Improved sensitivity for quantification of proteins using triply charged cleavable isotope-coded affinity tag...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Isotope-coded affinity tag (ICAT) methods coupled with LC/MS/MS are vital for accurate protein quantification.
• Sensitivity limitations hinder the analysis of low-copy proteins in complex biological samples.

Purpose of the Study:

• To investigate the electrospray ionization (ESI) and collision-activated dissociation (CAD) behavior of peptides modified with cleavable ICAT (cICAT) reagents.
• To optimize conditions for enhanced sensitivity in protein quantification using cICAT methods.

Main Methods:

• Synthesized and digested model proteins to generate cICAT-peptides.
• Analyzed ESI and CAD behavior of singly, doubly, and triply charged cICAT-peptide ions ([IP+2H]2+ and [IP+3H]3+).
• Optimized multiple reaction monitoring (MRM) conditions for triply charged precursor ions.

Main Results:

• Triply charged cICAT-peptide ions ([IP+3H]3+) exhibited higher CAD reactivity and fragment abundance compared to doubly charged ions ([IP+2H]2+).
• MRM assays using [IP+3H]3+ precursors demonstrated 2- to 8-fold increased sensitivity over [IP+2H]2+ without compromising quantitative accuracy.
• Improved signal-to-noise ratios were observed for [IP+3H]3+ quantification, enabling sensitive monitoring of protein induction in rat liver.

Conclusions:

• The study successfully optimized cICAT-peptide analysis by utilizing triply charged ions for enhanced sensitivity in LC/MS/MS.
• [IP+3H]3+ ions represent a superior precursor for sensitive and accurate protein quantification via MRM.
• This optimized approach significantly improves the detection of low-abundance proteins in complex biological systems.