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Multiple Reaction Monitoring

for absolute or relative protein quantification

The Alpha-Quant™ assays developed by Alphalyse

  • Do you want to quantify specific proteins in a complex mixture?
  • Do you not have an ELISA assay or suitable antibody for quantification?

The high sensitivity and specificity of Multiple Reaction Monitoring (MRM) is of great advantage. That is why we use it for selective quantification of specific proteins in very complex mixtures. This technique provides both identification and quantification of the specific analyte. Furthermore, it is complementary to rtPCR, Western blotting and ELISA.

Advantages of Multiple Reaction Monitoring (MRM)

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

  • Direct LC MS/MS measurement of analytes. – Not indirect measurement of protein-antibody binding.
  • Multiplex analysis. – We can measure multiple peptides/proteins in same LC MS run.
  • High reproducibility (5% CV) of relative quantification, when combined with internal standards.

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

Services/Products
Protein analysis by Alphalyse

Quantification of specific protein by MRM LC-MS/MS

  • Quantification of one specific protein in complex matrices
  • Identification and qualification of signature peptides
  • Selection and synthesis of Stable Isotope Peptides
  • Quantification by MRM LC-MS/MS

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Introduction
Alphalyse has developed absolute protein quantification assays for a variety of proteins and various applications in protein drug development. The Alpha-Quant™ assays have been applied in protein research/discovery, up- and down-stream process development in protein manufacturing, cell line development, fermentation optimization, protein purification, protein formulation, in-process control analysis, pharmacokinetics measurements in toxicology, and analysis for regulatory documentation.

Alpha-Quant™ is applied to quantitation of specific individual proteins, and can be used for single protein analysis and for multiplex quantitation of multiple proteins in the same analysis. This includes measurement in complex matrices such as serum/plasma and raw fermentation lysates with hundreds of other proteins, and measurement of purified natural proteins and recombinant proteins for non-medical and for medical use.

Specific application examples include:

  • Quantitation of formulated vaccine protein
  • Multiplex quantification of proteins in multivalent vaccine
  • Quantification of allergens for allergy vaccine development
  • Multiplex quantification of drug substance sequence isoforms
  • Multiplex quantification of antibody heavy and light chain ratios
  • Multiplex quantitation of antibody mixtures
  • Multiplex quantification of drug substance and contaminating host cell proteins
  • HTP quantification of therapeutic protein in serum samples for pharmacokinetics and toxicology clinical studies.

Quantification Workflow using stable isotope labeled internal standard peptide

  1. Select representative signature peptides
  2. Synthesize AQUA peptides with heavy isotope label (C13, N15)
  3. Optimize peptide purification and LC MS/MS conditions
  4. Add known amount of AQUA peptide to sample for quantification
  5. Digest protein with protease and purify peptides
  6. LC-MS/MS and MRM measurements for quantitative analysis

Protein quantitation using stable isotope labelled internal standards. Signature peptides are selected, synthetic ISTD peptides are added in known amount and used for normalization and quantification by MRM measurements.

Introduction
Protein quantification by mass spectrometry requires a protein in sufficient amounts to obtain good data. It is important that samples are prepared in a clean laboratory to avoid contamination with human keratin.

To develop MRM quantification assays requires the 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, but the best result are obtained with low buffer strength in volatile solvents without detergents
    3. Minimum amount ~ 1 mg
  2. Put the protein sample into microcentrifuge tube

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

Ship your protein samples by a courier company of your own choice

Place your samples into micro tubes and pack in a suitable transport box.

  • Contact a courier company of your choice (FedEx, UPS, DHL, TNT, World Courier etc.). Use the following sentence to describe the content: “Protein material – Non-toxic, non-hazardous, for research purpose only“.
  • Use cool packs or cooling units if your samples should be stored below 5°C/40°F
  • Ship the samples to the office closest to you (Canadian customers, please ship to Europe):
Customers in the USA
Alphalyse, Inc.
200 Page Mill Road, Suite 100
Palo Alto, CA, 94306
USA
Att: Jette Williams
Tel: (650) 543-3193
Customers outside of USA
Alphalyse A/S
Unsbjergvej 4 D
DK-5220, Odense
DENMARK
Att: Anette Rasmussen
Tel: +45 6310 6500
Order a prepaid FedEx Air Waybill < 1 lb./ 500g

A prepaid FedEx Air Waybill can be used for fast and easy shipment of samples in packages up to 1 lb./ 500g.
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack in suitable transport box.
  • Print the PDF document and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to send your samples stored at room temperature, this option is usually the right choice. If you fill your box with cooling packs, the weight will most often exceed 1 lb./500g.

Order a prepaid FedEx Air Waybill < 10 lb./5kg (e.g. for packages with cooling packs)

This prepaid FedEx Air Waybill can be used for shipment of samples in packages up to 10 lb./ 5kg, with or without cooling packs (do not use dry ice!).
We will generate a prepaid Air Waybill, prefilled with the correct information, and send it to you by e-mail (PDF-format) within 24 hours.
Then follow these simple instructions:

  • Place your samples into micro tubes. Pack them in a suitable transport box. Fill with cooling units/cold packs (do not use dry ice!).
  • Print the PDF and stick it on the transport box or just hand it to your FedEx officer upon pickup.
  • Call to request a pickup from your local FedEx office.

If you plan to fill your transportation box with cooling packs, select this shipping option. If you plan to use dry ice, you cannot use this Waybill!

Get a quote for shipment by World Courier

The World Courier service can be used for shipment of samples of all types. If you select this option, we will contact you to provide a quotation based on location and shipping conditions. You will receive a prepaid Air Waybill which will be invoiced together with your analysis results. This is usually a good option if FedEx does not service your location.
With the prepaid World Courier service you get:

  • GxP compliant processes to ensure maximal security and traceability
  • Door-to-door solution on a dedicated vehicle
  • Possibility of providing qualified packaging, temperature monitoring and dry ice
  • Shortest possible transit-time through “next flight out”-procedure
  • Customs clearance and help with paperwork

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