The Proteomics Shared Resource (PSR) provides technical expertise and full proteomic services to The Ohio State University Comprehensive Cancer Center (OSUCCC) and to the cancer research community across Ohio. It offers cancer investigators the critical tools and expertise needed for identifying proteins, protein modifications and protein biomarkers.
The PSR’s services include:
1. Protein Sample Preparation: The PSR accepts cell pellets, tissue and biological fluids for proteomic analysis, offering all the protein extraction services to properly prepare samples for downstream analysis. This includes:
- Cell lysis (whole cell or specific cellular)
- Protein precipitation (TCA and/or chloroform precipitation)
- Protein quantitation using standard Bradford, Lowry, BCA, and fluorescence assays
- Protein complex mixture separation by 1D SDS-PAGE (separate complex protein mixture by molecular weight) or 2D SDS-PAGE (separation by both isoelectric point [pI] and molecular weight) eletrophoesis
- Gel staining utilizing commassie, LavaPurple, Sypro for total proteins, multiplex staining for phosphorylation (ProQ-Diamond) and glycosylation (ProQ-Emerald) analyses
- Western Blotting: The PSR will perform a standard or 2D western blot experiment as long as the researcher can provide or suggest an appropriate antibody.
2. Protein Purification and Expression: Using its specialty in molecular biology and protein chemistry, the PSR offers technical assistance for complicated clinical and/or protein associated projects. The PSR helps develop custom assays for:
- Protein interaction studies (Immunoprecipitation and Co-Immunoprecipitation with provided antibodies)
- Isolation of protein expressed in media
- Protein salt fractionation
- Protein isolation and enrichment via different chromatographic approaches (tag purification, ionic exchange, HIC reverse phase, size exclusion, Heparin affinity and Protein A/G affinity column)
- Exdotoxin or albumin removal and protein refolding screening
3. Protein Identification: The PSR provides identification(s) for protein or a mixture of proteins in either solution or gel phase using a bottom-up approach. Protein in solution or in gel can be digested and the peptides analyzed by capillary LC-MS/MS. Peptide fragments generated by tandem MS are searched on MASCOT to internally sequence the protein. This method produces numerous sequences from low fmole of material and enables us to identify hundreds, even thousands, of proteins in a single run. Protein Identification includes these steps:
Enzyme Digestions: Protease digestion breaks large proteins into small peptides that are more amenable to mass spectrometry analysis. Trypsin predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine or arginine, generating positively charged peptides that fragment well by CID. Thus, trypsin is the most common enzyme used for proteomics; however, chymotrypsin, Arg-C, Lys-C and many other enzymes can be used as needed.
LC/MSMS analysis: The digested peptides are separated by caplliary C18 reverse phase column before being sent to the mass spectrometer for further analysis. A Thermo LTQ, Thermo LTQ-XL Orbitrap, Bruker amaZon or Bruker maXis is used for acquiring data. The length of LC graidents will be determined by the complexity of the final samples analyzed by MS/MS.
Database Search on MASCOT: Collected data is searched on MASCOT against the the latest version of NBCI or SwissProt database. Data can also be searched against databases provided by the researchers in fasta format.
2D LC/MSMS Analysis (optional): For complex mixtures, the PSR recommends performing a 1D fractionation prior to digestion and LC/MSMS analysis. In this approach, the complex mixtures of proteins are separated on an online fraction column first to reduce the complexity of samples submitted to LC/MSMS analysis. Each fraction is trapped online and separated by a separation column for LC/MSMS analysis, separately; the results are then merged prior to bioinformatics analysis. As many as 2-3000 proteins in one sample have been identified using this approach.
4. Quantitative Proteomics: The PSR offers both gel-based and non-gel-based methods to monitor protein differentiation expressions between samples. The PSR provides several approaches for the quantitative proteomics investigation. At least four biological replicated are required if statistical evaluation is needed.
Label-Free Quantitation using Spectral Counting: Spectral counting is a label-free quantitation technique, used in mass spectrometry, in which the number of spectra, identified for given peptides from the same protein in different biological samples, are counted and later integrated to determine the relative amount of the given protein. Quantitation also can be done by comparing peak areas for the protein from different biological conditions.
iTRAQ: iTRAQ (isobaric tag for relative and absolute quantitation) is a non-gel-based labeling technique used to identify and quantify proteins/peptides from different sources in one single experiment, by using isotope-coded covalent tags that label the N-terminus and side chain amines of peptides from protein digestions. The ratios obtained by comparing the intensity of reported ions are used to calculate the ratios of a given peptide/protein.
