Quantitative Proteomics

Dundee Cell Products provide a range of quantitative proteomic analysis services. We have state-of-the-art mass spectrometers and expertise in the latest applied proteomics technology.

Our Quantitative Proteomic Services Includes:

For phosphorylation site mapping please see our non-quantitative mass spectrometry page (http://www.dundeecellproducts.com/services/mass-spectrometry).

SILAC Quantitative Proteomics

SILAC-based quantitative proteomics is a novel and exciting technology used for high throughput quantitative analysis of large protein complexes and protein-protein interactions. This approach provides a quantitative and unbiased strategy that can reveal how specifically either inhibitors, or other perturbations, affect the dynamic properties and/or cellular distributions of proteins. It can also be used as a sensitive and effective method to determine the specific interaction partners of proteins under study. Dundee Cell Products offers a custom SILAC service that includes consultancy, experimental design, sample processing, mass spectrometry, data analysis and interpretation of results.

SILAC studies are performed using cells with the normal isotopes 12C and 14N in which the amino acids arginine and lysine are to be replaced with the heavy isotopes 13C and/or 15N. Heavy isotope substitution is made on the basic amino acids arginine and lysine because these are the sites of trypsin cleavage, thereby conveniently generating a set of tryptic peptides each with increased mass that can readily be detected and quantitated by mass spectrometry. When mammalian cells are grown using SILAC reagents, the proteins become substituted with the heavy forms of arginine and lysine. Subsequent MS analysis resolves and quantitates the relative ratios of each isotopic form of every peptide. This determines whether the level of each protein in the complex has increased, decreased or remained at the same level after the treatment used. The accuracy of the quantitation is enhanced because the measurements are made at the level of individual peptides, while typically multiple (2-10+) peptides are analysed for each protein. In this way the response of large numbers of endogenous, untagged proteins (many hundreds to thousands) in the cell or a specific complex can be evaluated quantitatively at multiple time points in a single experiment.

iTRAQ, TMT and Label-Free Quantitative Proteomics

ITRAQ, TMT and Label-Free Quantitative proteomics provide an unbiased and untargeted method of protein quantification. All of these techniques are suitable for drug analysis, time course studies, biomarker identification, protein expression profiling and functional characteristation of protein components in non-cell based assays.

For iTRAQ and TMT studies samples from different treatments or conditions are lysed to extract the proteins. Protein concentration is then determined through a standard protein assay and enzymes such as trypsin used to digest the proteins, generating proteolytic peptides. Each sample is then labelled with a different isobaric reagents / tags which labels the N-terminus and primary amines of peptides and proteins. The labelled digests are then pooled, separated via our nanoflow LC system and then analysed using a state-of-the-art Thermo Orbitrap mass spectrometer (LTQ Orbitrap Velos Pro). MS/MS analysis of the sample produces the relative abundance ratio of the peptides present which are then quantified via fragmentation of the label. Data base analysis identifies the labelled peptides and corresponding proteins. Data is provided in an electronic format. ITRAQ and TMT vary by the number of samples / different labels that can be analysed at once. For example the two main reagents used for iTRAQ analysis are 4-plex (4 samples) and 8-plex (8 samples) while with TMT you can use 6-plex (6 samples) or 10-plex (10 samples) reagents.

Whilst metabolic labelling has become the gold-standard of quantitative proteomics, it is not always appropriate, or is difficult to use for some analyses e.g. projects using human or animal tissues, patient samples, serum and primary differentiated cells that are not able to undergo multiple cell division cycles in culture. Therefore label-free quantitation may be more appropriate. We are always happy to discuss alternative approaches that can be taken to provide clients with the best possible quantitative proteomic approach for their specific research project.

Post-Translational Modification Proteomics

Methylation proteomics:

Dundee Cell Products methylation proteomics service allows the customer to obtain information on the methylation status of their biological samples. Our workflow involves using mono-methylated arginine specific antibodies to enrich for modified peptides in protease digested cell or tissue extracts. The antibody-purified modified peptides are then analysed by LC-MS/MS to determine sites of modification and to quantify peptides/proteins thus generating quantitative profiles of modified proteins within the sample.

Acetylation proteomics:

Our acetylation proteomics service provides the customer with information on the acetylation status of their biological samples. Our workflow involves using high affinity acetylated-lysine (Ac-K) specific antibodies to enrich for acetyl-modified peptides in protease digested cell or tissue extracts. The antibody-purified modified peptides are then analysed by LC-MS/MS to determine sites of modification and to quantify peptides/proteins in order to obtain quantitative profiles of modified proteins within the sample.

Ubiquitination proteomics:

The ubiquitin proteomics service we offer uses the K-GG motif specific antibody to enrich for ubiquitinated substrates by pulling down ubiquitinated peptides in protease digested cell or tissue extracts. The antibody-purified modified peptides are then analysed by LC-MS/MS to determine sites of modification and to quantify peptides/proteins to obtain quantitative profiles of modified proteins in the sample.

Phosphoproteomics:

Post-translational phosphate modifications occur within one-third of all cellular proteins. Our phosphoproteomics service can identify and quantify proteins containing a phosphate group by means of different quantitative mass spectrometry approaches, such as SILAC and iTRAQ labelling. Our service can identify which protein or pathway are activated by phosphorylation or indicate which proteins might be potential drug targets.

For phosphorylation site mapping please see our non-quantitative mass spectrometry page (http://www.dundeecellproducts.com/services/mass-spectrometry).

Benefits of quantitative proteomics:

Apart from the use of quantitative proteomics in basic investigative research benefits in other areas include:

  • Significant cost savings can be realised by removing compounds from the drug discovery and development process that are non-specific or may cause side effects prior to initiating clinical trials
  • Reliable identification of protein interaction partners in cell extracts by eliminating non-specific interaction signals, hence faster and cheaper determination of novel protein function(s) in the cell
  • High throughput quantitative protein expression profiling
  • Faster identification of drug targets from phenotypic screens
  • Faster and better characterisation of fusion proteins used in cell-based assays)
  • Better and faster hit-target profiling in mammalian cells hence better target validation
  • Investigative toxicology: faster determination of mechanism of action of xenobiotics in human cells
  • Faster and more efficient novel biomarker identification

Benefits of using Dundee Cell Products:

  • Our service includes consultancy, experimental design, sample processing, mass spectrometry, data analysis and interpretation
  • UK-based company, contactable during normal lab hours
  • Project-managed by postdoctoral scientists, from design to delivery
  • Competitively priced and cost-effective, no hidden charges
  • High customer satisfaction rate – rapid reliable service (usual turnaround time for standard proteomics 3-4 wks)
  • Complete client confidentiality, 100% IP retained by client

What information do I need to send to Dundee Cell Products Quantitative Proteomics Service?

Please provide us with a brief summary as to what you would like to do. This allows us to understand the background to the project underpinning the proposal and what you want to achieve. This will then allow us to look at the scientific approach, evaluate the project to ensure feasibility and then advise as to the best strategy to accomplish this.

Frequently Asked Questions