The Center for Virus Research (CVR)
The University of California at Irvine
3221 McGaugh Hall
Irvine, CA 92697-3906
Phone: 949.824.9314
Fax: 949.824.9437
Services:
Advanced Technology & Resources
The Center for Virus Research
Directed by Dr. Luis P. Villarreal
Oversight committee chaired by Dr. Bert Semler.
Established in July, 2000 as an Organized Research Unit
within the University of California, Irvine.
Proteomics Mass Spectrometry Facility
*This shared resource is affiliated with the Chao Family Comprehensive Cancer Center, an NCI-designated comprehensive cancer center.
This facility currently has two tandem mass analyzers: An AB/Sciex 4700 MALDI-TOF/TOF and an LTQ linear ion trap instrument running permanently as a nanospray LC-MS instrument.
Complex protein mixtures
For peptide fractionation, we have four older stand-alone Waters quaternary HPLC stacks, along with a nanoflow/nanocapillary MDLC from LC Packings (UltiMate pump, UltiMate Plus pump, Famos autosampler, Switchos loading pump/switchable valves, Probot MALDI plate spotter).Combinations of these instruments provide various options for peptide fractionation depending on sample complexity and abundance, and mass analyzer preference. Peptide fractionation options for the LTQ include (for online nanospray work) reversed-phase LC-MS, MudPit and divorced-MudPit. Various forms of (offline) LC-MALDI have been tried so far for the 4700 mass analyzer, including simple reversed-phase 1D, simultaneous salt-step 2D, simultaneous dual-gradient 2D, divorced dual-gradient 2D or divorced triple gradient 3D.
The four older Waters 600E HPLC clusters also provide additional front-end capacity for alternative applications (such as phosphopeptide isolation) and applications development.
In addition to the above, we have a Beckman PF2D "Proteome Lab" for two-dimensional fractionation of complex mixtures at the protein level, to be followed if required by LC-MS or LC-MALDI.
Mass analyzers, HPLC control PCs and client PCs are networked within the lab to one another and to two servers, allowing searches by Mascot or Sequest (or both) with universal TPP validation of any dataset if required.
For relative protein quantitation, the above LC-MALDI workflows have been combined with 4-plex iTRAQ for isotope-based relative quantitation of each protein identified in up to four mixed samples from different (related) sources. With a little added expense, these workflows could go 8-plex if required.
Simple mixtures - gel spots/bands
In addition to the above, we are committed to protein identification from simpler samples such as stained gel spots or slices cut from SDS polyacrylamide gels.Samples numbering in hundreds if necessary (2D gel spots or slices from entire 1D gel lanes) can be rapidly analyzed using the 4700, and/or individual samples can be analyzed more slowly but with depth and sensitivity using the LTQ.
Gel slices/spots are generally subjected to in-gel trypsinization prior to mass analysis.
Sample preparation, submission and recharge
Investigators are generally recharged as a contribution toward instrument maintenance, and also personnel time for the larger experiments.
Protein Identification from SDS PAGE gel bands
(1) Currently, sample preparation (band excision from SDS PAGE gel), in-gel digestion, sample clean-up is performed by the submitting investigator.
(2) Please refer to this downloadable In-Gel Digestion Protocol for details on sample preparation.
(3) Since the protocol is quite involved, people often come to my lab to do it.
(4) Please DO NOT submit your most precious sample until you first have this protocol working in your hands for a non-precious sample.
(5) We request images of the gel before and after band cutting, with the cut positions in the gel labeled by sample name, and details of staining procedure.
(6) We will typically try protein ID by MALDI first, then nanospray LC-MS for any that fail by MALDI. Please think about whether you would wish to either (a) cleanup only half of your in-gel digested for MALDI and keep the remainder in reserve, or (b) at a later time, re-run a gel for samples that did not give an answer by MALDI, and repeat the in-gel digestion.
(7) I will typically try automated MALDI first, then follow up manually for any samples that did not give strong hits.
Recharge rates for samples prepared by submitting investigator:
Recharge rates
MALDI-TOF/TOF (automated): $15/band
MALDI-TOF/TOF (manual): $25/band
-For sample prep by yourself, in my lab, we may charge a little extra for consumables.
Nanospray LC-MS: $50/band
Protein ID from more complex mixtures
Recharge Rates
NanoLC-MALDI (eg. protein mixture from pulldown): $500/sample
PF2D two dimensional protein fractionation: $600/run (consumables only)
Additional iTRAQ isotope tagging: $350/group of four samples (consumables only)
Mass Spectrometry Facility Director: Paul D. Gershon, Ph.D.
Call: 949.824.9606 or 949.824.7954
E-mail: pgershon@uci.edu
