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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 specific protein in complex matrix – by MRM

The high sensitivity and specificity of Multiple Reaction Monitoring (MRM) using mass spectrometry are of great advantage for selective quantification of specific proteins in very complex mixtures.

First and foremost, the technique provides both identification and quantification of the specific analyte. In that way, it can be used for monitoring H...

Absolute quantification of specific protein in complex matrix – by MRM

The high sensitivity and specificity of Multiple Reaction Monitoring (MRM) using mass spectrometry are of great advantage for selective quantification of specific proteins in very complex mixtures.

First and foremost, the technique provides both identification and quantification of the specific analyte. In that way, it can be used for monitoring Host Cell Proteins (HCP’s) 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, not 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 quantification when combined with internal standards.

Need help?

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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    Protein characterization for optimization of manufacturing processes.

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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 quantification of specific protein in complex matrices are based on the selection of specific peptides as signature peptides. We use it for assay development based on Multiple-Reaction-Monitoring (MRM). A project is typically divided into three parts:

Part 1: Feasibility study

The purpose of the feasibility study is to investigate the possibility to develop an assay that can be used for the quantification of 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. This next step ensures that specific signature peptides are formed and that this/these peptides do not appear as part of miss-cleaved peptides or modified peptides (oxidized, deamidation etc.). The digest will be performed with urea as denaturation buffer and using the proteases Lys-C and trypsin.
  3. We then prepare a short report evaluating if it is possible for Alphalyse to develop an assay that can be used for the purpose of the project. It also includes an estimation of LOQ and LOD.
Calibration curve for MRM quantification by mass spectrometry

Standard curve for MRM quantification of specific HCP

Part 2: Assay development

After we agreed to start development of an assay we usually follow these steps:

  1. Optimization of sample preparation and digest of the proteins for mass spectrometric identification of released peptides.
  2. Ordering of isotope labeled peptide standards (heavy peptides).
  3. Sample preparation and digest of the protein with 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. We set up quantitative measurements using heavy peptides and external calibration curves on the protein reference standard.
  5. Finally, we plan qualification of quantification of the assay; demonstration of measurement range, linearity, precision and accuracy.

Part 3: Sample analysis

When the final assay is ready, we measure real samples and prepare the report. We also offer to go over the results at a telephone conference so you can get answer to possible questions regarding the data.

MRM chromatogram - mass spectrometry

MRM areas of the corresponding non-isotope peptides

Sample preparation

Sample requirements for MRM quantification

Protein quantification by mass spectrometry requires a protein in sufficient amounts to obtain good data. It is thus important that samples are prepared in a clean laboratory to avoid contamination with human keratin. To develop MRM quantification assays requires a protein in pure form and a matrix sample without the protein similar to the matrix of the real samples to be analyzed.

Protein samples can be submitted 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 can be done 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 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. This 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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