Research Overview

Changes in the quantitative pattern of proteins associated with aging processes are providing insight into disease pathologies and may help in early diagnosis of disease. We applied various mass spectrometry-based approaches to perform quantitative measurements on human cells and tissues.

 

MRM approach has been developed to measure specific peptides in complex biological mixtures. Proteases are first used to cleave the proteins into peptides. This approach then uses the selectivity of the triple quadrupole mass spectrometer by first allowing only ions with a specific mass, that of the intact peptide (MS1), to be transmitted from the first quadrupole to the second quadrupole in the mass spectrometer. The intact peptide ions are fragmented in the second quadrupole producing fragment ions. In the third quadrupole, only a single fragment ion (MS2) is transmitted to the detector, resulting in the signal that ultimately is used for quantification. The pair of m/z values for the intact peptide and specific fragment from this peptide (MS1-MS2 pair) is called a MRM transition. MRM transitions that represent many proteins can be monitored in a single analytical run. Using this approach, the absolute protein quantification is based on the selection of specific MRM transitions to be used as stoichiometric representatives of a protein. Once these ratios are determined, the protein content can be quantified by comparing the peak area with that of a stable isotope-labeled chemically synthesized internal peptide standard.

 

Amide H-D exchange MS is a sensitive technique that can be used to study the changes in protein structure and dynamics due to protein-protein interaction, ligand binding, and protein modification. The general procedure includes equilibration of control and treated proteins in D₂O allowing isotopic exchange of amide hydrogens, then quenching of exchange, pepsin digestion, and MS analysis. The level of amide-based deuterium incorporation into each pepsin-generated peptide can be determined by the change of molecular mass as a function of time. This information is essential for localization of regions of proteins that are changed and for defining the dynamic processes that proteins undergo on a local or global scale.
 

To estimate the relative amount of a specific protein in two separate samples (control versus experiment), the proteins in control sample are alkylated with light (non-deuterated, d₀) acrylamide while the proteins in experiment sample are alkylated with heavy (deuterated, d₃) acrylamide. After mixing these samples at 1:1 ratio, the proteins are separated using various analytical techniques. The proteins of interest are then digested with trypsin and identified using MS. The following relative quantification is accomplished by comparing a ratio between intensities of the light and heavy mass components of the cysteine-containing peptides.