Novel post-digest isotope coded protein labeling method for phospho- and glycoproteome analysis

Area of Science:

• Proteomics
• Biochemistry
• Molecular Biology

Background:

• Quantification of post-translational modifications (PTMs) in proteomics remains challenging due to a lack of reliable methodologies.
• Accurate PTM quantification is crucial for understanding protein function and cellular processes.

Purpose of the Study:

• To develop and validate a novel post-digest Isotope-Coded Protein Labeling (ICPL) quantification strategy for characterizing phosphorylated and glycosylated proteins.
• To compare the efficacy of the novel ICPL method against classical ICPL and label-free MS quantification.

Main Methods:

• The study employed protein digestion, ICPL labeling, sample mixing, and PTM enrichment (TiO(2) for phosphorylation, hydrazide chemistry for glycosylation).
• Enriched and non-enriched peptides were analyzed using 2D-(SCX-RP)-Nano-HPLC-MS/MS.
• Method validation involved alternative protein quantification strategies and label-free MS quantification.

Main Results:

• The novel ICPL method significantly improved protein identification (∼600 proteins) and quantification (>95%) compared to the classical ICPL method.
• Comparison of prostate cancer cell lines (PC3 and LNCaP) revealed specific protein modulations related to metastasis.
• Preliminary analysis of autopsy cases supported the biological relevance of the identified proteins.

Conclusions:

• The developed post-digest ICPL strategy offers a significant advancement for quantifying PTMs, particularly phosphorylation and glycosylation.
• The method provides valuable insights into the molecular mechanisms of cancer metastasis by comparing distinct cell lines.
• The findings highlight the potential of this approach for identifying clinically relevant protein biomarkers.