MASS SPECTROMETRY-BASED PROTEOMICS RESOURCES IN THE DEPARTMENT OF PATHOLOGY, UNIVERSITY OF MICHIGAN

The mass spectrometry-based proteomics resource in the Department of Pathology occupies primarily serves members of the Department of Pathology. The facility occupies about 1500 sq feet of space in Rm 4204 at the Medical Science I Building. The services offered by the resource include identification of proteins from multiple sources such as bands from Coomassie and Silver-stained gels, shot-gun proteomics analysis of complex mixtures using multidimensional proteomic identification technology (MudPIT), identification of post-translational modifications, and quantitative proteomic profiling using stable isotope labeling of amino acids in culture (SILAC), 16O/18O, and isotope-coded affinity tags. The mass spectrometry laboratory is equipped with three mass spectrometers exclusively devoted to proteomic analysis. These include an LTQ linear ion-trap mass spectrometer with electron-transfer dissociation (ETD) module, an LTQ-Orbitrap XL tandem mass spectrometer and an TSQ Quantum Ultra triple quadrupole mass spectrometer. The LTQ with ETD permits identification and quantification of proteins in complex mixtures. In addition, post-translational modifications such as phosphorylation, ubiquitination, acetylation and methylation can be analyzed if required. The LTQ-Orbitrap XL is utilized for higher mass accuracy analyses of proteins in complex mixtures, mapping of post-translational modification sites, and relative quantification experiments. The dynamic range of quantification is in the order of two orders of magnitude. The TSQ Quantum Ultra provides highly sensitive and selective multiple protein quantitation in a background of complex mixtures by using high resolution selected single and multiple reaction monitoring assays. The instrumental fast scan speed (short duty cycle) of >300 MRM transitions/second permits unique high resolution quantitation with high sensitivity (4 orders of magnitude) and exquisite specificity. In addition, the resource can flexibly provide specific functionalities to aid departmental investigators achieve aims that relate to their unique biological questions. Technical expertise in mass spectrometry is provided by dedicated staff namely Dr. Venky Basrur (Lab Manager) and Mr. Kevin Conlon, both of whom have extensive technical expertise in biological mass spectrometry. The mass spectrometry data are analyzed through the Trans-Proteomic Pipeline which includes analytical tools for sequence matching such as X!Tandem, and tools for assessment of data quality and false discovery rates using concatenated forward and reverse database searches and Protein/Peptide PROPHET algorithms. Analysis and data storage are performed on a 16-node cluster maintained by the Proteomics Informatics Group directed by Dr. Alexey Nesvizhskii supported by Dr. Damian Fermin. Results of analyses are delivered electronically by email and and are accessible via a secure web-based interface.