What's News at Nucro-Technics

Protein-Specific Bioanalytical Services

Protein-Specific Bioanalytical Services

Protein-Specific Bioanalytical Services

List of Classical Protein Biomarkers of Toxicity Quantified in Rat Plasma

Protein Biomarker Name Type of Test/Mechanism
Alanine aminotransferase (ALT/ALAT1) Hepatocellular/Liver function
Aspartate aminotransferase (AST/AATC) Hepatocellular/Liver function
L-lactate dehydrogenase A chain (LDHA) Liver function
Serum albumin (ALB/ALBU) Liver function
5′-nucleotidase (CD73/5NTD) Hepatobiliary/Liver function
Sorbitol dehydrogenase (SDH1/DHSO) Hepatocellular
Glutamate dehydrogenase (GLDH/DHE3) Hepatocellular
Glutathione-S-transferase alpha-1 (GST/GSTA1) Short-term pathological change / hepatocarcinogenicity
Glutathione-S-transferase alpha-3 (GST/GSTA3) Short-term pathological change / hepatocarcinogenicity
Haptoglobin (HPT) Liver hypertrophy, bile duct damage, inflammation

 

Intact Protein and Peptide Mass Determination
This service enables accurate determination of the molecular weight of a purified or recombinant protein, including protein/antibody drug conjugates (ADC).

Protein Identification
This service allows identification of protein components in a complex mix. Protein mixtures will be precipitated, reduced, alkylated and subjected to either in-solution digestion or in-gel digestion of proteins in 2D gel spots, SDS-PAGE bands or non-denaturing gel bands. The resulting peptides are analyzed by LC-MS-MS and fragmentation spectra matched to protein sequence databases elucidating the list of proteins present in the mix.

Profiling of Complex Protein Mixtures/Protein Biomarker Discovery
In order to catalogue protein components of complex protein mixtures, such as blood plasma, cell culture or tissue lysates, complexity of these mixtures need to be reduced. Using HPLC or other fractionation techniques allows going deeper into proteome resulting in more good quality identifications. Protein mixture profiling is the basic assay for biomarker discovery. If sample loading was normalized by weight, the crudest way to estimate abundance of specific proteins is spectral counting. This technique relies on counting all MS/MS spectra assigned to (and used to identify) the protein of interest.

Immunodepletion is a technique often used for in-depth profiling of blood plasma. This technique allows for depletion of albumin and several other high expressing proteins that comprise about 90% of plasma proteome. Depletion of these components instantly enriches all other plasma proteins which lead to a better coverage of the proteome in an MS experiment, allowing identification of more low-abundant proteins, otherwise masked by these major plasma proteins.

Immunoprecipitation is a useful enrichment technique when studying low abundant proteins. It allows several orders of magnitude enrichment of a target protein, enabling MS quantification of even very low abundant proteins.

Cell membrane (cell surface) proteome enrichment can be achieved using a method we developed for tissue culture cell lines. This method allows enrichment, characterization and quantification of receptors that transduce extracellular signals inside the cell, many of which are anticancer drug targets.

Protein Structural Analysis
This service allows for experimental determination of modification (conjugation) sites. The protein will be in-solution digested and the resulting peptides monitored for presence of post translational modification or for a known drug (small molecule) modification. Specific proteases other than trypsin may be used to generate peptides which may be used to monitor the modification sites. This service requires a high purity protein sample.

Protein Quantification by SRM/Protein Biomarker Validation
Selected Reaction Monitoring (SRM/MRM) is a well-established technique initially developed for quantification of small molecules. This technique is recognized for its robustness, accuracy and speed both by the bioanalytical industry and the regulators (FDA, EMEA, Health Canada etc.) In this approach a representative peptide resulting from proteolytic digestion of a protein is selected. Since the peptide is derived from the protein to be quantified, the quantification relies on assumption of 1:1 molar ratio of the peptide to protein. Such a peptide is quantified similarly to a small molecule. Therefore, regulatory schemes used for the validation of small molecules can be directly applied to validate the quantification of proteins. Adapting SRM technology to proteomic analysis allows for several significant benefits:

  1. High sensitivity and exceptional selectivity puts this technology in direct competition with ELISA;
  2. Dynamic range of four to five orders of magnitude is significantly better than that of ELISA;
  3. Rapid method development does not depend on material availability (no antibody required);
  4. Ability to multiplex up to 1000 analytes in a single analytical run;

This technique is perfectly suited for protein biomarker validation, projects where mid-to-high expressing proteins are present in a highly complex biological matrix, where multiple proteins or multiple fragments/variants of a single protein need to be measured.

There are two strategies for protein quantification using SRM technique:

  1. Relative quantification, whereby the response of a peptide present in an experimental sample is compared to that in a control sample (i.e. treated vs. untreated). The method is rapid and inexpensive because it can be developed based on the experimental sample alone.
  2. Absolute quantification, where a heavy isotope labeled peptide (Stable Isotope Labeled – SIL, AQUA peptide) otherwise identical to an endogenous peptide is generated. The response of heavy and light peptides is directly comparable as they are measured concomitantly in the same analytical run. If highly purified heavy labeled peptide is used, this method provides for ultimate confidence and allows for very accurate measurement of molar concentration of protein in question. Method development takes longer, as time of generation of a heavy isotope labeled peptide needs to be considered.

MS1-based Quantitation of Proteins/Protein Biomarker Discovery
Proteins are quantified based on a response measured in the MS1 scan of a high resolution, high mass accuracy instrument (Q-Exactive Orbitrap). Such quantification can be performed either relative to a separate run of a control sample or in a same analytical run using various labeling techniques to distinguish the samples (SILAC, iTRAQ, etc.) This technique allows identification and simultaneous quantification of a large portion of proteins present in the mix. Providing benefits of discovery and speed, this technique generates extremely rich datasets, highly suitable for protein biomarker discovery studies. However, it is not as precise and sensitive as the SRM technology, especially in samples that require large dynamic ranges.

For more general information regarding Nucro-Technics’ Bioanalytical capabilities, click here.

For further information about our protein-specific bioanalytical services, to receive a quotation, or to schedule a test, please contact NUCRO-TECHNICS.