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Intact Mass Analysis

  • Characterize modifications and degradation products
  • Identify coeluting proteins or shoulders
  • Suitable for proteins and monoclonal antibodies
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Technical details, sample info, experts etc.

Do you need the exact intact mass of proteins or antibodies?

Intact mass analysis of proteins by ESI-MS and correlation with the sequences enables a detailed understanding of the protein heterogeneity. Also, you can use it to confirm correct protein expression.

Furthermore, the intact mass of co-eluting proteins/antibodies and integration of HPLC peak ar...

Do you need the exact intact mass of proteins or antibodies?

Intact mass analysis of proteins by ESI-MS and correlation with the sequences enables a detailed understanding of the protein heterogeneity. Also, you can use it to confirm correct protein expression.

Furthermore, the intact mass of co-eluting proteins/antibodies and integration of HPLC peak areas allow a semi-quantitative analysis of protein contaminants and degradation products.

When can you benefit from intact mass analysis?

  • Detection of post-translational modifications, e.g., oxidations
  • Glycoprofile characterization
  • Identify contaminating proteins that are not well separated by UV-HPLC. (coeluting proteins or shoulders etc.)
  • Stability study – characterize degradation products
  • Drug substance batch comparison

PROTEIN MASS

Intact mass analysis by LC ESI-MS is the golden standard for proteins up to 250 kDa.

ANTIBODY MASS

It is possible to conduct the intact mass analysis with and without reduction. We also offer protein mass analysis before and after removing N-glycans and after enzymatic cleavage into specific sub-units.

Please send us a request so we can adjust the protein mass analysis to meet your specific needs.

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The process:

  • 1 You provide us with at least 10 µg protein or 100 µg antibody
  • 2 Analysis of your sample with LC ESI-MS to determine the intact mass with high accuracy
  • 3 Optional: We apply a custom MS method, specifically for your project
  • 4 You receive the report within 8-20 work days, depending on the analysis

More information:

See an example of how results are presented

Get sample report Intact mass analysis by ESI-MS of protein or antibody

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More information

We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

Technical details

Intact Mass Analysis by ESI-MS

By ESI-MS analysis, we first spray the sample in liquid phase through a capillary at high voltage into the MS instrument. This way, we measure the mass over charge ratio (m/z). For large biomolecules, the electrospray process results in multiply protonated molecules with a distribution of ion species at m/z range from 1-3000.

We then determine the protein mass of the molecule using a deconvolution algorithm that calculates the mass of the intact non-protonated protein, peptide, or antibody. The ESI-MS process requires that the sample preparation is quite pure without interfering with salts or detergents.

We can also purify the sample by reversed-phase HPLC (Agilent 1200 system) using a short C8 column before analyzing it on a Q-TOF mass spectrometer (Bruker Maxis Impact system). The mass of the sample is determined by deconvolution of the obtained raw spectra using the MaxEnt algorithm. The high resolution and accuracy of the Q-Tof instrument thus result in a very accurate mass determination within 0-3 Da of the theoretical mass.

For antibodies, we analyze the sample in the intact form and after reduction with DTT.

Protein HPLC chromatogram - intact mass analysis by ESI-MS

HPLC chromatogram with protein mixture

Sample preparation

For intact mass analysis, submit protein or antibody samples in liquid or as lyophilized material.

The chromatographic purity should be >90%.

Minimum sample amount: ~ 10-50 microgram protein, ~ 100 microgram antibody

Avoid detergents and keep buffer concentration at a minimum. LC ESI-MS analysis works on samples containing small amounts of salts or urea, but the best result is obtained with low buffer strength in volatile solvents without detergents.

  • Put the protein/peptide/antibody into a microcentrifuge tube.
  • Use Eppendorf Safe-Lock tubes or similar tubes from your lab.
  • Lyophilize or speed vac the antibody to a solid sample to ensure stability during shipment.
  • Alternatively, freeze the liquid sample for cold shipment.

Attention

Please note that intact mass analysis of intact proteins/antibodies by mass spectrometry does NOT work on samples from gels or PVDF membranes.

If your sample contains detergents of any sort, send us a request through the contact form to hear about the analytical capabilities.

Related services

You can combine this analysis with other analyses such as:

Meet the experts

For this type of analysis our experts include:

Do you need help?
Li Peng Lundgren

PhD in Protein Chemistry

Read more
Do you need help?
Martin Villadsen

MSc in Engineering within Medical Biotechnology

Read more

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