The Facility has capacity to analyze over 100,000 DNA sequencing samples per year using Applied Biosystems model 3730 sequencers. Samples can range in size, from PCR product to BAC, and are accepted individually or on 96 well plates. Data is returned via web browser.
How to order using the LIMS
Identification of proteins in gel bands, excised from 1D or 2D gels, or in solution. Gel bands can be stained and most commercially available Coomassie, silver or fluorescent stains are compatible with mass spectrometric analysis. Proteins are digested (usually with trypsin), proteolytic peptides are separated using high performance nanoflow HPLC and mass spectrometric data are acquired with either the ThermoFisher LTQ ion trap mass spectrometer or with the AB Sciex QSTAR Elite mass spectrometer. Protein identification is carried out by database search using the Mascot® or Sequest® database search software. Database search results are assembled using the Scaffold® software; this enables users to review their data using a free Scaffold browser.
Detailed sequence information such as identification of post-translational protein modifications, amino acid sequence heterogeneities or chemical modifications (e.g. protein cross-linking). The protein is digested with an enzyme suitable for the type of characterization desired, peptides are analyzed with nanoflow HPLC and mass spectrometry and the data (molecular weights and fragment ion mass spectra of peptides) are extensively analyzed to identify and characterize specific modifications. Often characterization may involve the measurement of the protein molecular weight with high mass accuracy (better than 50 ppm or 1 u for a 20 kDa protein).
Comparison of the relative abundances of proteins in different samples. Relative quantitation of protein abundance can be carried out without any biological or chemical labeling of proteins or peptides (label-free quantitation) or by labeling proteins metabolically with amino acids incorporating stable isotopes (e.g. SILAC) or by labeling proteolytic peptides using chemical tags that incorporate stable isotopes (e.g. ICAT, iTRAQ). We use the Scaffold® software for label-free quantitation and the Mascot® or ProteinPilot® software for quantitation of samples labeled with isotopic or isobaric tags.
We use an Applied Biosystems model Voyager DE-STR matrix assisted laser desorbtion time-of-flight mass spectrometer to measure mass of a wide range of samples including protein, DNA/RNA, conjugates, synthetic polymers, lipids, dendrimers, carbohydrates and small molecules.
We use the Intavis multiple peptide synthesizer operated on a 1-10 micromole scale that allows simultaneous synthesis of up to 192 peptides. Peptides containing rare or expensive residues may be synthesized at relatively low cost because of the small amounts of materials required. This application enables synthesis of peptides in milligram quantity for proof-of-principle experiments. Peptides up to 60 residues in length can be synthesized.
The Applied Biosystems model 430A instrument, which permits real time synthesis progress information and synthesis of high quality crude product, is used for larger-scale orders and difficult syntheses. Peptides up to 60 residues in length can be synthesized.
We use the Intavis spotter instrument, which permits the synthesis of an array (10x15 cm) of up to 600 distinct peptides immobilized on a cellulose solid support. Modifications such as phosphorylation and acetylation may be included. Such arrays are useful for mapping modification sites, delineating protein-protein binding sites or antibody recognition sequences, and delineating other sequence motifs, such as protease cleavage or DNA- and metal-binding motifs. Most epitopes synthesized are under 30 residues.
This service enables the analysis, isolation and purification of individual proteins, protein-protein or protein-DNA complexes, other conjugates and small molecules. It can also supplement gel electrophoresis and gravity/peristaltic pump column systems to map regions within proteins or DNA. The Facility uses several analytical and preparative scale HPLC instruments (for purification up to 100 mg) and also provide training for HPLC operation to interested researchers.