crosslinked sample three fractions were measured in technical duplicates. The elution fractions 1.0-1.1, 1.1-1.2 and 1.2-1.3 ml containing the largest peptides were pooled and the two elution fractions 1.3-1.4 ml and 1.4-1.5 ml were also analyzed by LC-MS/MS. Absorption levels at 215 nm of each fraction were used to normalize peptide amounts prior to LC-MS/MS analysis.
3
Enrichment and Analysis of Crosslinked Peptides
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