Proteomics

 

Proteomics is the study of the complete set of proteins that is expressed at a given time in a cell, tissue, organ or organism, i.e. the proteome. Proteins are the machinery of the cell, responsible for performing the functions essential for cells to operate, survive, grow and divide. Proteins are also e.g. responsible for relaying signals or messages within and between cells that drive biological and metabolic processes. In addition, proteins are the main constituent of the physical structures of cells and organism.
There is a general interests to proteomics in life sciences, since as compared to e.g. genomics/functional genomics, proteomics is able to give much more complete understanding of the studied biological system. Due to events like alternative splicing and post-translational modifications (like phosphorylation, glycosylation, ubiquitinylation, myristyolation, palmityolation etc.), the number of different proteins expressed in an organism is by far greater than the number of its functional genes. The expression level of functional proteins is regulated at many post-transcriptional levels, like action of inhibitory RNA species, protein localization and degradation, in addition, post translational modifications, like phosphorylations, can have the role of switching an inactive protein to active. Due to this reasons, simple mRNA transcript measurements by functional genetics, may give a biased reading for the actual protein activity.

In most cases, changes at protein level determine the differences between sickness and health and can be used to monitor the progression of disease. Almost all drugs on the market and being developed act by trying to alter a protein (mal)function. Therefore, typically one of the first steps in drug discovery project is the identification and validation of the protein target(s) or markers associated with a disease (one type of biomarkers).

Our expertise in proteomics

We offer our customers a wide spectrum of proteomics expertise and services. Our services are based on nanoLC-MS/MS and other instrumentation platforms, coupled with bioinformatics and computational systems biology. The samples can be fluids, like serum, plasma, urine, cell lysates or solid tissue samples as well as processed samples like SDS gel plugs.

Below are some of our proteomics methods described. Please contact us to discuss your projects and how proteomics could help you.

Peptide mass fingerprinting for protein identification with mass spectroscopy

Peptide Mass Fingerprinting (PMF) is a fastest and cheap method of protein identification. The procedure involves optional protein enrichment by e.g. PAGE followed by excision of the band from the gel, alternatively protein identification can also be done directly from any protein sample in dissolved form.
Samples are digested with trypsin and the peptides are analyzed by a nanoLC-MS. MS analysis of the tryptic peptides will be conducted and the PMF data will be searched against protein database.
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Peptide mass fingerprinting for protein identification with tandem mass spectroscopy (MS/MS)

This mass spectrometry technique is an extension of peptide mass fingerprinting technique which is used mainly for confirmation of identity or to identity post- translational modification like phosphorylation, glycosylation etc.
We utilize a nanoLC-MS/MS instrumentation, which is a sensitive and reliable method for protein identification, allowing identification of unknown protein at low fmol levels. The second mass detector is analyzing the fragmentation of a particular precursor ion at different amide bonds and generating spectra of daughter ions. This allows the generation of the actual sequence data of the unknown peptide for its identification.
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Protein characterization – intact molecular weight analysis

The accurate mass analysis of proteins is typically needed when studying the post-translational modifications (PTM). Examples include analysis of the mass heterogeneity of glycoproteins, different phosphorylated variants, presence of ubiquitinylation and sumoylation, attachments of lipids or presence of disulphide (Cys-Cys) bridges. Accurate mass analysis can also be used to study the presence of alternatively spliced forms of the proteins as well as e.g. to check the batch to batch consistency/variability of therapeutic proteins or monoclonal antibodies.
The mass profile of a chosen protein is measured by nanoLC-MS. The mass spectra will be deconvoluted and further used to report the accurate observed mass values.
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Protein modification monitoring or monitoring of selected proteins by MRM mass spectroscopy

Multiple Reaction Monitoring (MRM) assay is considered the most selective and sensitive mass spectrometry technique to detect proteins of interest with enhanced specificity using selective ion monitoring scanning mode. During this process, complex biological protein samples will be enzymatically digested and peptides of specific masses are selected and all others are excluded followed by fragmentation of the selected peptides in MS and quantification of the known daughter ions. LC/MRM (SRM)-MS is a versatile tool for the discovery phase, quantitation of proteins during validation phase or post-translational modification profiling in complex biological samples.