SILAC: SILAC (stable isotope labeling by/with amino acids in cell culture) is a mass spectrometric technique that detects differences in protein relative abundance among samples using stable isotopic labeling. Briefly, cells are differentially labeled by growing in light, medium or heavy medium that substitutes certain amino acids with heavy isotope labeled ones (usually Arg, Lys and Leu). Metabolic incorporation of the amino acids into the proteins results in a mass shift for corresponding peptides. When the two samples are mixed, digested and analyzed by LC/MSMS, the ratio of peak intensities in the mass spectrum for the doublets of unlabeled and labeled peptides reflects the relative protein abundance.
5. Molecular Weight Measurement and Tandem MS Analysis: The PSR provides both accurate and nominal molecular weight measurements using both electrospray and MALDI. ESI is used to protonate/deprotonate small molecules, peptides and small intact proteins to determine their molecular weights. MALDI-TOF(TOF) analysis is available for small peptides and for the MS analysis of intact proteins, lipids, small DNA fragments and synthetic polymers.
Nominal Mass Measurement: A simple molecular weight analysis can determine the presence or absence purity, relative concentration and molecular weight of a compound. The MS&P can measure molecular weights as low as 50 Da and as high as 150,000 Da (and higher, but no one has submitted anything bigger). ESI, EI and MALDI can all be used for this type of sample. Polymers, peptides, proteins and oligonucleotides are typically analyzed by simple molecular weight analysis. Most of our instruments are high resolution, so isotope information is still available with the simple molecular weight analysis.
Accurate Mass Measurement: Accurate mass is used to determine the molecular weight and chemical composition of a sample to within 5 ppm or, in other words, accurate to the third decimal point for most of the peptides studied. It is used to verify a predicted molecular formula of a pure compound (required by most of the scientific journals).
Tandem MS analysis: Tandem MS analysis is routinely used to provide fragmentation information to decode the structure of small molecules and sequential information for peptides and proteins
Intact MS determination and top down.
6. MALDI Image: MALDI tissue image is a mass spectrometric imaging technique in which mass spectrometry is used to visualize and compare the spatial distribution of proteins, peptides, drug candidate compounds and their metabolites in thin slices of tissue samples. Briefly, a suitable MALDI matrix is sprayed on a thin slice of the tissue prior to mass spectrometric analysis. The spatial distribution of molecular species of the tissue is recorded by MALDI mass spectrometer and image processing software is used to visualize, compare and characterize the optical image of the sample.
7. GC-MS: Gas chromatography (GC) and mass spectrometry (MS) make an effective combination for chemical analysis. Among its uses are drug testing and environmental contaminant identification. The GC instrument vaporizes the sample and then separates and analyzes the various components. The size of the peaks is proportional to the quantity of the corresponding substances in the specimen analyzed. MS identifies the separated substances by using an electron impact ionization source (EI), which breaks the molecules into charged fragments that are detected by the mass analyzer. The compound can be identified by the GC retention time, the parent ion and fragmentation pattern as searched by a database of known compounds.
8. LC-MS: LC-MS is a very effective technique for combining the separation and identification of certain compounds. Combining chromatography with mass spectrometry allows the chromatographer to "see inside" the chromatographic peak and to resolve co-eluting compounds of different molecular weights. Molecular weight information can identify predicted unknowns with better certainty and identify true unknowns by obtaining fragmentation information from CID and searching against commercial databases of spectra.
9. Post-Translational Modification Analyses: Post-translational modification (PTM) is the chemical modification of a protein after translation. These modifications induce a shift in mass, therefore can be identified using mass spectrometry. Common PTM analysis includes phosphorylation, methylation, acetylation and oxidation. Samples may be enriched prior to digestion and then analyzed on the ultra-high-resolution LTQ-Orbitrap using LC-MS/MS, determing the sequence of the peptide and the location of the modification. The PSR has extensive experience working with researchers to identify and characterize customized post-translational modifications, amino acid mutations and peptide crosslinks.
10. Consultation: PSR personnel provide assistance to individual investigators with the design and implementation of proteomics studies, in particular those involving mass spectrometry. The benefits of mass spectrometry-based proteomics methods are numerous, including routine femtomole range sensitivity, rapid analysis speed and, most importantly, the ability to precisely and accurately determine protein identity and characterize modifications. Using methods like differential gel electrophoresis (DIGE) and Label -Free Quantitation, the PSR can also track the entire differential output of proteins and protein complexes by systems under disease, drug or environmental challenge. The PSR provides a unique shared resource that is both cutting-edge and affordable for the entire OSUCCC membership.
Equally important is the desire of PSR faculty and staff to educate the OSUCCC membership about the potential applications for mass spectrometry and proteomics in cancer research and assist members in acquiring the most advanced equipment coupled with the knowledge to operate it. A proteomic project is never a “one size fits all” experiment, so each project begins with a meeting involving key personnel on the project. At the meeting, the researcher describes the goals of the project and Drs. Zhang and Somogyi recommend the specific experimental plan and discuss the details of the sample preparation the researcher will need to follow for a successful experiment. Possible pitfalls and alternative solutions are also discussed.
