K-State Core Analytical Facility for Proteomics

A centralized state-of-the-art proteomics laboratory has been established at K-State for the detailed structural analysis of proteins/peptides by mass spectrometry. Facility is equipped a 2-D PAGE apparatus supported by robotic workstations that image, excise, digest, and stage protein samples for MALDI-TOF or MALDI-TOF/TOF analyses. The core facility will house an MS instrument, which will have the capability to generate peptide mass fingerprint (PMF) data on proteolytic digests and reference peptide signatures against spectral libraries for identification. If identifications are inconclusive, the MS instrument has the capability to isolate specific peptide precursor ions for further dissociation and generation of amino acid sequence information for characterizing structure and post-translational modifications (PTM’s).

In addition to implementing the above 2d-PAGE (gel-based) strategy, two additional sample preparation workstations are available that utilize a new proteomics strategy of separating digested protein mixtures by nano-flow liquid chromatography, followed by a post-column 1:1 split of effluent for analysis by ESI and MALDI - one half of the split effluent is delivered directly on-line to an ESI-Ion Trap MS/MS system - the other half is fraction collected onto a MALDI sample target at timed intervals for MALDI-TOF and/or MALDI-TOF/TOF analysis (typically known as LC-MALDI). The benefit of this new strategy is threefold: 1) the LC separation prior to MALDI analysis eliminates the ion-suppression/charge competition effects typically seen with peptide mixtures and, as a result, leads to a greater number of positively identified proteins; 2) the MALDI target with time-collected fractions becomes a “snapshot” in time of the LC run and, by having no temporal constraints, can be re-analyzed at any time using different experimental parameters to mine the data for additional proteomic content; 3) by obtaining both ESI-MS/MS data and MALDI-TOF/TOF data in a single nano-flow LC run, complementary sequence information is obtained due to different MS/MS dissociation mechanisms. Combining ESI and MALDI data as merged files increases amino acid residue coverage over what is typically seen with ESI-MS/MS or MALDI-TOF/TOF separately. This combined capability greatly enhances the ability to identify proteins by library search or through de novo sequencing methods.

Another important feature of the MALDI TOF/TOF instrument is its ability to be used for MALDI-Imaging of specific proteins in tissues.  This new technique can be used to identify specific proteins with mass ranges of 500-70,000 Da.

Workflow Management and Data Interpretation:  The Bruker system that we have purchased allows for complete software intergration for all of the system components, including sample tracking through transponder reading in the MALDI targets.  There are also a comprehensive set of  software tools for validation and reporting of results. There are separate programs for the quick evaluation of entire LC-MS/MS runs; a protein browser program to identify peptides and proteins; BioTools TM software for automated and interactive interpretation of MS and MS/MS data that help identify mutations, polymophisms; post-translational or chemical modifications; and de novo sequencing. There is also a program called ProteinScape TM that provides database solutions for proteomics projects. This program will send and retrieve data from our in-house server that has the mass spectral protein data base-- Mascot TM.  Print outs can be generated that label 2d-gel pictures with the proteins’ identities for the various spots.

For questions regarding the KSU Biotechnology Core's Services, please
email John Tomich at jtomich@ksu.edu

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This page last updated:
Janurary 10, 2006