Title

Substituted Phenyl Isothiocyanates for Improved Protein Quantification by Multiple Reaction Monitoring Mass Spectrometry

Date of Completion

January 2012

Keywords

Chemistry, Analytical|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Multiple reaction monitoring mass spectrometry (MRM-MS) is a powerful approach used in proteomics quantification. This method can selectively monitor gas phase transitions of target peptides. Consequently, ion signals from precursor and fragment ions limit the detection and quantification purpose of the approach. By peptide derivatization, MRM signals will be enhanced. Major contributors to the signal enhancement of derivatized peptides include (1) increased hydrophobicity of the derivatized peptide and (2) active participation of the derivatizing group for preferentially fragmentation of the derivatized peptide. CFTR01 peptide (NSILTETLHR), a signature peptide of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, was used to study (1) the site-preferential fragmentation of phenylthiocarbamoyl peptides and (2) the relative contributions of precursor and fragment ions for the increase in the MRM signal yields after phenylthiocarbamoylaton. A stable isotope-labeled CFTR01 [NSILTET( 13C6, 15N1 L)HR], peptide was used as a reference standard for the relative quantification of the signal yield obtained from derivatized peptide. Derivatization with 4-dimethylamino phenyl isothiocyanate (DIM), 4-diethylamino phenyl isothiocyanate (DIE), and 4-dipropylamino phenyl isothiocyanate (DIP) reagents were quantitatively investigated with percent chemical conversion (PCC), signal yield for tandem mass spectrometry (SYMS2) and signal yield for mass spectrometry (SYMS). The increased in MRM signal yield of phenylthiocarbamoyl CFTR01 peptide was contributed by both the increase in ionization efficiency of intact precursor and fragment ions relative to the underivatized peptide. DIM, DIE, DIP derivatizations showed ∼6, ∼7, and ∼15 times increase in signal yield, respectively for the combined precursor and fragment ions. ^