The 4700 Proteomics Analyzer is extensively used in our laboratory. We routinely use human post-mortem as well as non-human primate brain tissue to study complex mammalian behavior patterns in various neuropsychiatric disorders. The expression analyses of different phenotypes are studied by two dimensional-differential in gel electrophoresis as well as with iTRAQ™ (Applied Biosystems). The functional proteomes are characterized by various gel-staining technologies such as ProQ-Diamond™ Phosphoprotein Gel Stain (Invitrogen). For all the above proteomics-based studies, we use the 4700 Proteomics Analyzer for characterization of protein identities as well as their post-translational modification status.
The 4700 Proteomics Analyzer is a MALDI-ToF/ToF instrument. It is attuned with popular proteomics tools, including 1- & 2-D gel electrophoresis, as well as MDLC and downstream bioinformatics applications. It is also well-suited for the ABI ICAT™ and iTRAQ™ technologies for expression analysis. The coaxial tandem ToF/ToF coupled with CID makes it possible to achieve high quality fragmentation on a routine basis. The information generated from peptide fragments and immonium ions provides important structural information. The superior throughput of MALDI combined with the sensitivity and dynamic range of MS/MS gives unequivocal protein identification, even for low abundance proteins, with speeds well-matched for proteomics scale (approximately 200 MS/MS are acquired in 1 hour using 2000 laser shots per spectrum).
It is also feasible to archive the sample on the MALDI target plates. This permits a re-visit to the sample for mining additional structural information at a later time point, which is not possible with electrospray ionization LC-MS/MS. This is of importance for most proteomics projects where the modifications sought are classically unknown. For 1-and 2-D gel electrophoresis samples, the RDA can specifically select the precursors for MS/MS based on confident/less identifications from PMF. The RDA also makes it possible to re-analyze the samples for low abundance gene products by excluding the proteins identified in the 1st run. This can be achieved by increasing the intensity as well as the number of laser shots for subsequent runs. Although we don’t use it very often, high-energy fragmentation can also be used with the linear ion detection mode for primary analysis of proteins prior to digestion.
The pairing of LC with the 4700 Proteomics Analyzer decouples the rate of chromatographic separation from the rate of MS/MS acquisition. The traditional LC-MS/MS systems, such as electrospray ionization, are ‘on-the-fly’ systems with analytes being transitorily eluted for a restricted period of time. The coupling of LC with MALDI does not have such a limitation, allowing the acquisition to proceed as its unique rate without the fear of loosing valuable sample subsets as they elute during the LC run. The peak and adduct exclusion enables collection of non-redundant data at the same time, thereby optimizing the utilization of sample.
The option of automated synchronization of MS/MS acquisition with the chromatographic peak maxima facilitates increased sensitivity. The entire MS and MS/MS from a MDLC experiment comprising 10-15 cation exchange chromatographic fractions further separated by reverse-phase on to 10-15 x 192 spot MALDI target plates can be analyzed at a single time by the GPS Explorer™ software. The GPS Explorer™ characterization is based on the ‘proteomics standard’ MASCOT® search engine combined with ion intensities. Also included in this software is a de novo option for characterization of peptides from species for which annotations in the database are still incomplete.
Dr. Nilesh S. Tannu
Research Associate
Wake Forest University School of Medicine
Physiology and Pharmacology