SERVICES

We currently provide services only for investigators of Mayo Foundation and their allied health staff.

Bioinformatics

The MPRC currently employs two software engineers with responsibility for maintaining and developing tools for protein database searchs, user interfaces and developing and implementing algorithms utilized for biomarker discovery. Additionally, this group has developed tools for streamlined reporting of protein data base searches from multiple search engines.

Electrophoresis

The MPRC harbors a wide suite of tools for 1D and 2D polyacrylamide gel electrophoresis. This includes equipment for 1D and 2D analytical and large scale (20 x 20 cm) gels. Additionally, specialized staining protocols for phospho- and glycoproteins is available.

Silver stained and non-fluorescently stained gels and blots are scanned by a GS800 (Biorad) scanner. Fluorescent images are scanned by a FX Pro Plus scanner.

PDQuest 2D (Biorad) or Z3 (Compugen) gel analysis software is utilized for archiving gel images, as well as comparative analysis of multiple gels.

Manual or robotic spot cutting is utilized to excise spots for protein identification.

For more specific questions related to electrophoresis please contact Dr. M Cristine Charlesworth at 507-284-9269

Peptide Synthesis

The MPRC Peptide Synthesis Facility, formerly known as The Protein Core, was established as a shared research resource on the Mayo Rochester campus in 1984. Daniel McCormick, Ph.D. is the facility's founder and current director. The facility, along with the Mayo Clinic Cancer Center and the General Clinical Research Center are part of the Mayo genomic and proteomic effort. The facility is housed in Guggenheim 1633 and comprises part of the Mayo Proteomics Research Center (MPRC). The facility provides services in peptide synthesis to Mayo investigators and also conducts research and development of new methods in the field of solid phase peptide synthesis.

Mayo investigators interested in using specific services offered by the MPRC Peptide Synthesis Facility are encouraged to contact the facility's director, Dr. McCormick (507-284-4992 , or any of the facility's personnel (507-284-2457). For inquiries regarding peptide synthesis, investigators should contact Jane Peterson (507-284-9434) or Patricia Caffes (507-286-1997). E-mail inquiries regarding services may also be directed to personnel of the MPRC using the e-mail addresses, which are listed on the "Personnel" page of this website.

Peptide synthesis information
The primary purpose of the synthesis component of the MPRC Peptide Synthesis Facility is to provide the user with purified synthetic peptides and peptide nucleic acids. Peptide lengths can range anywhere from 3 to 70 amino acids. Custom synthesis is also provided for peptides requiring D-amino acids, phosphorylated amino acids, fluorescent labels, or disulfide bridge formation. Peptide immunogen design and production (KLH and BSA conjugation) is also available.

Solid phase peptide synthesis involves systematic amino acid chain assembly starting from the carboxy-terminus of the peptide, which is covalently linked to an insoluble polystyrene support and continuing toward the amino terminus. A peptide bond is formed between the deprotected alpha-amino group of one amino acid and the activated carboxyl group of the next amino acid.

Information required for synthesis

• Amino acid sequence of desired peptide
• Use of peptide
• Quantity of peptide desired

Sample protocol

• Using automated peptide synthesizer, desired amino acid sequence is made with the C-terminal residue attached to one of a variety of resins.
• Peptide is cleaved from solid support, and side chain protecting groups are removed by acid hydrolysis.
• Crude peptides are purified by high performance liquid chromatography.
• Peptide integrity is assessed by amino acid analysis, mass spectrometry, and/or sequence analysis.

Limitations

• Longer sequences possible with considerable lower yield and purity (>60 residues).
• Extremely hydrophobic peptides may be difficult to purify.
• Cysteine-rich peptides may be difficult to synthesize/purify.

To discuss a peptide synthesis project, contact the Ms. Jane Peterson (507-284-2457) or Dr. Dan McCormick (507-284-4992).

Protein Identification

A major function of the MPRC is the identification of proteins. Most frequently, this is done on proteins partially purified by 1D or 2D PAGE. However, proteins in solution are also amenable to analysis. N-terminal sequencing via automated Edman degradation is still available. MS/MS mass spectral analysis constitutes a modern alternative to protein sequencing and identification.

In MS/MS analysis, proteins are digested with an appropriate endoproteinases (e.g. trypsin or Lys-C). These polypeptide fragments are then introduced into the mass spectrometer and the mass of the intact peptide is measured. Additionally, the polypeptide can be fragmented in the mass spectrometer's collision cell and a ladder of polypeptides generated corresponding to the original polypeptide less N- and C-terminal amino acid(s). These fragment ions give a ladder of masses corresponding to the loss of individual masses corresponding to the twenty amino acids. These mass differences relate to the amino acid sequence which is then compared to a data base of known protein sequences (Swiss Prot) or expressed sequence tag (EST) databases, such as dbEST at the National Center for Biotechnology Information.

