Proteomics Specialized Equipment and Capability

Proteomics Specialized Equipment and Capability
Nano LC MS/MS
The LC MS/MS system contains of a nano HPLC with an auto sampler LC Packings/Dionex and a QSTAR XL mass spectrometer (Applied Biosystems and Sciex). This system is mostly used for identification of proteins. The proteolytic digests of electrophoretically separated bands (SDS-PAGE) or spots (2D electrophoresis) or complex protein solutions were digested with trypsin (or other proteolytic enzyme) and separated by nano-HPLC. The eluate from the reversed-phase (RP) column can be directly introduced onto the mass spectrometer, where candidate ion selection, fragmentation, data collection and protein identification were performed. For protein identification, ProteinPilot program for searching the human and RefSeq databases from NCBI are used.

Isobaric tag for relative and absolute quantitation iTRAQ is used for comparative analysis of protein levels. ITRAQ is a non-gel-based method for comparing protein contents from different sources in one single experiment. Following tryptic digestion and N-terminal labeling of each sample with a different mass tag, the samples are subsequently pooled, fractionated by nano-HPLC and analyzed by MS/MS to identify peptide and simultaneously generate a low mass reporter ion from the mas tage that can be used to determinate the relative amount of peptide in each sample. Isotope labeling can be optimally used to identify quantitative differences in protein composition in closely related samples (see Clifton et al, Electrophoresis 2009, 30, 3636-3646).

Figure 5: comparison between SDS-PAGE and SELDI-TOF

The ProteinChip Series 4000 System SELDI TOF Mass Spectrometer
The ProteinChip Series 4000 System allows the Proteomics Core to greatly facilitate biomarker discovery by taking advantage of important advances made in last time, namely, increased sensitivity, extended molecular weight range, higher resolution and the robust statistical analysis capabilities of the Express Biomarker Analysis Module. The multifunctional Series 4000 provides a complete solution for biomarker discovery as well as biomarker interaction analysis and assay development. This type of translational research is of critical importance to the COBRE CCRD and is a major objective of the National Institutes of health (NIH) Road Map Initiative. In addition to biomarker discovery, the upgraded system will provide improved capabilities for characterizing multi-component protein complexes, discovering receptor-ligand pairs, elucidating regulatory proteins and signaling pathways, discovering DNA-transcription factor interactions, and performing peptide mapping for protein identification and characterization interactions. In Fig. 5, comparison between SDS-PAGE and SELDI-TOF mass spectrometric analyses of urine of mice poisoned with aristolochic acid is shown.

Laboratory automation Workstation
The Laboratory Automation Workstation Beckman Coulter Biomek 2000 enables rapid, reliable and reproductible sample preparation on both protein chips and 96 vial ELISA plates.

Protein Chip System with SELDI/MALDI Interface to Mass Spectrometer 
In the near future, the ProteinChip interface will be installed on the Advanced Biosystems Q-Star Tandem Mass Spectrometer. This will provide the capability to conduct high performance protein identification, low molecular weight proteome phenotyping, and epitope mapping/protein interaction analysis. The interface affords superior mass accuracy thus differentiation between peptides, oligosaccharides, lipids, and EAM signals, while directly profiling for biomarkers in the low molecular weight proteome, can be accomplished.  Further, many peptides and small proteins can be directly identified, by performing ProteinChip ms/ms analysis. Elegant epitope mapping, receptor-ligand and other protein-protein interaction studies can also be performed when combined with ProteinChip Antibody Capture and pre-activated ProteinChip arrays.

Analytical and Preparative 2-D Gel Analysis
BioRad protean 2-D elecrophoresis sytem, FX Pro Plus Molecular Imager and PDQuest 2-D analysis software. Complex mixtures of cellular proteins or partially purified proteins will be resolved, imaged via radioisotopes, fluorescent dyes, tags or chemiluminescence and subsequently identified using protein SELDI-TOF chip technology, or LC/MS/MS.

For fast 2-D gel analysis of a small amount of protein mixtures a 2-D system (Invitrogen) will be used.

For very rapid IE-, RPC-, AC- and IA-chromatographic analyses, so-called CIM-disk and tube shaped monolithic columns will be used. Using this technology, analyses of even very complex protein mixtures can be performed in less than five minutes.

Hybridoma Facility 
The hybridoma facility in the division of medical oncology at Rhode Island Hospital, in operation for over 16 years, is part of the COBRE CCRD.

One Hoppin Street • Providence, RI 02903
Phone: 401-444-2871 • email: ESmith6@lifespan.org