The Proteomics Shared Resource (PSR) provides technical expertise and full proteomic services to The Ohio State University Comprehensive Cancer Center (OSUCCC) and the Ohio cancer research community. It offers cancer investigators the critical tools and expertise needed for identifying proteins, protein modifications and protein biomarkers.
The PSR’s services include:
NEW SERVICE: Protein Expression and Purification Services in conjunction with Mass Spectrometry and Proteomics Facility can help clone, express and purify our proteins as well as problem solve and identify protein to help determine your protein’s function and possible pathway.
Sample Preparation (Cell Lysis, Fractionation, Desalt)
The PSR will accept cell pellets, tissue and biological fluids for proteomic analysis. As such we offer all the protein extraction services to properly prepare samples for downstream analysis. This includes cell lysis, protein precipitation and other standard sample preparation techniques.
Protein spots of interest can be determined following excision from the polyacrylamide gel, protease digestion, and nano LC-MS/MS analysis of the peptides on either a LTQ or LTQ Orbitrap mass spectrometer. Nano-LC/MS/MS is used to provide internal sequence of the protein and has many advantages over traditional MALDI analyses. We have found this method to be sensitive and powerful, producing numerous sequences from low fmole of material and enabling us to identify proteins for which peptide mass mapping by MALDI was unsuccessful.
Protein quantitation is used to determine protein amounts in ?g amounts using standard Bradford Assay or fluorescence detection.
Post Translational Modification Analysis
The LTQ Orbitrap mass spectrometer allows for high resolution mass analysis of peptides for accurate post-translational modification studies including phosphorylation, acetylation, or any other modified amino acid of interest.
Intact Protein Molecular Weight Measurement
A simple molecular weight analysis can determine the presence or absence of a compound, purity, relative concentration and molecular weight. We can measure molecular weights as low as 50 Da and as high as 150,000 Da. ESI, EI and MALDI can all be used for this type of sample. Polymers, peptides, proteins, oligonucleotides are typically analyzed by simple molecular weight analysis. Most of our instruments are high resolution, so you can still get isotope information with the simple molecular weight analysis.
1D and 2D SDS-PAGE
Following protein preparation from cells or tissue, protein separation services using 1D or 2D-PAGE techniques are available. PAGE is used to separate complex protein mixture by molecular weight (1D PAGE) or by pI and molecular weight (2D PAGE).
Differential Gel Electrophoresis (DIGE)
Utilizing the Differential Gel electrophoresis (DIGE) method, investigators can track the appearance, disappearance or molecular weight shifts of proteins and identify the protein. The DIGE method of examining differential proteomics is quite sensitive and can be used to identify proteins and changes in protein expression levels.
iTRAQ (isobaric tag for relative and absolute quantitation) is a non-gel based technique used to identify and quantify proteins/peptides from different sources in one single experiment by using isotope coded covalent tags that will label the N-terminus and side chain amines of peptides from protein digestions.
Protein Purification of His and GST-Tagged Proteins
Using the Biorad Profinia, automated Purification of His or GST tagged proteins is enabled which yields purified protein in 30 – 45 minutes and replaces gravity flow techniques.
Coomassie Stain, Sypro Ruby Stain, Deep Purple Stain & Silver Stain
The gels can be stained to detect protein in the PAGE with various stains including Coommassie, Sypro Ruby, Deep Purple and Silver.
Multiplex Stain (Phosphorylation and Glycosylation Analyses)
Multiplex analysis by visualization of proteins and protein modifications using specialized fluorescent stains, and advanced gel image analysis are available to study phosphorylation, glycosylation and other modifications.
LC/MS is a very effective technique to combine peak detection with peak identification. Combining chromatography with mass spectrometry allows the chromatographer to "see inside" the chromatographic peak and to resolve co-eluting compounds of different molecular weights.
Albumin removal is used for serum samples to remove the 14 highest abundant proteins from the sample. Removal of the high abundant samples from serum allows for the detection of low abundant proteins in serum samples that are typically masked by the high abundant proteins.
iTRAQ and Quantitation
Quantitation in proteomics has become a popular area in recent proteomics research with the development of quantitation techniques such as DIGE, SILAC, ICAT, iTRAQ and AQUA. Among them, iTRAQ (isobaric tag for relative and absolute quantitation) is a non-gel based technique used to identify and quantify proteins/peptides from different sources in one single experiment by using isotope coded covalent tags that will label the N-terminus and side chain amines of peptides from protein digestions.
MudPIT/Shotgun Proteomics Analysis
Complex mixtures of proteins can be analyzed directly without the need for protein separation or fractionation. Whole protein mixtures are digested directly with trypsin from solution instead of in-gel. The peptide mixture is then analyzed on the ultrahigh resolution LTQ-Orbitrap mass spectrometer with advanced chromatography. As many as 3000 proteins in one sample have been identified using this approach.
The PSR will perform your standard western blot experiment as long as you can provide or suggest an appropriate antibody. We also will perform a 2D western blot as well.
ESI is used for small molecule or small intact protein analysis.
MALDI analysis is available for small peptide, intact protein analysis and polymer analysis. We also can analyze small DNA fragments by MALDI.
Nominal Mass Measurement
A simple molecular weight analysis can determine the presence or absence of a compound, purity, relative concentration and molecular weight. We can measure molecular weights as low as 50 Da and as high as 150,000 Da (and higher, no one has submitted anything bigger). ESI, EI and MALDI can all be used for this type of sample. Polymers, peptides, proteins, oligonucleotides are typically analyzed by simple molecular weight analysis. Most of our instruments are high resolution, so you can still get isotope information with the simple molecular weight analysis.
Accurate Mass Measurement
Accurate mass is used to determine the molecular weight of a sample to within 10 ppm or in other words, accurate to the third decimal point. It is used to verify a predicted molecular formula of a pure compound.
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 and 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.
LC/MS is a very effective technique to combine peak detection with peak identification. 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 a "fingerprint' mass spectrum or fragmentation from CID and searching against commercial databases of spectra.
Post Translational Modification Analyses
Posttranslational modification (PTM) is the chemical modification of a protein after translation. These modifications induce a shift in mass that can be identified using mass spectrometry. Common PTM analysis includes glycosylation, phosphorylation, acetylation just to name a few. Samples may be enriched prior to digestion with trypsin and the analyzed on the ultra-high resolution LTQ-Orbitrap LC-MS/MS. The PTM and specific amino acid residue in the protein sequence is determined using this method.
Protein digestion breaks large protein in to small peptides that are more amendable to mass spectrometry analysis. Trypsin is the most common enzyme used for proteomics, however chymotrypsin, Arg-C, Lys-C and many other enzymes can be used as needed. Trypsin predominantly cleaves peptide chains at the carboxyl side of the amino acids lysine or arginine, except when either is followed by proline.
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