| Proteomics Core Administrative | Protein Analytical | Imaging | Organic | Proteomics | Viral Production |
 |
||||||||||||||||||||||||||
|
DESCRIPTION OF THE PROTEOMICS CORE The Proteomics Core is an expansion and continuation of a core that was established during the last funding period. Since its inception in 2004, the Proteomics Core has been working with all COBRE junior PIs and other investigators to provide high quality proteomic analysis service in their projects. The quality of service and close interaction between the Proteomics Core and COBRE investigators will continue during the proposed funding period. Purpose of the Proteomics Core The main purpose of the Proteomics Core is to provide the COBRE investigators with state-of-the-art proteomic analysis capabilities. The types of proteomics service provided by the core include:
Utilization of the Proteomics Core The types of proteomics analyses that will be utilized by the individual COBRE junior PIs and other investigators are summarized in the table below. These procedures are well established and ongoing at the Proteomics Core and will be described in detail in a later section. Utilization of specific types of proteomics analyses offered by the Proteomics Core
Function and Specific Procedures Protein Analysis Using Two-Dimensional Gel Analysis Two-dimensional gel electrophoresis is a powerful technique for protein separation and can be used for protein profiling in combination with DIGE technique. The 2D gel based proteomics approach will continue to be used for two applications: (a) protein profiling, and (b) characterizing phosphorylated proteins. Samples for 2D gel analysis will be prepared by precipitation with ice cold acetone followed by centrifugation. This procedure removes the bulk of salts and lipids as well as concentrates the sample. We have found that this procedure gives better recovery of proteins than precipitation with 10% TCA and is necessary to obtain reliable and reproducible separation on isoelectric focusing. Precipitated samples are redissolved in 6 M urea, 2 M thiourea isoelectric focusing application (rehydration) buffer and used as described by Bio-Rad on an appropriate pH range. [In general, most focusing runs are performed using pH 3-10 IPG Strips. If greater resolution is required in a particular region of the pH gradient, multiple separations are performed on overlapping sets of narrow range IPG strips. Isoelectric focusing is performed using a Bio-Rad Protean IEF Cell. In general, focusing is performed through a series of timed voltage ramps up to a final voltage of 6,000 volts and for a fixed total of 30,000 - 50,000 volts/hr. The pH gradient is calibrated by inclusion of commercial OEF standards. Second dimension separations are performed by SDS-PAGE. For most applications, laboratory-cast 10 % SDS-PAGE gels that give excellent reproducibility at low cost are used. For quantification, 2D gels are stained with SYPRO Ruby, destained and scanned using the Typhoon Phosphorimager/Scanners. Where necessary, gels also can be destained and restained with silver stain protocols compatible with mass spectrometry. The data is analyzed using PDQuest software, and standard statistical analysis packages. This procedure can detect and quantify proteins at low nanogram quantities. For better reproducibility, comparison of intensities for a given spot from one gel to the next is done by normalizing spot intensities detected to total gel fluorescence, then comparing these relative spot intensities among different gels. As an alternative, quantitation can be performed using the Fluorescence 2D Difference Gel Electrophoresis (DIGE) technique which allows multiple samples to be co-separated and visualized on one 2D gel. Up to three protein extracts, for example one control and two treated, are individually labeled with different fluorescent dyes (Cy2, Cy3 and/or Cy5), then combined and co-electrophoresed by standard 2D PAGE method. The images are then merged, and differences between them can be determined using image analysis software. Another important application of 2D gels is to analyze protein phosphorylation. A protein with different phosphorylation statuses produces a series of protein spots with similar molecular weight but different pI values in the 2D gel analysis. This procedure, in combination with the phosphor-protein specific fluorescence dye ProQ Diamond, will assist in determining protein phosphorylation prior to the mass spectrometry analysis for locating phosphorylation site(s). Mass Spectrometric Analyses of Proteins and Peptides The Proteomics Core offers superior mass spectrometric analysis using three state-of-the-art mass spectrometers, Qstar XL quadrupole time-of-flight MS, 4800 MALDI-TOF-TOF MS, and 4000 Q-Trap MS. A number of different approaches are used to identify proteins and phosphoproteins by mass spectrometry. A rather general description is provided here and more specialized mass spectrometric analyses are described in individual projects. The protein of interest is subjected to gel electrophoresis (2D gel or SDS-PAGE) and protein spots or bands of interest are excised from the gel, destained, digested with trypsin or other chemical or enzymatic cleavage agents as needed, and the resulting peptides extracted. The peptides from each spot are analyzed by the 4800 MALDI-TOF-TOF mass spectrometer. Tryptic peptide masses as well as the tandem MS (MS/MS) spectra of selected peptides are acquired in an automated fashion. Another aliquot is placed in a 96-well autosampler and subjected to LC-MS analysis in an automated mode. Because both 4800 MALDI-TOF-TOF and Qstar XL offer high sensitivity, high resolution, and high quality in MS/MS experiments, a high coverage of sequences can be determined from these peptides. These combined approaches generally permit protein identification without any ambiguity. The 4800 MALDI-TOF-TOF and 4000 Q-Trap are particularly