The Proteomics Core at Mayo Clinic provides basic scientists and clinician-investigators with consultations, technologies and services for the analysis of proteins from tissues, cells or biological samples. By utilizing current and innovative proteomics techniques, the core advances scientific knowledge of disease biogenesis and contributes to improved patient care.
Services offered by the Proteomics Core include:
Peptide synthesis. Products produced by this service include synthetic peptides, peptide-conjugated immunogens, and 13C, 15N stable isotope-labeled peptides as target standards for multiple reaction monitoring mass spectrometry quantification. Phosphopeptides, glycopeptides, peptides with synthetic or custom amino acids, and biotinylated or fluorescently labeled peptides are also synthesized.
Most synthetic peptides comprise the 20 common D or L amino acids, but synthetic or custom amino acids can also be incorporated into the peptide. Peptides ranging in length from six to 60 amino acids can be ordered; longer peptides (greater than 60 residues) or peptides with multiple disulfide bonds are by special request.
- Molecular weight determination. Molecular mass determination over a wide molecular weight range is provided for peptide or protein confirmation. It is also used in determining truncations, mutations, disulfide bonds, modifications, impurities or degradants within samples.
Protein and peptide chromatography. Both fast protein liquid chromatography (FPLC) and high-performance liquid chromatography (HPLC) are included under protein and peptide chromatography:
- FPLC offers research-scale chromatographic fractionation of proteins from biological fluids, conditioned media, cell lysates and more. Isolation of monoclonal antibodies from ascites or hybridoma media is also available. A variety of protein separation chemistries are used; the most common are Protein A, Protein G, size-exclusion and ion exchange.
- HPLC is offered at a semi-preparative scale to provide peptide isolation, purification and characterization of noncomplex samples. It is also used to fractionate complex samples, as a step to reduce complexity for downstream processing. Available column chemistries include C4, C8, C18 and ion exchange.
- Protein electrophoresis. Gel-based separation of proteins of which proteins are separated by size and visualized via staining, either colloidal blue or silver. Protein electrophoresis is often used to determine the complexity of a sample, to separate proteins from one another, or as a tool to visualize a protein band of interest. Areas of interest in a gel band can be excised, digested by protease and identified via mass spectrometry techniques. Precast mini- and mid-sized gels are available.
Protein identification and characterization. Samples submitted for protein ID are generally digested with trypsin. The resulting peptides from the protein are examined using mass spectrometry, then analyzed with proteomics software from which a list of proteins is reported. Protein characterization includes the identification and location of post-translational modifications (PTMs), the detection of mutations or truncations, and the determination of intact molecular mass.
PTMs are site specific. To have the best chance of identifying these sites, larger amounts of starting protein material are often needed. This is to be able to use multiple proteases, mass spectrometry runs and maximize protein coverage. Additional personnel effort, experimentation, instrument time and manual data analysis are needed for these sample-dependent projects.
Targeted quantitation. Absolute quantitation of targeted peptides and proteins is an approach that uses labeled internal standards and labeled standard curves. Relative quantitation of targeted peptides and proteins is accomplished by comparing two measurements against each other within a sample.
A considerable amount of development time and effort is required to determine the best way to process samples, to build instrument methods for mass spectrometry and to determine the data-mining methods for analysis. It is very important to consider control samples. A meeting is required prior to starting this type of project.
Differential proteomics. Mass spectrometry-based differential proteomics aims to define differences between groups or treatments and for the initial "discovery" of potential biomarkers. It involves comparing distinct proteomes, such as cells, tissues or cell lines that are normal, diseased or treated. Generically, almost all differential projects involve a certain number of process steps that include enzymatic digestion of the proteins, often a fractionation step at either the protein level (gel electrophoresis, for example) or the peptide level (such as strong cation exchange), followed by data acquisition of the sample or its fractions by liquid chromatography-tandem mass spectrometry, and analysis of the data using bioinformatics software.
The success of a differential proteomics project starts with a good experimental design. The number of samples and the sample acquisition, stabilization and storage methods are critical for success. A meeting or teleconference is required prior to starting this type of project.
Proteomics Core staff members are available to meet with investigators and research teams to provide advice and consultation regarding project design and discuss effective implementation of core services. Additionally, the core provides letters of support, documentation and methodology information for grant applications that include some aspect of protein analysis.
Acknowledging the core
When publishing data or other work that has been facilitated by the Proteomics Core, please provide recognition to the group, person, or people responsible for the work, either by authorship or by acknowledgment. By doing so, you help to secure future resources and funding to the Proteomics Core, enabling the facility to maintain cutting-edge technology and promote further proteomic research.
Suggested text: This publication was made possible with support from the Medical Genome Facility Proteomics Core, Mayo Clinic. The core is a shared resource of the Mayo Clinic Cancer Center (NCI P30 CA15083).