Glycoprofiling was performed as previously published (Grav et al., 2015 (link)). In short, exponentially growing cells were seeded at 1×106 cells/mL and supernatants harvested after 4 days by centrifugation. Supernatants were filtered and proteins contained in the sample were concentrated by centrifugation using Amicon Ultra columns (Merck Millipore, Merck KGaA, Darmstadt, Germany) with 3000 Da cutoff. N-glycans from retained proteins were released and fluorescently labeled with GlykoPrep Rapid N-Glycan kit (ProZyme Inc., Hayward, CA). Labeled N-glycans were analyzed by LC-MS on a Thermo Ultimate 3000 HPLC with fluorescence detector coupled on-line to a Thermo Velos Pro Iontrap MS. Glycan abundance was measured by integrating the areas under normalized fluorescence spectrum peaks with Xcalibur software (Thermo Fisher Scientific) giving the relative amount of the glycans. All annotated sugar structures are peaks with correct mass (Suppl. Fig. 6, Suppl. Table S2 ) and at least a signal to noise value of 20:1 as calculated with Xcalibur. In total, the analyzed secretome consisted of more than a thousand proteins.
Velos pro iontrap ms
Manufactured by Thermo Fisher Scientific
The Velos Pro Ion Trap MS is a high-performance mass spectrometer designed for a variety of analytical applications. It features a linear ion trap technology that provides high-resolution, accurate mass measurement and tandem MS capabilities. The instrument is capable of rapid data acquisition and can be configured with different ionization sources to accommodate diverse sample types.
Automatically generated - may contain errors
Lab products found in correlation
Ultimate 3000 hplc, by Thermo Fisher Scientific (3 mentions)
Xcalibur software, by Thermo Fisher Scientific (2 mentions)
Glycoworks rapifluor ms n glycan kit, by Waters Corporation (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Amicon ultra column, by Merck Group (1 mentions)
Thermo xcalibur software, by Thermo Fisher Scientific (1 mentions)
Mono q 5 50 gl column, by GE Healthcare (1 mentions)
Hitrap mabselect column, by GE Healthcare (1 mentions)
5 protocols using velos pro iontrap ms
1
Glycoprofiling of Cell Secretome
2
Engineered CHO Cells for Protein Purification
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Example 5
1 engineered CHO Cell line with KO of the Sppl3 gene and CHO-S wt cells were both transiently transfected using chemical transfection with plasmids encoding either a Erythropoietin or Clinhibitor gene fused to a HPC4-affinity purification tag. The transfected cells were grown for 72 hours in CD CHO+8 mM L-gln using standard conditions as described previously, after which the supernatant was harvested, sterile filtered and stored at −80° C.
For protein purification, the supernatants were thawed and purified by affinity chromatography using a 1-mL anti-protein C affinity column for EPO and C1inhibitor, and the fractions containing the EPO and C1inhibitor respectively were pooled.
N-glycan analysis was performed on the purified samples, with GlycoWorks RapiFluor-MS N-Glycan Kit (Waters, Milford, Mass.) according to the manufacturer's instruction. In this case 12 μl of purified protein sample were used for each. Labeled N-Glycans were analyzed by a LC-MS system using a Thermo Ultimate 3000 HPLC with fluorescence detector coupled on-line to a Thermo Velos Pro Iontrap MS. Separation gradient 30% to 43% buffer and MS was run in positive mode.
3
N-Glycan Quantification via LC-MS
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N-Glycans were fluorescently labeled and quantified via LC-MS as described previously in (35 (link)). Briefly, the supernatant was concentrated using Amicon® Ultra‐4 Centrifugal Filter Units. Secretome proteins were fluorescently labeled with GlycoWorks RapiFluor‐MS N‐Glycan Kit (Waters, Milford, MA). N‐linked glycan analysis was performed by LC‐MS using a Waters Acquity Glycan BEH Amide 130 Å, 2.1 mm × 150 mm, 1.7 μm column (Waters, Milford) with a Thermo Ultimate 3000 HPLC with the fluorescence detector coupled on‐line to a Thermo Velos Pro Iontrap MS (run in positive mode) and a separation gradient of 30–43% buffer. The amount of N‐glycan was measured by integrating the areas under the normalized fluorescence spectrum peaks with Thermo Xcalibur software (Thermo Fisher Scientific) giving normalized, relative glycan quantities.
4
Glycoprofiling of Purified Proteins
5
Monoclonal Antibody Purification and Glycan Analysis
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Cell culture samples were taken from the cultures. After centrifugation and filtration to remove the cells and cell debris, the secreted mAbs in the culture supernatants were purified by protein A affinity chromatography (recombinant protein A agarose, Pierce, Rockford, IL), according to the manufacturer's protocol. Purified mAbs were fluorescently labeled with GlykoPrep Rapid N-Glycan kit (ProZyme, Hayward, CA), according to the manufacturer's protocol. N-linked glycan analysis was performed by LC-MS system using a Thermo Ultimate 3000 HPLC with fluorescence detector coupled on-line to a Thermo Velos Pro Iontrap MS, as described previously [22] .
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