The search results yield a list of proteins containing the proposed sequence(s) derived from the MS/MS data and a confidence score indicating the statistical significance of the match. In most searches multiple peptides from the same protein are identified adding to the confidence of the identification. For help in interpreting search results the following summary at Matrix Sciences is a good review for appropriate interpretation of the search results as is the entire help section for issues concerning data base searching. It should be noted that "all search programs will produce a top ranked answer; however, only the credulous are willing to accept them carte blanche." Johnson et al., "Informatics for protein identification by mass spectrometry". Methods 35:223-236, 2005.

For more specific questions related to protein identification, please contact Ben Madden at 507-284-9406 or email to madden.benjamin@mayo.edu

Protein/Peptide Purification

The MPRC has the capability to purify peptides and proteins by all modern chromatographic methods including: affinity, ion exchange, reversed phase and gel filtration.
Equipment utilized for purification includes:

1. Beckman 421 & Waters 600 preparative RP-HPLC systems
2. Two Bio-Rad Biologic DuoFlow FPLC systems for small to large scale protein purification
3. Michrom 2002 HPLC with Gilson FC203B fraction collector for off-line SCX fractionation
4. Two Savant SpeedVacs

For more specific questions related to protein / peptide purification, please contact Melinda Miller at 507-538-3675 or email at miller.melinda@mayo.edu

Proteomics

"In general, proteomics deals with large-scale determination of gene and cellular function directly at the protein level."⁽¹⁾ The term "proteome" was coined by Marc Wilkins in 1995 for the PROTEin complement of a genOME.⁽²⁾ Basically, this science involves the analysis of all the proteins (gene products) produced by a genome. The tools required to do this are mass spectrometers (MS). MS systems have been available for many years in many forms but it wasn't until the advent of two ionization techniques (electrospray (ESI) and matrix assisted laser desorption ionization (MALDI)) that the ability to rapidly and routinely analyze proteins and peptides with a MS system became widely available. The importance of these two ionization methods was recognized by the Nobel committee by chemistry awards for both techniques in 2002.

Proteome analysis usually begins with proteins isolated from appropriate tissue, cells or other biological fluid (tears, plasma etc.). These proteins are usually fractionated by either 2D electrophoresis or solution techniques (e.g. cation exchange, size exclusion chromatography, etc.). The fractionated proteins are then digested with an endoproteinase, usually trypsin, that cleaves at specific amino acid residues ( carboxy side of a lysine or arginine for trypsin). These proteolytic fragments are then introduced into a mass spectrometer and subsequently analyzed. This analysis typically includes a determination of the mass of the intact peptide as well as the mass of peptide fragments (daughters) produced in the MS/MS system. The intact mass and mass of the daughters produce a sequence tag which can be submitted for a data base search. Protein(s) with corresponding sequence(s) are identified and returned to the submitter. A typical MS/MS spectra of a peptide is shown here with the corresponding sequence information.

The MPRC employs a wide range of proteomics techniques including but not limited to: electrophoresis, 2D-LC (e.g. "MudPit": Multidimensional Protein Identification Technology), protein I.D. from gels or solution samples, as well as differential protein experiments.

Differential proteomics experiments can be performed with or without stable-isotope labeling for quantitative proteomics.

 

1. Stable-isotope labeling techniques include:
SILAC (Stable Isotope Labeling with Amino acids in Cell culture) where cells grown in culture are labeled metabolically by growth in ¹⁵N salts or ¹³C labeled amino acids.

Isotopically encoded reagents. Examples of these types of reagents include ICAT (Isotope-Coded Affinity Tags) and iTRAQ^™ a novel reagent for differential protein quantification.

Enzyme catalyzed incorporation of H₂¹⁸O and subsequent comparison to a nonlabeled control.

2. Nonisotopically labeled samples can be compared by the differential analysis of control and case matched pairs by 2DGE images or direct comparison of retention time/mass pairs of tryptic peptides or low molecular weight protein fractions.

(1) Aebersold and Mann, Mass Spectrometry-Based Proteomics, Nature, 422:198-207, 2003.
(2) Wilkins, M. et al., Progress with Proteome Projects: Why all proteins expressed by a genome should be identified and how to do it, Biotech. Gen. Eng., 13:19-50, 1996.

For information regarding any of these analysis please contact Bob Bergen at 8-0381.