suitable for protein phosphorylation analysis, The 4800 MALDI-TOF-TOF has high sensitivity and works with the immobilized samples on plate, thus provide better opportunity for analysis compared to LC-MS/MS. The 4000 Q-Trap is valuable because of its multiple reaction monitoring (MRM) capability. Gel-free Protein Quantification Using Stable Isotope Labeling and iTRAQ Reagents 2D gel electrophoresis is a widely used standard protocol, new technologies using stable isotope labeling and iTRAQ reagents. Stable isotope labeling techniques are well developed as well. Dr. Zhu's has been one of the groups pioneering the amino acid specific stable isotope labeling in cell culture. The iTRAQ reagents permit quantitative proteomics analysis using reagents that are isobaric, with a different distribution of isotopes between a reporter and balance groups in tandem MS/MS spectra. Thus labeled peptides are identical in MS mode, but upon fragmentation in MS/MS mode produce strong, diagnostic, low-mass MS/MS signature reporter ions. The peak areas of the reporter ions allows quantification of up to four different samples simultaneously. Other stable isotope labeling approaches (ICAT and SILAC) only allow binary pair-wise quantification of two samples and thus the iTRAQ approach is preferred and will be used by all biomedical projects and will replace 2D gel electrophoresis wherever appropriate. Both the 4800 MALDI-TOF/TOF and 4000 Q-Trap mass spectrometers are suited for the iTRAQ quantitative analysis. Mass Spectrometric Analyses of Phosphoproteins and Phosphopeptides Protein phosphorylation will be mainly studied using two approaches. The first approach is to utilize the ability of 2D gel electrophoresis to separate phosphorylated isoforms due to their different electrofocusing points as described earlier. The second approach is to use immobilized metal affinity chromatograph to enrich the phosphopeptides from tryptic digests of a particular protein or a mixture of proteins from a subcellular compartment. The peptides eluted from the IMAC column are subsequently subjected to online LC-MS/MS analysis using 4000 Q-Trap and/or offline LC-MALDI-TOF-TOF analysis. This approach has proven to be successful to detect phosphoproteins more comprehensively and to identify the phosphorylation sites. LC-MALDI-MS/MS Platform A new proteomic technology LC-MALDI-MS/MS is available to investigators. This technology marries the power of HPLC separation with the in-depth MALDI-TOF-TOF analysis. A capillary HPLC system (Ultimate from LC Packings) is utilized to separate complex tryptic peptides of an isolated subcellular fraction (e.g. caveolae) using a C18 RP column. Peptides eluted are collected to a MALDI plate using with a micro-fraction collector and subjected to MALDI-TOF-TOF analysis. The LC-MALDI-MS/MS platform is critical to investigators who will analyze complex samples that require HPLC separation prior to the mass spectrometry analysis. Equipment The Proteomics Component is equipped with the following major items of equipment: Two-Dimensional Gel Separation Apparatus The Proteomics Core currently has available a Bio-Rad Protean IPG System for electrofocusing separation of up to 12 samples simultaneously on 7, 11 or 17 cm pre-cast IPG strips coupled with corresponding Protean and Criterion Gel apparatuses for 2nd dimension SDS-PAGE in 7, 11 or 17 cm formats. The latter apparatuses will accommodate up to 4 gels simultaneously. ABI QStar XL Q-TOF Mass Spectrometer The QSTAR XL Hybrid LC/MS/MS System is a high-performance, hybrid quadrupole time-of-flight mass spectrometer designed specifically for protein identification and characterization. A dedicated nano-flow HPLC system from Eksigent is configured with the NanoSpray source to allow online LC-MS/MS analysis. Specific scan modes such as precursor ion scanning using patented collision cell technology permits determination of peptide sequence and the type and location of post-translational modifications with outstanding specificity and sensitivity. ABI 4800 MALDI-TOF-TOF Mass Spectrometer The core has recently purchased a matrix-assisted laser desorption ionization (MALDI) tandem time-of-flight (TOF-TOF) mass spectrometer through a Shared Instrumentation Grant from NIH/NCRR. The 4800 MALDI-TOF-TOF mass spectrometer offers high sensitivity, efficient fragmentation of singly charged precursor ions generated by MALDI, and superior mass accuracy and resolution in both MS and tandem MS/MS modes. In addition, the Proteomics Core has a separate capillary HPLC system (Ultimate from LC Packings) that is configured with a micro-fraction collector (ProBot from LC Packings). As discussed earlier, this system provides the new powerful LC-MALDI-MS/MS technology for investigators. ABI 4000 Q-Trap Mass Spectrometer The Proteomics Core was also awarded another Shared Instrumentation Grant from NIH/NCRR to purchase a 4000 Q-Trap linear ion trap mass spectrometer with a Shimadzu UFLC (HPLC). This new LC-MS/MS system offers enhanced sensitivity. Particularly, the Q-Trap mass spectrometer has the multiple reaction monitoring (MRM) capability, which will be extremely valuable in characterizing phosphorylated peptides. This new LC-MS/MS will help with the characterization of protein phosphorylation, which many COBRE investigators desire to do. Broader Impact The Proteomics Core has served all COBRE junior PIs in the past funding period. It also served all investigators associated with COBRE, namely the mentors and the PIs of the pilot projects. In addition, the Proteomics Core has served investigators across the entire University campus, many of whom have NIH-supported research programs. We expect to continue to provide quality service to the COBRE investigators, as well as others if resources are available. The University covers the majority of costs associated with this core (>75%) and will continue to fund the core in the future.
| |||||||||||||||||||||||||||
