Description of Research in Analytical Chemistry:
My research has used mass spectrometry along with stable isotopically labeled tracers and kinetic models to answer questions of human physiology and biochemistry. The research program of my group in chemistry focuses on developing new methods and techniques of mass spectrometry to measure the biological molecules and,using stable isotopically labeled tracers, their rates of production and disposal.
Much of our research is focused upon amino acid and protein metabolism in humans. Students are encouraged as part of their thesis work to apply methods developed to clinical studies of metabolism in humans. Many simple metabolic questions have never been answered in humans. We do not know which pathways regulate the metabolism of several important amino acids in humans, and we do not know how protein and amino acid metabolism is regulated in the body to maintain protein stores or why the body accelerates oxidation of amino acids in states of stress, trauma, or sepsis using protein stores to the point of being life threatening.
Proteins have many functions from being the basic unit of our muscles to enzymes to being key signaling molecules. The regulation of all of this functions is complex and often involves posttranslational modification (PTM) of proteins through phosphorylation and other covalent changes.
Mass spectrometry has been widely applied for compound identification, but precise measurement of isotopes in biological compounds has received less attention. Gas chromatography-mass spectrometry (GCMS), liquid chromatography-mass spectrometry (LCMS), and isotope ratio mass spectrometry (IRMS) instrumentation are all available and are used for research in our group to measure stable isotope ratio tracers in biological samples. LCMS has been particularly useful for developing methods in our group to quantify PTM of proteins for both phosphorylation and glycosylation.
Work in Analytical Chemistry:
The figure below is from the Ph.D. thesis of Michael Previs (see MJ Previs, P VanBuren, KJ Begin, JO Vigoreaux, MM LeWinter & DE Matthews: Quantification of protein phosphorylation by liquid chromatography mass spectrometry. Anal. Chem. 80: 5864-5872) and highlights the scheme we have developed to quantify phosphorylation of proteins at multiple sites using a tryptic digest of proteins and LCMS analysis. The key to the method is to perform the phosphorylation quantification without having to measure the phosphorylated peptide per se. This approach greatly simplifies the measurement scheme and does not require use of isotopically labeled standards.
- The key to the method is to first split the sample into two aliquots (step 1).
- One half of the protein sample is digested with trypsin (step 2).
- The peptide digest is then measured by electrospray ionization (ESI) LCMS operated to collect both mass spectra (MS) of parent ions, but also product ions via MS/MS in a standard mode for proteomics experiments (step 3). The MS and MS/MS data obtained are searched using SEQUEST to identify peptides and proteins from the peptides. The peak area of the time v. intensity MS plot is determined for the peptides that could be phosphorylated (X) and one or more reference peptides (Z) in the protein that are not modified. Only the unphosphorylated species of the potentially phosphorylated peptide (X) is measured.
- The other half of the protein sample is first treated with alkaline phosphatase to dephosphorylate the proteins (step 2a), then the sample is digested with trypsin (step 2b).
- This sample is also measured by ESI-LCMS/MS, peptides identified by SEQUEST, and the peak areas of the unphosphorylated peptides (X) and the reference peptides (Z) measured (step 3).
- The ratio of the peak area of the unphosphorylated peptide to the reference peptide(s) (X/Z) is obtained for both the untreated aliquot A and the dephosphorylated aliquot B.
- Because aliquot B is a measure of the total amount of peptide (phosphorylated and unphosphorylated), dividing the X/Z ratio of A/B gives the degree of phosphorylation in peptide X in the sample (step 4).
- Our original work also included a derivatization scheme to propylate the C-terminus carboxyl-group of every peptide (which also propylates glutamate and aspartate residues). This scheme allows aliquot A to be treated with unlabeled n-propanol after step 2 and aliquot B to be treated with labeled propanol, e.g. n-[1,1,2,2-2H4]propanol, after step 2b.
- The two aliquots are then combined and run as a single sample in step 3.
Last modified January 19 2015 08:33 AM