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.

Complementary Protein Profiling Approaches Currently in use in the MS & Proteomics Resource
Method
Labeling
Separation
Sample Comparison
Limits
Pros
2D LC*
None
2D chromato- focusing & RP HPLC
quantify pair wise RP fractions by UV
best for comparison of 2 samples at >500μg
ideal for serum/plasma discovery
iTRAQ
Isobaric tags at N-terminus & ε-N of Lys
CEX & LC-MS/MS
4 & 8-plex MS/MS quantitation based on intensity of reporter ions
quantitation on MS/MS selected peptides only
8 plex allows greater multiplexing
ICAT
C12/C13 at Cys
CEX, Avidin & LC-MS/MS
2-plex MS quantitation
only detects Cys-containing proteins
simplifies complex mixtures; no PTMs
SILAC
Stable isotope labeled peptides
CEX & LC-MS/MS
2-plex MS quantitation
difficulties with software quantitation
Labeling occurs early on in the sample prep
Label Free Quantitation
None
LCMS
LC-MS based disease biomarker discovery tool
limited or no initial protein identification. Repeat run often used for identification
countless samples can be compared
MudPIT
None
CEX & LC-MS/MS
2D LC approach used to catalogue proteins from complex samples
additional pre-fractionation aids in identification of >proteins in the proteome
no direct quantitation

*DIGE and 2D LC are performed at the protein level; all other protein profiling approaches are done at the peptide level