MALDI-TOF Core

An MDS Sciex 4000 QTrap, an MDS Sciex/Applied Biosystems 4800 MALDI TOF-TOF, an Applied Biosystems 4700 Proteomics Analyzer (MALDI TOF-TOF), and a reflectron, delayed extraction MALDI-TOF mass spectrometer (Voyager DE-PRO, Perseptive Biosystems/Applied Biosystems) are available for analyses of protein, peptide, CHO, small molecule, and oligonucleotide masses. Multiple sample preparation services are also available, including complete 1D and 2D LC separation services (BioCad LC, an Eksigent 2D Nanoflow LC system coupled to an LC Packings/Dionex ProBot MALDI plate spotter for peptide level separations, an ABI LC-TEMPO separation/spotting system, and a Beckman-Coulter PF-2D system for whole protein level separations).

1D and 2D gels (standard or DIGE gels) can be analyzed on our BioRad FX Pro Plus Fluorimager/PhosphorImager/Densitometer using Quantity One and PDQuest software, with subsequent automated spot excision, proteolytic digestion, and MALDI plate spotting using our LEAP Technologies 2DiDx spot-cutting robot. DIGE gels can also be analyzed with the Typhoon Gel Imager , Decyder Software, and the GE-Amersham Spot Cutter by contacting Dr. Bill Freeman in the Department of Pharmacology (x4037)

Funding for the 4800 MALDI TOF-TOF, 4700 Proteomics Analyzer, the Eksigent 2D nanoflow LC system, the LC Packings/Dionex ProBot MALDI plate spotter, the BioRad FX Pro Plus Fluorimager/PhosphorImager/Densitometer, the LEAP Technologies 2DiDx spot-cutting robot, the GE-Amersham Spot Cutter & DeCyder licenses, Phenyx Proteomic Analysis software, and the Beckman-Coulter PF-2D system was provided by Tobacco Settlement Funds (TSF) through the State of Pennsylvania during the period 2001-2006.

In addition to standard quality control assessment of synthetic peptides and oligonucleotides from our Macromolecular Core, the MALDI-TOF's can be used for analyses of complex mixtures of peptides, for example from proteolytic digests of proteins purified by column chromatography, by metal affinity chromatography, by SDS-PAGE or 2D gel electrophoresis, or from PVDF/nitrocellulose membranes, with a microliter of sample at micromolar concentrations (=detection of sub-picomole amounts in a single MALDI spot) of materials sufficient for analysis in most cases, with detection of femtomole and sub-femtomole amounts possible in many cases. These types of analysis can be used, for example, to detect and analyze phosphoproteins and other post-translational modifications, for isozyme analysis, and for identification of unknown proteins.

Quantitative analyses of differences in protein expression between different states can be performed using (1) standard 2D gel analysis comparing spot-densities on two gels; (2) DIGE gel analysis using a single gel with proteins from different samples labeled with different fluors; (3) whole protein 2D LC separations and comparing fraction densities between 2 samples using our Beckman-Coulter PF 2D system; (4) Quantitation at the peptide level using differential heavy isotope ICAT or iTraq labeling followed by gel and/or 2D LC separations, and finally differential relative quantitation on the MALDI instrument itself using GPS Explorer 3.5 software, or Phenyx Proteomic Software.

Unknown proteins can be further characterized by partial sequence determination of proteolytic fragments using tandem MS/MS ion fragment analysis. Additional analyses (e.g., partial sequence determination utilizing Collision-induced Dissociation (CID) fragmentation analysis with our MALDI TOF-TOF are available in our facility; alternately, electrospray ionization TOF spectrometry can be coordinated with the Mass Spec facility at PSU Main Campus (http://hils.psu.edu/stf/pmcf/home.html), or nanospray ionization studies can be performed on our MDS Sciex 4000 QTrap instrument.

PRICES: There is no charge for initial consultations and feasibility discussions, nor for training on the Voyager DE-PRO MALDI-TOF self-service instrument. Hourly fees for trained users are $25 per hour (Voyager DE-PRO only), or simple samples such as purified proteins or in-gel digestions of single spots can be analyzed on our DE-PRO or 4800 MALDI TOF-TOF by Mass Spec Core personnel for $15 per sample (mass within 0.05%) or $25 per sample (mass within 0.015% using external/internal calibrants), with an additional $10 charge per sample for removal of interfering salts or detergents if necessary. Trypsin or other proteolytic digestions done by Mass Spec Core personnel have a $25 set-up fee plus $15 per sample digested (includes ZipTip cleanup and plate spotting). Staff time is charged at $60 per hour, and more complex samples and analyses such as user-provided tryptic digests for MudPit or iTRAQ analyses, or post-translational modification analyses, are between $100 and $1250 per sample depending on the extent of additional processing and staff analysis time needed. Please discuss your specific project and experimental goals with Dr. Stanley to determine the price for these more complex services.

Note also that the quoted prices are discounted prices for INTERNAL Penn State University and College of Medicine personnel and reflect institutional subsidies to defray portions of the actual costs of these analyses. External users are welcome, but will be charged the full costs of the analyses, usually 25-35% higher fees.

Samples must be accompanied by a completed "MALDI Sample Submission form", which you can download by clicking here --> "Sample Submission Form"

IT IS CRUCIAL FOR ACCURATE ANALYSIS THAT YOU RECORD ON THE "Sample Submission Form" WHAT REAGENTS & PROCEDURES YOUR SAMPLES HAVE SEEN!

Anne Stanley runs the lab day-to-day

Room C1735, Tel. (717) 531-4055 (astanley@psu.edu)

When using data or tools generated here in publications or grants, please cite "Anne Stanley and/or Bruce Stanley (as appropriate) of the Proteomics/Mass Spectrometry Core Facility of the Section of Research Resources, Penn State College of Medicine".

Please also let us know about publications or grant applications that make use of the services of this facility.

Other Mass Spec Resources & Information:

We have a good basic MALDI-TOF library and articles available for copying

MALDI-TOF Tutorial (WEB-based)  (excellent introduction to what MALDI-TOF is and can do)

Tutorial - Using MS for Proteomics (good introduction to Protein ID using MS, including limitations)

Protein Prospector at UCSF (find proteins from masses, theoretical digests, etc.)

PROWL pages at Rockefeller (find proteins from masses, theoretical digests, etc.)

Peptide Mass at Expasy (find proteins from masses, theoretical digests, etc.)

EMBL Mass Spectrometry Page (software, companies, reference masses, etc.)

Other Mass Spec Resources on the Internet  (software, companies, reference masses, etc.)