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Proteomics Shared Resource
|Darryl Pappin, Ph.D.||Keith Rivera - Research Assoicate
Tahmid Hassan - Laboratory Technician
The Proteomics Shared Resource provides services to elucidate the primary structure of novel proteins and to analyze protein/protein interactions, post-translational modifications and protein levels. The facility is being upgraded with state-of-the-art mass spectrometry equipment and will be able to provide analysis using microcapillary LC-MS/MS on linear ion traps and Orbitraps, hybrid quadrupole-TOF mass spectrometers and MALDI-TOF.
All samples are processed prior to mass spec analysis. Sample preparation includes:
- solubilization of tissue or cell pellet
- in-gel trypsin digestion
- in-solution trypsin digestion
- on bead digestion
- protein concentration measurements
Separation and protein characterization
Various methods are available:
MudPIT: a technique based on integrated 2D liquid chromatography for online separation of digested proteins from 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 individual RP gradients (Washburn et al, 2001).
Protein phosphorylation analysis: IMAC and TiO2 are used for enrichment of phosphopeptides (Pinske et al 2004). Typically, samples are digested independently by three different proteases (trypsin, elastase and subtilisin) and pooled for single analysis for higher sequence coverage of the modification site (Shimogawa et al, 2006).
iTRAQ: iTRAQ -labeled peptides (Ross et al, 2004) are separated by OFFGEL based on pI (Horth et al, 2006). Each OFFGEL-separated fraction is then analyzed by either nanoLC (ChipCube)-QTOF or nanoLC-LTQ Orbitrap.
SILAC: SILAC-labeled peptides (Ong and Mann, 2007) are analyzed by MudPIT using linear ion trap-orbitrap.References:
- Ong and Mann, 2007. Methods Mol Bio 359:37-52 PMID: 17484109
- Pinsek et al, 2004. Anal Chem 76(14):3953-43 (PMID: 15253627)
- Ross et al, 2004. Mol Cell Proteomics 3(12):1154-69 (PMID: 15385600)
- Shimogawa et al, 2006. Curr Biol 16(15):1489-501 (PMID: 16890524)
- Hoth et al, 2006. Mol Cell Proteomics 5(10):1968-74 (PMID 16849286)
- Washburn et al, 2001. Nat Biotechnol 19(3):242-7 (PMID: 11231557)
The facility has five mass spectrometers
- Thermo Finnigan Linear ion trap (LTQ Thermo)
- 2 Thermo Scientific Linear ion trap-orbitrap (LTQ Orbitrap)
- Agilent 6520 Quadrupole time-of-flight
- Thermo Scientific Vantage Triple Quadrupole mass spectrometer (TSQ)
Sample preparation for the instruments is supported by a number of capillary liquid chromatography systems from Agilent and Proxeon.
Proteomics data is analyzed with the following data analysis packages:
- Mascot and Mascot Integra