Improving the Sensitivity of Protein Quantification by Immunoaffinity Liquid Chromatography─Triple Quadrupole Mass...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• Advancements in liquid chromatography-tandem mass spectrometry (LC-MS/MS) aim to lower limits of quantification for low-abundance proteins.
• Immunoaffinity (IA) enrichment and nanoflow LC (nLC) are established methods to enhance sensitivity.
• Further improvements in signal-to-noise (S/N) ratios are often necessary for sensitive protein analysis.

Purpose of the Study:

• To evaluate the applicability of echo summing for protein quantification using IA combined with nLC-MS/MS (IA-nLC-MS/MS).
• To assess the impact of echo summing on accuracy, precision, and sensitivity for protein assays in human serum.

Main Methods:

• Applied echo summing, a technique involving repeated collection and summation of single selected reaction monitoring (SRM) transitions.
• Utilized sequential IA enrichment coupled with nLC-MS/MS for protein analysis.
• Quantified beta nerve growth factor (NGF) and soluble asialoglycoprotein receptor (sASGPR) in human serum samples.

Main Results:

• Five iterations of echo summing improved accuracy and precision for NGF quantification compared to traditional methods.
• Echo summing approximately doubled the functional sensitivity for sASGPR peptides.
• The minimum quantifiable level for sASGPR was extended 4-fold lower using echo summing.

Conclusions:

• Echo summing is directly applicable and beneficial for protein quantification in IA-nLC-MS/MS assays.
• The technique offers significant improvements in sensitivity, accuracy, and precision for low-abundance protein targets.
• Similar sensitivity gains are anticipated for most protein IA-nLC-MS/MS applications.