IIRMES is pleased to announce the opening of CEPA - a proteomics center for student training and research - made possible by a $500,000 grant obtained from the W.M. Keck Foundation.  These funds, together with supplemental matching funds provided by the College of Natural Sciences and Mathematics at CSULB have been used to purchase an Applied Biosystems 4800 Matrix Assisted Laser Desorption Ionization, tandem Time of Flight Mass Spectrometer (MALDI-TOF/TOF-MS) for protein and polypeptide analysis and identification (Figure 1).  Now fully operational, CEPA is managed by Dr. Ashraf Elamin. who operates the MALDI-TOF-MS and the center for intruction, training, research and contract analyses (see competitive pricing).  It is anticipated that CEPA will become part of IIRMES's Facility for Elemental Micro Chemical Analysis (FEMCA), a  CSU system-wide analytical core facility supported by CSUPERB.  To the best of our knowledge, this facility is the only one of its kind in the USA that focuses on the use of this technology for undergraduate training and research.

Figure 1.  AB 4800 Double focusing MALDI-TOF/TOF showing flight path of ions through the ion optics and the reflectron.

The AB 4800 MALDI-TOF/TOF-MS is a double focusing instrument with a linear flight tube and a reflectron with an intervening collision cell for analyte fragmentation. The instrument was installed in June 2006 and forms the the analytical center piece of a suite of instruments designed for automated sample preparation and handling that includes a 2D gel system, spot cutter, liquid sample handler with digestion and target spotting and software for spot recognition and peptide digestion mass finger print mapping (table 1).  

Table 1:  Equipment Purchases for Proteomics Facility

MALDI-TOF has revolutionized the study of non-volatile compounds such as peptides, oligosaccharides, phospholipids, bile salts and post translational modifications of proteins such as states of glycosylation and phosphorylation. The inherent attributes of TOF-MS are excellent and comprehensive sensitivity, an almost unlimited mass range and speed of analysis. Since the discovery of matrix assisted laser desorption in the 1988, products with molecular weights exceeding hundreds thousands of daltons, such as polymers, proteins, glycans and nucleotides, have been analyzed by TOF-MS detectors.  For analysis, samples are spotted together with a UV absorptive matrix onto a target surface that is introduced into the vacuum system of the mass spectrometer.  Unlike our GBC Optimass ICP-oTOF-MS which uses an Argon plasma generated by an inductive radio-frequency as a strong ionization source to fragment, atomize and then render the sample to an ion stream, MALDI-TOF-MS utilizes a soft-ionization procedure using a pulsed laser beam to minimize molecular fragmentation.  The irradiated sample forms ions of the type [M+X]⁺where X= H, Li, Na, K, etc.). In addition to causing ionization, the light energy absorbed by the sample results in a rapid heating and expansion that causes sublimation of the matrix and supersonic expansion of the analyte away from the target surface at a velocity that varies between ~200 to 1000 meters per second (m/s). Once generated in the laser source, a potential, is applied across the source to extract and accelerate the ions from the source into the field-free 'drift' zone of the instrument. Theoretically all the ions are given the same initial kinetic energy by the extraction pulse and then drift along the field free drift zone where they are separated so that all ions of the same m/z arrive at the detector at the same time. In practice, a kinetic energy distribution is obtained for each discrete m/z species. This is corrected by the application of a series of electric fields in a reflectron that repulse the ions back along the flight tube resulting in the refocusing of ions with the same m/z value on the reflectron detector (figure 1).

A number of preparative steps are typically necessary prior to MALDI-TOF analysis (figure 2). 

Complex mixtures of proteins will initially be run on 2D gels.  The stained proteins will be identified and located in the X-Y position using the software and robotically removed for controlled digestion in a liquid handler. They can then be either analyzed directly (Figure 3) to determine the molecular weight of the parent molecule or subjected to digestion using a variety of different such as glycosylases, phosphatases and proteases.  These can be used diagnostically to determine post-translational modifications or determine limited information on the primary amino acid structure.  For example enzymes, such as trypsin, cleave the polypeptide backbone at lysine and argenine residues, except when they are next to proline residues (Figure 4).

Figure 3.  Software identification and analysis of a 2D gel of a cell lysate showing numerous separated proteins

Protease digests will be subsequently deposited onto a MALDI target using a liquid handler and automated spotter for analysis.  The mass fingerprint of the digested protein will be uniquely identified by matching the experimental peptide masses with known sequence database values (figure 3).

One of the limitations of this approach is that unequivocal identification of proteins using 2D gels and MALDI digests alone necessitates identification of the fingerprint in the digest map in the instrument database.  At this moment there are only a few species for which a complete proteome with associated annotated fragment digest database is known.  One of the major advantages of having a double focussing TOF-TOF with an intervening collision cell is that fragments from a protein digest can be selectively transmitted through the first TOF MS analyzer for subsequent fragmentation and de novo sequencing in the second TOF MS.  Therefore, the ability of this instrument for de-novo sequencing of fragment digests will be invaluable for organisms whose genomes have not been fully annotated and would allow degenerate primers to be generated, allowing a cDNA approach for protein identification. 

Perhaps the most exciting aspect of funding of the MALDI-TOF is that it will be used in teaching.  A number of existing classes in the Biological Sciences and Chemistry and Biochemistry curricula at CSULB aim to use the instrument within class exercises for demonstrations in proteomics (table 2). 

able 2.  Current CSULB Classes that will incorporate MALDI-TOF

Table 3.  CSU Undergraduate Courses Incorporating MALDI-MS Instruction and Analysis