Protein Quantification

Need to know the concentration of specific proteins in a complex sample ?

Alphalyse offers a unique protein quantification service for specific proteins in complex matrices. The protein quantification is based on isotope dilution LC-MS/MS and Multiple Reaction Monitoring (MRM) of specific peptides from the protein(s) of interest. Absolute protein quantification by isotope dilution is well-established in the pharmaceutical industry for quantification of small molecules, and has recently been adapted for absolute quantification of proteins.

The Alpha-Quant™ assays developed by Alphalyse have several significant advantages over other protein quantification methods, including HPLC and antibody based ELISA assays.

Advantages:

Short assay development time – 6-12 weeks
Accurate within 10-15%
Sensitive down to 100 ppm
Specific for selected analytes
Multiplex measurements – multiple proteins and individual protein forms can be quantified in same analysis

We develop the protein quantification assay for your specific proteins, and analyze your samples on a fee-for-service basis.

Please contact us to discuss your specific project in more detail. We will provide you with a full project proposal, including analysis strategy, timelines and costs.

Quantification Workflow using stable isotope labeled internal standard peptide

Select representative signature peptides
Synthesize AQUA peptides with heavy isotope label (C13, N15)
Optimize peptide purification and LC MS/MS conditions
Add known amount of AQUA peptide to sample for quantification
Digest protein with protease and purify peptides
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.

More About Alpha-Quant™

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.

Alpha-Quant™ method development matrix

Development of the quantification assay for your specific proteins requires optimization of experimental conditions for the specific protein amino acid sequence and the matrix where the protein concentration is measured. At Alphalyse we apply our know-how and experience from various quantification projects using the method development matrix described below.

Process step		Method description
Protein purification		The proteins of interest may be analyzed directly, or after additional cleanup from the sample matrix. Cleanup may for example include protein depletion, ultrafiltration, size-exclusion chromatography, antibody affinity purification, protein A or protein G antibody purification.
Protein denaturation		For maximum enzymatic cleavage efficiency the protein may be denatured by addition of organic solvents, buffers, detergents, chaotropic agents and heat treatment.
Cysteine reduction/alkylation		Proteins containing disulfide bonds may be further denatured by cysteine reduction and alkylation.
Enzyme digestion		Enzymatic protein cleavage is often obtained by trypsin digestion that usually results in peptides that are easily purified and measured by mass spectrometry. Other enzymes and cleavage agents may also be used, it is important that the cleavage is specific, complete and reproducible, and that the peptides subsequently can be purified. Other enzymes include; Lys-C, Glu-C, Asp-N, and chymotrypsin.
Isotope labeled peptides		Isotope labeled internal standard peptides are selected based on experimental data showing that the peptide is generated by enzymatic cleavage. Peptides are chosen for optimal sequence and MS measurements.
Peptide purification		The natural and isotope labeled peptides can be purified and cleaned up prior to the LC-MS/MS analysis. Typical techniques include on-line liquid chromatography (reverse phase material, Hilic material, ion-exchange material, solid-phase-extraction material), as well as off-line cartridges and 96-well plate systems.
LC-MS/MS		LC-MS/MS conditions are optimized for LC separation and MS/MS fragmentation for each peptide.
MRM measurements		Optimal and quantitative MRM measurements are setup for the natural peptides and for the stabile isotope labelled internal standard peptides.
Quantitation		Quantitative measurements and calculations are setup for each MRM measurement, each peptide and each protein to be quantified.
External calibration		External calibration curves are generated for each peptide based on measurement the reference protein standard protein spiked in known amounts into the relevant measurement matrix.
Qualification of quantification assay		The final assay with all the optimized assay parameters is qualified, including concentration measurement range, precision, and accuracy.