Protein Profiling
The MS & Proteomics Protein Profiling unit has seven different complementary state-of-the-art protein expression analysis approaches in place for discovery of protein biomarkers that underlie the disease or treatment of interest. These protein profiling approaches include differential (fluorescence) gel electrophoresis (DIGE), Multiplexed Isobaric Tagging Technology for Relative Quantitation (iTRAQ), Isotope-coded affinity-tag-based protein profiling (ICAT), Stable Isotopic Labeling by Amino Acids in Cell Culture or SILAC, ProteomeLab PF2D (2D LC), Multi-dimensional Protein Identification Technology (MudPIT) and Label Free Quantitation. Several of these protein expression approaches can also be utilized in combination with locating post translational modifications (PTMs) (e.g. phosphopeptide quantitation using SILAC) in order to further understand the differential changes of PTMs and their impact on disease. The below table briefly describes these approaches.
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Complementary Protein Profiling Approaches Currently in use in the MS & Proteomics Resource
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Method
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Labeling
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Separation
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Sample Comparison
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Limits
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Pros
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2D LC*
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None
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2D chromato- focusing & RP HPLC
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quantify pair wise RP fractions by UV
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best for comparison of 2 samples at >500μg
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ideal for serum/plasma discovery
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iTRAQ
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Isobaric tags at N-terminus & ε-N of Lys
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CEX & LC-MS/MS
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4 & 8-plex MS/MS quantitation based on intensity of reporter ions
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quantitation on MS/MS selected peptides only
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8 plex allows greater multiplexing
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ICAT
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C12/C13 at Cys
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CEX, Avidin & LC-MS/MS
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2-plex MS quantitation
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only detects Cys-containing proteins
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simplifies complex mixtures; no PTMs
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SILAC
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Stable isotope labeled peptides
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CEX & LC-MS/MS
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2-plex MS quantitation
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difficulties with software quantitation
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Labeling occurs early on in the sample prep
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Label Free Quantitation
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None
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LCMS
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LC-MS based disease biomarker discovery tool
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limited or no initial protein identification. Repeat run often used for identification
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countless samples can be compared
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MudPIT
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None
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CEX & LC-MS/MS
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2D LC approach used to catalogue proteins from complex samples
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additional pre-fractionation aids in identification of >proteins in the proteome
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no direct quantitation
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*DIGE and 2D LC are performed at the protein level; all other protein profiling approaches are done at the peptide level