The trypsin-digested peptide mixtures were adjusted to 0.065% TFA by addition of 10% trifluoroacetic acid (TFA). The mixture was set at room temperature for 30 min followed by centrifugation at 10,000 g for 10 min. The supernatant was manually injected into a 100 µL loop. The peptide fragments were separated by reverse phase chromatography through a Sephasil Peptide C18 5 µ ST 4.6/100 column (GE Healthcare Life Sciences, Piscataway, NJ) using an AKTAbasic system equipped with UNICORN 3.10 version (GE Healthcare Life Sciences, Piscataway, NJ). The peptide mixtures were loaded onto the column from the sample loop with 500 µL of buffer A (0.065% TFA in 2% acetonitrile). The column was washed with 5-bed volumes of buffer A. The peptides were eluted from the column at 0.5 mL/min with a 20-bed volume of a linear gradient from 100% buffer A to 100% of buffer B (0.05% TFA in 100% acetonitrile). The fractions were collected at 0.25 mL/well in 96-well plates with Frac-950 fraction collector (GE Healthcare Life Sciences, Piscataway, NJ). The radioactivity of every fraction was counted using MicroBeta JET (PerkinElmer Life Sciences, Gaithersburg, MD).
Frac 950 fraction collector
Manufactured by GE Healthcare
Sourced in Germany
The Frac-950 fraction collector is a laboratory instrument designed to automatically collect fractions of samples during chromatographic separations. It can be used with a variety of chromatography systems to collect multiple fractions of interest. The Frac-950 features precise timing and fraction volume control to ensure accurate sample collection.
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4 protocols using frac 950 fraction collector
1
Reverse Phase Chromatography of Trypsin-Digested Peptides
2
Fractionation and Quantification of GOS
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Vivinal GOS was fractionated on DP by size-exclusion
chromatography (SEC) using an AKTA purifier system (GE Healthcare,
Chicago, IL, U.S.A.). GOS (20 mg/mL, 5 mL) was injected into three
serially connected Hiload 26/60 Superdex 30 preparative grade columns,
each with a column volume of 300 mL (GE Healthcare). The temperature
was set at 35 °C, and the flow rate was set at 2.6 mL of H2O/min. A refractive index (RI) detector (Shodex RI-72, Yokohama,
Japan) was used to monitor the elution. The eluent containing GOS
was collected in 5 mL fractions using a Frac-950 fraction collector
(GE Healthcare). Subsequently, fractions were analyzed using matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS)
as described previously26 (link) to determine
the molecular weight of GOS present in the fractions. The fractions
with a purity of at least 90% according to the signal intensity as
measured by MALDI–TOF–MS were pooled. Fractions containing
DP3, which eluted between 720 and 740 mL, of multiple runs were pooled
and freeze-dried. On the basis of the elution pattern and m/z, also DP1, DP2, DP4, DP5, DP6, and
DP ≥7 were combined per DP and freeze-dried. Weights obtained
per DP were used to determine the relative abundance of the different
DPs in purified Vivinal GOS.
chromatography (SEC) using an AKTA purifier system (GE Healthcare,
Chicago, IL, U.S.A.). GOS (20 mg/mL, 5 mL) was injected into three
serially connected Hiload 26/60 Superdex 30 preparative grade columns,
each with a column volume of 300 mL (GE Healthcare). The temperature
was set at 35 °C, and the flow rate was set at 2.6 mL of H2O/min. A refractive index (RI) detector (Shodex RI-72, Yokohama,
Japan) was used to monitor the elution. The eluent containing GOS
was collected in 5 mL fractions using a Frac-950 fraction collector
(GE Healthcare). Subsequently, fractions were analyzed using matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry (MALDI–TOF–MS)
as described previously26 (link) to determine
the molecular weight of GOS present in the fractions. The fractions
with a purity of at least 90% according to the signal intensity as
measured by MALDI–TOF–MS were pooled. Fractions containing
DP3, which eluted between 720 and 740 mL, of multiple runs were pooled
and freeze-dried. On the basis of the elution pattern and m/z, also DP1, DP2, DP4, DP5, DP6, and
DP ≥7 were combined per DP and freeze-dried. Weights obtained
per DP were used to determine the relative abundance of the different
DPs in purified Vivinal GOS.
3
Protein Separation by Size Exclusion Chromatography
4
HILIC Separation of Tryptic Peptides
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Hydrophilic interaction liquid chromatography (HILIC) was performed as described by Singer et al. (2010) (link) with modifications. In detail, tryptic digests (100 µg) were reconstituted in 40 µl of 0.1% (v/v) aq. formic acid before 260 µl of acetonitrile was added in three portions (60, 100 and 100 µl) followed by 30 s of vortexing after each addition. Of the sample, 290 µl was separated by a Luna 3u HILIC (silica, 100 × 2 mm, particle size 3 µm, Phenomenex, Aschaffenburg, Germany) using an Äkta Purifier HPLC system, equipped with A-905 autosampler, P-903 gradient pump binary pump, UV-900 UV-Vis detector, and Frac-950 fraction collector (GE Healthcare, Solingen, Germany). The separations were performed by linear gradients of aq. ammonium acetate in acetonitrile using the conditions summarized in Supplementary Table S3 . The collected fractions (5 × 1 ml) were dried under reduced pressure, reconstituted in 60% (v/v) acetonitrile in 0.1% (v/v) aq. formic acid and diluted with 0.1% (v/v) formic acid (Supplementary Table S4 ).
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