The Proteomics Shared Resource (PSR) provides OSUCCC – James members with complete proteomic support using a wide range of state-of-the-art mass spectrometers based analytical platforms. The PSR offers extensive technical expertise, leading mass spectrometers and supporting equipment to identify and characterize proteins, protein complex (via Native MS analysis), protein modifications, protein interactions and protein biomarkers as well as protein quantitation studies in cancer samples from various sources such as serum, urine, BAL fluid, saliva, frozen tissues, cell culture media, formalin-fixed tissues and cell lysates. The PSR also provides qualitative/statistical downstream analysis and visualization of data.
Agilent 6545 Quadrupole Time of Flight LC-MS System: The Agilent 6545 QTOF is equipped with two Agilent 1290 UHPLC systems, one for reverse phase and one for normal phase separations. This instrument is designed for high resolution and broad profiling of unknown compounds in untargeted metabolomics studies
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 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.
This platform combines nano-LC separation (liquid phase) with ion mobility separation (gas phase) to enhance the separation of peptides generated by digestion of complex protein mixture. By using these two “orthogonal” separation methods we can reliably identify large numbers of proteins (>3,000) with shorter analysis time (compared to 2D LC measurements).
Bruker Ultraflextreme MALDI TOF-TOF instrument: This instrument is used for survey experiments to determine purity of protein samples, identification of lipid components and MALDI tissue imaging (proteins, lipids).
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 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. A FAIMS ion mobility device is under installation and will be used in near future.
Thermo QE Plus 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 QE Plus Orbitrap: This is a recently acquired and installed ultra-high-resolution instrument with micro-HPLC capability. It will be used mostly (but not exclusively) for metabolomics studies.
The OSUCCC – James Shared Resources and core facilities provide specialized services to cancer researchers. They offer cost-effective, state-of-the-art technology and instrumentation; expert guidance and training; and clinical, administrative and technical support.
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. Quantitative proteomics 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 the proteomics team (Drs. Arpad Somogyi, Liwen Zhang, Sophie Harvey, Andrew Reed and Miranda Gardner) will 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.
Data Quantitation and Visualization – PSR can provide both typical and specialized data analysis methods for quantitative proteomics, and untargeted lipidomics and metabolomics. Services for quantitative proteomics include data manipulation and pre-processing (QC/QA with missing value assessment, clustering, filtering, etc), differential expression analysis, statistical analysis, pathway analysis (Gene Ontology, KEGG, IPA, etc), and data visualization with plots generated in R. Services for untargeted metabolomics and lipidomics include pre-data processing (missing value imputation, normalization, ect…), mass annotation, univariate statistical analysis, supervised and unsupervised multivariate statistical analysis, and pathway analysis.
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.
MALDI Imaging: MALDI tissue imaging is a 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. MALDI imaging is available on a Bruker Ultraflextreme MALDI TOF-TOF instrument (large molecules, e.g., proteins) and on the Bruker 15 T FT-ICR ultrahigh resolution instrument (small molecules with very accurate mass measurements).
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 2 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.
Native MS analysis- Native MS (nMS) is a technique which enables the analysis of intact non-covalent complexes including protein-protein and protein-ligand complexes. nMS can provide insight into the oligomeric states of protein complexes, heterogeneity of samples (e.g presence of truncations and PTMs) along with information on stoichiometry and specificity of binding. Native MS can also be used to study RNA and DNA samples. By performing these experiments on high-resolution instruments accurate masses can be determined.
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 Orbitrap Fusion or timsTOF PRO using LC-MS/MS to determine 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.
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 nano 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 HCD. Thus, trypsin is the most common enzyme used for proteomics. However, chymotrypsin, Arg-C, Lys-C, Asp-N and many other enzymes can be used as needed.
LC-MS/MS analysis: The digested peptides are separated by nano C18 reverse phase column before being sent to the mass spectrometer for further analysis. A Thermo orbitrap Fusion, Thermo QE Plus and Bruker timsTOF Pro is used for acquiring data. The length of LC gradients 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 latest version of Uniprot or NCBI database. Data can also be searched against databases provided by the researchers in fasta format. MASCOT results will be compiled using Scaffold so the users can view and compare the data more easily.
2D LC-MS/MS Analysis (optional): For complex mixtures, the PSR recommends performing pre-fractionation prior to LC-MS/MS analysis. In this approach, the complex mixtures of proteins are digested and fractionated on an online or offline fractionation column first to reduce the complexity of samples submitted to LC-MS/MS analysis. Each fraction is collected and then separated again by a separation column for LC-MS/MS analysis, separately; the results are then merged prior to downstream informatics analysis. As many as several thousands of proteins in one sample have been identified using this approach.
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/peptide quantitation using standard Bradford, fluorescence assays and NanoDrop
Protein cleanup (ziptip desalting)
S-trap digestion for proteins in high concentration of SDS or Urea. Protein complex mixture separation by 1D SDS-PAGE (separate complex protein mixture by molecular weight) and commassie blue staining
Peptide enrichment such as phosphopeptide enrichment
Protein depletion to remove abundant proteins in certain samples such as serum
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 replicates 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.
TMT: TMT (tandem mass tag) 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 media 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-MS/MS, the ratio of peak intensities in the mass spectrum for the doublets of unlabeled and labeled peptides reflects the relative protein abundance.
Matrix Science Mascot Server: A powerful search engine for mass spectrometry data to identify proteins and peptide modifications.
Proteome Software Scaffold 4.9.0: Used to visualize complex proteomic data and to calculate spectral counting for label-free experiments.
ProteomeDiscoverer 2.2: Powerful platform that can be used to search the data by MASCOT and sequest for protein identification as well as protein quantitation using TMT or SILAC.