Quantitative proteomics using ultralow flow capillary electrophoresis-mass spectrometry

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Quantitative proteomics is crucial for understanding cellular processes.
• Existing workflows face challenges in sensitivity and throughput for complex samples.
• Capillary electrophoresis-mass spectrometry (CE-MS) offers high separation efficiency.

Purpose of the Study:

• To evaluate an ultralow flow interface for coupling CE-MS with reversed-phase high-pressure liquid chromatography (RP-HPLC) fractionation for quantitative proteomics.
• To establish a workflow combining stable isotope labeling by amino acids in cell culture (SILAC) with CE-MS.
• To assess the capability of CE-MS for identifying and quantifying low-abundance modified peptides.

Main Methods:

• Proteins from SILAC-labeled and unlabeled yeast strains were mixed and digested.
• Peptides were fractionated using RP-HPLC.
• Analysis was performed using CE-MS with a neutral capillary coating at ultralow flow rates (<10 nL/min).

Main Results:

• Quantified 28,538 peptides corresponding to 3,272 proteins.
• Identified 1,371 phosphopeptides, with 49 differentially regulated between strains.
• Identified a total of 8,106 modified peptides (including acetylation, phosphorylation, deamidation, oxidized forms) and 33,854 unique peptide sequences.

Conclusions:

• The developed workflow combining SILAC labeling with CE-MS is effective for quantitative proteomics.
• CE-MS, particularly at ultralow flow rates, is a powerful tool for identifying low-abundance modified peptides without enrichment.
• This approach provides a robust method for comprehensive proteome analysis and differential expression studies.