Shared LC-MS/MS for Quantitative Targeted Proteomics

Univ Of North Carolina Chapel Hill, Chapel Hill NC

Investigators

Abstract

Project Summary/Abstract: This request, as a resubmission from UNC Chapel Hill, is for an upgrade from an older nanoLC-MS/MS, a Sciex 5500 QTRAP, that was acquired in 2008 with a SIG. A microLC-MS/MS triple quadrupole is requested, the Sciex 7500 QTRAP Ready that is coupled to a Waters M-Class microLC. This upgrade will increase peptide sensitivity relative to the 5500 by approximately 40-fold and permit faster throughput. Our laboratory has operated the Sciex 5500 coupled with a Waters NanoAcquitity for about 13 years, over time building an extensive group of collaborators within the Schools of Pharmacy and Medicine, as well as scientists from other universities, nationally and internationally. Some collaborations have also been with the pharmaceutical industry within drug discovery and metabolism. The focus has been almost entirely quantitative targeted absolute proteomics (QTAP) using proteotypic stable isotope labeled (SIL) peptides as standards for calibration. Our lab is established as a leader in the development and application of QTAP for multiplex measurements of proteins involved in the absorption, distribution, metabolism and excretion (ADME) of drugs. This has led to the creation of an extensive library of SIL peptides standards permitting the accurate and precise quantification of 50 or more proteins in a single sample, usually enzymes and transporters, in as little as 50,000 cells (e.g. human hepatocytes), with methods validated for about 200 proteins across humans and preclinical species. The application of QTAP within biomedical research is almost limitless and more recently we have developed methods for integrin surface adhesion proteins in exosomes (Users: Kabanov, Batrakova) as well as phosphorylation events on ITAM signaling proteins in macrophages (User: Vilen). Given the broad potential for use of QTAP in medical research, it is still largely untapped due to the lack of familiarity of its potential by many biomedical researchers and the need to establish and centralize SIL peptide standards. Our experience has created a facility that is growing on the UNC campus and providing a service that is unique compared to other MS facilities on this campus and at other universities, now including collaborations with the Lineberger Cancer Center at UNC to examine cancers with QTAP (User: Emanuele). As a result of our experience and unique capabilities, some of our collaborators are from other universities (User: Aleksunes) within the US and abroad. With our success and increasing instrument capabilities, the need for greater throughput and sensitivity is needed, thus the request to obtain a Sciex 7500. As part of this request the new instrument and our staff will be integrated into the Nanomedicine Characterization Facility Core (NCCF), under the Center for Nanotechnology and Drug Delivery (CNDD) within the UNC Eshelman School of Pharmacy. This will include a training component within a T32 grant to educate fellows, graduate students, faculty, and the attendees at Workshops conducted by the Center (open to all) on the use and benefits of QTAP for biomedical research.