Services

Basic

Protein Profiling With GeLC/MS/MS. The PBMS Core Facility uses high-throughput nano liquid-chromatography tandem mass spectrometry to identify proteins from digested gel slices. Typically, prior sample preparation includes running the sample out on a gel (Invitrogen), cutting the Coomassie-stained gel, and digesting the gel pieces with trypsin. See Sample Protocol and Karim et al., 2005, for details of sample preparation.

Advanced

Isotope-Coded Affinity Tag (ICAT). ICAT is an established differential labeling technique which uses stable isotope labeling to permit quantitative analysis of paired protein samples. The isotope labels, which bind to cysteine-containing peptides, possess identical chemical and structural properties but exist in a heavy or light state. The mass difference between the two isotopic states can be detected by MS analysis and used to determine the relative abundance of each cysteine-containing peptide pair. See Gygi et al., 1999, and Han et al., 2001, for details of sample preparation for ICAT analysis.

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC). SILAC is a relative quantitation technique in which specific, essential, labeled amino acids are incorporated in vivo into all mammalian proteins in a cell line. Cells are grown in either normal media or media containing an isotopically-labeled form of the specific essential amino acid(s). SILAC was introduced by Ong et al., 2002. Recently Dr. Hwang, Director of the PBMS Facility at UCHC, has improved the SILAC methodology by double amino acid labeling, a refinement which significantly increases the coverage of sequencing (Hwang et al., 2006).

Phosphoproteomics. Phosphorylation events are post-translational modifications of proteins which play a critical role in signal transduction within the cell. The development of methodology for the enrichment and quantification of the phosphoproteome is requisite to an understanding of signal propagation through transduction pathways. Dr Hwang and colleagues in the Center for Vascular Biology at UCHC are actively involved in phosphoproteomic research (Extended Abstract, ASMS Conference, 2005).

Multidimensional Protein Identification Technology (MudPIT). MudPIT is a popular proteomics technique in which 2D liquid chromatography is used to separate and identify proteins in complex mixtures. The chromatographic column comprises a strong cation exchange material in series with a reversed phase material, and the chromatography proceeds in cycles consisting of increasing salt concentration followed by increasing hydrophobicity. See Washburn et al., 2001, and Han et al., 2001, for two examples of the use of MudPIT in high-throughput proteomics analyses.

Direct Tissue Proteomics (DTP). A major roadblock to early detection and treatment of cancer has been the lack of sensitive and robust technology for the detection of cancer cell signatures from minute quantities of available tissue or serum. A promising new methodology, termed Direct Tissue Proteomics (DTP), has recently been developed in the research lab of Dr. David Han, Faculty Director of the PBMS Core Facility at UCHC. DTP identifies proteins directly from formalin-fixed, paraffin-embedded tissue samples, allowing full-scale proteomic analysis of minute biopsy sections. An exciting contribution of this shotgun proteomics approach to cancer diagnosis and treatment is its ability to detect and identify potential biomarkers from archival cancer tissue. See Sunil et al., 2006, for details of the Direct Tissue Proteomics approach.

Laser Capture Microdissection (LCM). The crucial task of identifying molecular changes in cancer cells is seriously hampered by the fact that even minute quantities of tissue often contain a mixture of cells. LCM enables researchers to automatically isolate pure cancer or healthy cell populations by using images from a live video camera to definitively purify cells of interest. A Laser Capture Microdissection instrument is available through the PBMS Core Facility for scheduled use by UCHC members, with on-site training provided as required. First-time users should contact Dr. Karim Rezaul to arrange for a mandatory training session. Established users may also contact Dr. Rezaul to schedule time on the instrument, subject to availability.

Absolute Quantification of Proteins (AQUA). AQUA (Gerber et al., 2003) is a recently developed technology which permits direct quantification of proteins and their modification states through the use of synthetic peptides which are chemically identical to their naturally occurring counterparts. The internal standard peptides, synthesized with stable isotopes, are identified by selected reaction monitoring and tandem MS, and then used to precisely quantify naturally occurring levels of the corresponding proteins and their modification states. Viveka Mayya, currently a graduate student in the lab of Dr. David Han, has worked extensively with AQUA technology (see Mayya et al., 2006).

Label-Free Quantification of Proteins. While ICAT, SILAC, and AQUA quantification methodologies rely upon stable isotope labeling, label-free quantification methods have also been described. Examples include spectral count (Liu et al., 2004) and emPAI (Ishihama et al., 2005). Compared with stable isotope methods, label-free abundance measures have the advantages of ease of use and applicability to all proteins identified in an MS analysis. For these reasons it is likely that label-free measures will come to be routinely reported in proteomics studies. The PBMS Core Facility can provide spectral count calculation for protein profiling experiments, as well as a full statistical analysis based on replicate analyses.