Proteome analysis of intact proteins in complex mixtures

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Analysis of intact protein mixtures using electrospray ionization mass spectrometry (ESI-MS) presents challenges due to complex, overlapping multiply charged envelopes.
• Moderate resolution mass spectrometers are often evaluated for their ability to deconvolute such complex spectra.

Purpose of the Study:

• To assess the capability of a moderate resolution Q-TOF mass spectrometer in resolving intact protein mixtures.
• To analyze the soluble proteome of adult chick skeletal muscle, a tissue with specialized protein expression.

Main Methods:

• Proteins from adult chick skeletal muscle were separated using SDS-PAGE and identified via matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and electrospray ionization tandem mass spectrometry (ESI-MS/MS).
• Intact protein mixtures were analyzed using a Q-TOF mass spectrometer, with samples introduced via direct infusion or a C4 desalting trap.
• Mass spectrometry data were processed to resolve complex peak patterns into accurate molecular masses.

Main Results:

• The Q-TOF mass spectrometer successfully resolved complex electrospray ionization mass spectrometry data into distinct molecular masses.
• Identified masses were often consistent with known protein sequences, accounting for common co- and post-translational modifications like N-terminal methionine loss and acetylation.
• The analysis confirmed the presence of glycolytic enzymes, consistent with the specialized nature of skeletal muscle.

Conclusions:

• Moderate resolution mass spectrometry, specifically the Q-TOF instrument, is effective for resolving complex intact protein mixtures.
• This capability is valuable for protein expression profiling and for verifying post-translational modifications in mature proteins.
• The study demonstrates the utility of ESI-MS in detailed proteomic analysis of complex biological samples.