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High-Resolution Liquid Chromatography MS Analysis
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High-resolution Liquid Chromatography Mass Spectrometry (LC-MS) provides an accurate determination of the molecular weight of a protein sample or its subunits up to 200 kDa. LC-MS requires high purity samples and provides unmatched accuracy. The sample is detected in multiple charge states (as shown in the figure below). Using deconvolution software the exact mass of the intact molecule can be calculated with a high degree of accuracy.
CovalX utilizes the latest mass spectrometry instrumentation and software for electrospray (ESI) analysis. We have access to a full range of high-resolution electrospray instruments including the latest Orbitrap instrumentation including high mass detection (intact mAb).
Typically we request the highest purity and concentration possible for each sample, but sample prep can be customized to a customer’s needs as well. All samples are injected using liquid chromatography separation prior to mass spec ionization. Optimization of instrumental operating parameters for protein/peptide analysis is also required for providing proper data.
Intact
The most common and direct analysis of the molecule is done first by an intact analysis. This can also be done for even larger molecules and protein complexes (upto 2MDa) using the CovalX High Mass MALDI mass spectrometry analysis. The intact LC-MS measurement is done directly on the sample using minimal sample modification. This analysis can give an initial overview of the molecules structural heterogeneity from post-translational modifications due to the multiple species detected. More advanced analysis of Post-Translational Modifications (PTMs) is also possible. Analysis of the Deglycosylated and/or Reduced samples can provide additional insight into the molecules structure.
Deglycosylated
By removing the glycans from the antibody it is possible to have a direct determination of the molecular weight of the protein after deglycosylation. This measurement is a first step in the characterization of the glycosylation pattern of a monoclonal antibody. Using other mass spectrometric methods, it is possible to analyse the glycosylation site and patterns with higher resolution.
Reduced
The LC-MS analysis of a monoclonal antibody after reduction allows the characterization of the Light Chain (LC) and Heavy Chain (HC) with high resolution. This analysis allows the measurement of the molecular weight of the LC and HC with high accuracy. This analysis is a complement of the intact mass measurement.
CovalX utilizes the latest mass spectrometry instrumentation and software for High Resolution LC-MS analysis.
Typically we request high purity sample, but sample prep can be customized to a customer’s needs. All samples are injected using liquid chromatography separation prior to mass spectrometry ionization.
- Sample types: Recombinant proteins, purified proteins
- Material request: 50-100 µg of protein at 1-2mg/mL if possible
- Data turn around: Two weeks or less depending on project size
- Deliverable: Full written report and discussion. Raw data available upon request.