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Multiple Reaction Monitoring

  • Quantify specific proteins in a complex mixture
  • Identify up- or down-regulation of specific proteins
  • Save time for development of ELISA assay
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Absolute quantification of a specific protein in a complex matrix – by MRM

The high sensitivity and specificity of Multiple Reaction Monitoring (MRM) using mass spectrometry are of great advantage for selective measurement of specific proteins, e.g. host cell protein, in very complex mixtures.

First and foremost, the technique provides both ID and absolute amount of the specific analyte. In that way, it can be used f...

Absolute quantification of a specific protein in a complex matrix – by MRM

The high sensitivity and specificity of Multiple Reaction Monitoring (MRM) using mass spectrometry are of great advantage for selective measurement of specific proteins, e.g. host cell protein, in very complex mixtures.

First and foremost, the technique provides both ID and absolute amount of the specific analyte. In that way, it can be used for the measurement of Host Cell Proteins (HCPs) of concern either in individual process steps or in the final drug substance. Furthermore, it is complementary to rtPCR, Western blotting, and ELISA.

What are the advantages of Multiple Reaction Monitoring (MRM) by mass spectrometry?

The MRM assays developed by Alphalyse have several advantages over other protein quantification methods. Advantages compared to methods like HPLC analysis and antibody-based ELISA assays include:

  • Direct LC-MS/MS measurement of analytes. Additionally, indirect measurement of protein-antibody binding.
  • Multiplex analysis. We thus measure multiple proteins in the same mass spectrometry run.
  • High reproducibility (5% CV) of relative protein quantification when combined with internal standards.

Need help with the measurement of a specific host cell protein?

We develop the MRM LC-MS/MS analysis specifically for your protein and project. As we analyze your samples on a fee-for-service basis, please contact us to receive a quote.

MoreLess details

The process:

  • 1 Contact us to discuss your project and receive a project proposal
  • 2 Analysis phase lead by Alphalyse appointed principal investigator
  • 3 Report w. the absolute quantity of individual proteins
  • 4 Follow-up by phone or email

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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 description

Introduction to Multiple Reaction Monitoring

The absolute amount of a specific protein in complex matrices is based on selecting specific peptides as signature peptides. We use it for assay development based on Multiple-Reaction-Monitoring (MRM). A typical project consists of three parts:

Part 1: A feasibility study

The purpose of the feasibility study is to investigate the possibility of developing an assay that can be used to quantify the specific protein in the matrix. The feasibility study includes:

  1. Planning phase. First, we evaluate potential sample preparation protocols and signature peptides, i.e., specific marker peptides, based on the protein sequences.
  2. Digest and LC-MS/MS analysis. We perform the digest with urea as a denaturation buffer and the proteases Lys-C and trypsin. This next step ensures that specific signature peptides form and that/these peptides do not appear as part of miss-cleaved peptides or modified peptides (oxidized, deamidation, etc.).
  3. We then prepare a short report evaluating if it is possible for Alphalyse to develop an assay for the project. It also includes an estimation of LOQ and LOD.
Calibration curve for MRM quantification by mass spectrometry

Standard curve for MRM measurement of specific HCP

Part 2: Assay development

After we agree to start the development of an assay, we usually follow these steps:

  1. Optimization of sample preparation and digestion of the proteins for mass spectrometric identification of released peptides.
  2. Ordering of isotope-labeled peptide standards (heavy peptides).
  3. Sample preparation and digestion of the protein with the addition of heavy peptides for quantitative measurements. Includes selection of optimal product-ions for each peptide. We also evaluate quantitative results and select quantitative peptides for quantification assay.
  4. Integration of workflow; sample prep. incl., reduction/alkylation, protein digestion, peptide cleanup, LC-MS/MS analysis, and MRM measurement of, e.g., host cell protein. We set up quantitative measures using heavy peptides and external calibration curves on the protein reference standard.
  5. Finally, we plan the assay qualification and demonstration of measurement range, linearity, precision, and accuracy.

Part 3: Sample analysis

We measure actual samples and prepare the report when the final assay is ready. We also offer to go over the results at a telephone conference so you can get answers to possible questions regarding the data.

MRM chromatogram - mass spectrometry host cell protein measurement

MRM areas of the corresponding non-isotope peptides

Sample preparation

Sample requirements for MRM measurement

Protein measurement by mass spectrometry requires a protein in sufficient amounts to obtain good data. Developing MRM quantification assays requires a protein in pure form and a matrix sample without the protein similar to the matrix of the actual samples to be analyzed. Thus, please prepare the samples in a clean laboratory to avoid contamination with human keratin.

You can submit protein samples in liquid or lyophilized.

  1. Purify the protein

    1. The chromatographic protein/peptide purity should be >90%.
    2. Avoid detergents and keep buffer concentration at a minimum. LC-ESI MS works on samples containing small amounts of salts, urea, or detergent. However, we obtain the best result with low buffer strength in volatile solvents without detergents.
    3. Minimum amount ~ 1 mg

  2. Put the protein sample into a microcentrifuge tube

    1. Use an Eppendorf Safe Lock tube or similar tubes from your lab.
    2. Lyophilize or speed vac the protein to a solid sample. It ensures protein stability during shipment.
    3. Alternatively, freeze or refrigerate to +4 oC for cold shipment of the liquid sample.
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Meet the experts

For this type of analysis our experts include:

Do you need help?
Janne Skaarup Crawford

BSc in Cell Biology and Biochemistry

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Do you need help?
Rikke Raaen Lund

PhD in Biomedicine

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