The University of North Carolina conducts both fundamental and disease-oriented research to enhance current therapies and design new interventions. To understand many diseases, it is essential to characterize the alterations they cause in protein expression or protein modification. State-of-the-art proteomic methods couple 2D PAGE, or ICAT (isotope coded affinity tags) methods followed by capillary HPLC to mass spectrometry (MS) for quantitation and identification of differentially expressed proteins. The Proteomics Core Laboratory (PCL) has a triple quadrupole (3Q) MS (without data dependant switching) arid will soon receive a MALDI/TOF as well. The MALDI/TOF will identify proteins by their peptide mass fingerprint, after in-gel digestion. In general, however, MALDI/TOF instruments cannot sequence individual peptides routinely and the method is not compatible with on-line separations (LC-MS/MS). The poor mass accuracy, resolution and sensitivity of the 3Q limit its applicability in proteome analyses. To overcome these limitations, we are requesting a Quadrupole/Time Of Flight (Q/TOF) instrument because it can automatically isolate and sequence a series of selected proteolytic peptides as they emerge from capillary LC. These capabilities are crucial to identify differentially expressed membrane proteins, very acidic or basic proteins because these are difficult to separate by 2D PAGE, and low molecular weight proteins because only a few tryptic peptides are extracted from the gel, which might not be sufficient for unambiguous identification using peptide mass fingerprinting. Furthermore, the Q/TOF can identify and characterize post-translational modifications, such as phosphorylation and glycosylation, by specifically isolating and sequencing modified peptides, even at low- abundance and in the presence of a complex mixture. This proposal includes investigators from the Schools of Medicine and Pharmacy, the Cystic Fibrosis Center and the Lineberger Cancer Center, illustrating the broad applicability and future use of the instrument. The Q/TOF will be mainly used to identify differentially expressed proteins from SDS PAGE gels or directly with on-line LC- MS/MS separations after ICAT, and to identify and characterize post- translational modifications. To a minor extent, the Q/TOF will also be used to characterize biomolecules, such as heparan sulfate oligosaccharides and oligonucleotides. These methods will be directed toward complex problems that hamper research on breast cancer, cystic fibrosis, autoimmune blistering diseases, and herpes simplex virus among others. The Q/TOF will enable biomedical scientists to rapidly characterize and quantify biomolecules in complex matrices with high fidelity, thus enhancing our understanding and accelerating our advances in medicine, molecular biology, and chemistry.
| Danell, Ryan M; Ouvry-Patat, Severine A; Scarlett, Cameron O et al. (2008) Data Self-Recalibration and Mixture Mass Fingerprint Searching (DASER-MMF) to enhance protein identification within complex mixtures. J Am Soc Mass Spectrom 19:1914-25 |