Following the meeting, the researcher prepares a small trial sample set used to generate preliminary data, to perform any necessary method development and to determine the likely success or failure of the project. If the trial sample is successful, the researcher prepares the full sample set required for the experiment. If problems are encountered with the trial sample, the PSR can often determine the particular pitfall in this sample and assist the researcher to solve the problem quickly. This process has been very successful in supporting the PSR’s ability to generate high-quality, publishable data in a timely manner. An added goal of the PSR—to develop even more advanced methods for sample pre-fractionation prior to mass spectrometric analysis—is also highly beneficial to the membership. Gel-free electrophoresis equipment is available for protein separation, especially for less soluble hydrophobic membrane proteins, post-translationally modified proteins and proteins that express at a low level. These resources also provide analyses of complex mixtures of proteins independent of 2D gel electrophoretic separations.
The Proteomics Shared Resource (PSR) provides OSUCCC members with complete proteomic support using state-of-the-art electrophoresis and imaging equipment, robotic sample handlers and mass spectrometers. The PSR offers extensive expertise in identifying proteins from protein complexes utilizing leading mass spectrometer instrumentation and supporting equipment for a wide range of proteomic analysis, protein identification and post-translational modifications identification.
The PSR’s instruments include:
Bruker 15 T SolariX FT-ICR offers ultra-high resolution/mass accuracy as well as the isotopic footprint of small molecules, the instrument has a dual source (MALDI and ESI) that can be used for proteomics/metabolomics investigation, ion-molecule reactions and MALDI imaging.
Bruker ultrafleXtreme MALDI-TOF/TOF equipped with Proteineer fc II for LC-MALDI and ImagePrep for Tissue imaging sample preparation – 2 KHz speed TOF and smartbean-II laser for ultra-high data acquisition in both MS and MS/MS mode. Currently, this instrument is mainly used for protein profile analysis, peptide mass mapping and synthetic polymer and lipid analyses.
Bruker maXis Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometer for ultra-high-resolution exact mass and true isotopic measurements in both MS and MS/MS mode with a Dionex U3000 RSLC system. This instrument is widely used for accurate mass measurements of small molecules, lipid analyses and protein/peptide analysis.
Bruker amaZon ETD Ultra-performance, high-sensitivity proteomics ion trap (unit resolution) with dual funnel ion guide and advanced ETD/PTR capabilities (electron transfer dissociation and proton transfer reaction) with a Dionex U3000 RSLC system.
Thermo Orbitrap Fusion Ultra-high-resolution LC-MS equipped with different fragmentation methods (CID, HCD and ETD), the system is also equipped with Dionex nano-LC system for advanced proteomics, including bottom-up proteomics, post-translational modifications, relative quantitation, targeted quantitation and intact protein characterization.The nano-LC can be switched between 1D and 2D LC for to identify more proteins.
Thermo QE Orbitrap Ultra-high-resolution LC-MS complete with Dionex nano-LC system for advanced proteomics, including bottom-up proteomics, post-translational modifications, relative quantitation, targeted quantitation and intact protein characterization. The nano-LC can be switched between 1D and 2D LC to benefit the identification of proteins from more complex system.
Thermo LTQ Orbitrap XL for ultra-high-resolution LC-MS complete with Dionex comprehensive cap-LC system for advanced proteomics, including bottom-up proteomics, post-translational modifications, relative quantitation, targeted quantitation and intact protein characterization.
Thermo Linear Quadrupole (LTQ) Ion Trap MSn instrument for tandem mass spectrometric analysis, with many stages of CID MS analysis of liquid samples introduced by a Dionex U3000 Nano-LC or infusion for high-throughput (targeted) proteomics.
Thermo DSQ-II GC-MS instrumentation for GC-MS analysis of volatile samples, sometimes products of biochemical/enzymatic reactions (e.g., fatty acids and derivatives).
Ettan Proteomic Workstation and Typhoon Imager for automated, high-throughput sample handling, 1D and 2D SDS-PAGE analysis and differential image electrophoresis (DIGE).
Matrix Science Mascot Server, a powerful search engine for mass spectrometry data to identify proteins and peptide modifications.
Proteome Software Scaffold 4.8.3 to visualize complex proteomic data and to calculate spectral counting for label-free experiments.
ProteomeDiscoverer 2.2, a powerful platform that can be used to search the data by MASCOT and sequest for protein identification as well as protein quantitation using iTRAQ or SILAC.
Please see http://www.ccic.ohio-state.edu/MSP-Rates for our current rates.