Quantitative Top-Down Proteomics by Isobaric Labeling with Thiol-Directed Tandem Mass Tags

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Top-down proteomics (TDP) struggles with quantifying intact proteoforms in complex mixtures.
• Label-free quantification in TDP has limited multiplexing and faces challenges with multidimensional separations.
• Isobaric labeling is a standard in bottom-up proteomics, offering solutions to these limitations.

Purpose of the Study:

• Introduce thiol-directed isobaric labeling for quantitative TDP.
• Evaluate labeling efficiency and optimize mass spectrometry parameters for accurate proteoform identification and quantification.
• Assess quantification accuracy and potential ratio compression using different separation techniques and sample types.

Main Methods:

• Applied thiol-directed isobaric labeling to intact proteoforms.
• Optimized tandem mass spectrometry for fragmentation and reporter ion generation.
• Utilized gel-eluted liquid fraction entrapment electrophoresis × liquid chromatography-mass spectrometry (LC-MS) and high/low-pH LC-MS.
• Analyzed Escherichia coli (E. coli) and combined E. coli/yeast proteomes.

Main Results:

• Achieved accurate quantification of proteoforms, comparable to bottom-up proteomics.
• Quantified 876 proteoforms with high accuracy in an E. coli lysate.
• Demonstrated the method's utility in a two-proteome interference model to assess ratio compression.
• Noted that labeling is restricted to cysteine-containing proteoforms.

Conclusions:

• Thiol-directed isobaric labeling is a viable method for quantitative TDP.
• The approach provides high accuracy and overcomes limitations of existing methods.
• This technique expands the capabilities of proteoform analysis in complex biological samples.