The main advantage of this technology is in screening of biological samples for biomarker verification or quantitation. It rules out the need for specific antibodies or running any gels for protein separation or Western blotting. Transitions e.g. for potential serine/threonine or tyrosine phosphorylations will be predicted for a given protein in a given biological sample and quantified by MRM triggered scanning on nanoLC-MS/MS.
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Characterization of Protein Post-translational Modifications (PTM)

Post-translational modification (PTM) is a chemical modification of a protein after its translation often extending the range of functions of the protein by attaching to it other biochemical functional groups such as acetate, phosphate, various lipids and carbohydrates, changing the chemical nature of an amino acid or by structural changes via the formation of disulphide (Cys-Cys) bridges.
Among different types of PTMs, phosphorylations and glycosylations are the most common modifications. Other common PTMs include e.g. ubiquitinylation and sumoylation.
PTMs are analyzed in various applications, like studying signal transduction cascades, protein degradation tagging and analysis of the occupancy of the glycosylation sites.
This technique involves MS/MS fragmentation to determine the site and/or percentage of post-translational modification. PTM site are mapped with MS/MS fragmentation using a nanoLC-MS/MS.
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Protein quantitation by iTRAQ- isobaric Tag for Relative and Absolute Quantitation

Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) is a non-gel based multiplexed protein quantitation technique that provides relative and absolute measurements of in theory all peptides from different samples/treatments.
Trypsin digested iTRAQ labelled samples are pooled, fractionated and analyzed by liquid chromatography and analysed by tandem mass spectrometry (nanoLC-MS/MS). A database search is then performed using fragmentation data to identify the labelled peptides and hence the corresponding proteins whilst the iTRAQ mass reporter ion is used to relatively quantify the peptides. Quantitation of protein from multiple samples can be achieved in the same run.
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Proteome mapping (Global protein profiling) with Gel-LC-MS/MS

The main objective of proteome mapping is to profile the protein content of a given biological sample. Application of this technique will be mainly used to identify differentially expressed proteins different samples.
Gel-nanoLC-MS/MS (GeLC/MS) is a powerful and simple approach for proteomic analyses. Samples are run on 1-D SDS-PAGE to resolving proteins into protein bands of different molecular weights. After separation, protein bands are excised from the gel lane into equally sized segments. The greater the number of protein bands, the deeper the coverage of the sample. Each protein band is then in-gel digested with trypsin, and peptides analyzed by nanoLC-MS/MS. If further separation is needed, an additional strong cation exchange (SCX) liquid chromatography is used in addition to the reversed phase (C18) liquid chromatography (2D-LC) followed by identification by nanoLC-MS/MS known as MudPIT 2D-LC-MS/MS method.
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Proteome mapping (Global protein profiling) with LC-MS/MS (label-free mass spectrometry)

Label-free mass spectrometry is a totally non-gel based technique that can be also use for global protein profiling using full scan mode of LC-MS/MS. This technique also provides relative quantitation in expression of protein. This technique has advantages in terms of simplified sample handling and processing, low sample amount requirement (in μl) and no need of metabolic or chemical labeling.
Trypsin digested peptides are separated by strong cation exchange (SCX) liquid chromatography followed by reversed phase (C18) liquid chromatography (2D-LC) and identified using nanoLC-MS/MS, known as MudPIT 2D-LC-MS/MS method.
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Proteomics for biologics discovery

We offer our proteomics expertise also for customers discovering protein based biological drugs. We provide customized services in e.g. protein stability and kinetics in different biological fluids and tissue extracts, protein translocation like placental perfusion transport. These studies are always tailored to each project.

To discuss more, please contact us from this link.