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Palmitoylation Site Identification Service

The most common protein translation modification processes are classified as phosphorylation, glycosylation, acetylation, methylation, ubiquitination, etc. Among them, palmitoylation is one of the most well-known and the only reversible translation modification processes. The increase in hydrophobicity of palmitoylated proteins plays an important role in protein translocation, organelle localization, and functions, as well as confers diverse physiological functions to proteins. Hence, in-depth research on the mechanism of protein palmitoylation modification is of great significance for understanding complex signaling pathways. Although some progress has been made in studying the mechanism of protein palmitoylation, up to hundreds of palmitoylated proteins lack clear evaluation on modification sites and greatly hinder the investigation of palmitoylated proteins' functions.

S-palmitoylation, an important post-translational protein modification, is a specific protein S-acylation that affects protein structure and function. In addition to S-acylation, a few palmitoyl salts can be attached to various amino acid groups (e.g., glycine, cysteine, lysine) to form N-palmitoylation, or the hydrolysis of serine and threonine to form O-palmitoylation.

Advances in Research Methods of Palmitoylation

The widespread use of traditional targeted mutagenesis and protein palmitoylation inhibitors has promoted the elucidation of protein palmitoylation modifications, meanwhile research process remains time-consuming and laborious, and significant breakthrough has not been achieved. Therefore, our current knowledge of palmitoylation modifications is fairly limited compared to other translational modifications, such as phosphorylation and glycosylation.

With the development of mass spectrometry and proteomic technologies, an increasing component of palmitoyl proteins and their modification sites have been identified, which has greatly contributed to understanding protein palmitoylation. In addition, with the widespread use of mass spectrometry for directly detecting palmitoylation sites in proteins and the development of acyl-biotinylexchange (ABE) and metabolic labeling with palmitate analogs, more palmitoylation modification sites have been elucidated for further research.

Recently, click-chemistry and ABE have brought additional breakthroughs in the identification of palmitoylated proteins. Combined with high-throughput protein profiling, these two techniques have opened up more possibilities for discovering palmitoylation sites in various cellular tissues and clinical samples.

Our Palmitoylation Site Identification Service

Relying on a professional HPLC-MS/MS mass spectrometry platform and extensive project experience, Creative Proteomics is capable of providing efficient and professional palmitoylated protein identification services for various eukaryotic and prokaryotic samples.

Service Workflow

Click Chemistry

Labeling of target modified proteins with palmitic acid analogs (metabolic labeling)

Catalytic reaction to induce biotin labeling of palmitoyl-modified proteins (click chemistry)

Enrichment of modified peptides with affinity streptomycin beads (peptide enrichment)

Mass spectrometry (LC-MS/MS) to analyze modified peptides

Click Chemistry

Biotin Exchange Method

  • Alkylation of free thiol groups
  • Incubation of protein depalmitoylation
  • Biotinylation of SH group
  • Incubation of enriched biotin-labeled peptides (peptide enrichment) with affinity streptomycin beads
  • Mass spectrometric detection of peptides (LC-MS/MS analysis)

Biotin Exchange Method

Service Advantages

Easy and simple operation: no isotope labeling required; simple experimental procedure

High precision and accuracy: specific modification sites can be identified and analyzed; the Thermo Fisher Mass Spectrometer can be used to obtain additional peptide information and modification sites

Wide range of detection: palmitoylation sites can be detected in various cells or tissues

Sample requirements

  • Fresh animal tissue: ≥600 mg
  • Fresh plant tissue: ≥6 g
  • Cell culture: ≥1×107 cells/tube x 3 tubes
  • Fungi and bacteria: ≥600 mg
  • Serum, plasma: 450 μL × 4 tubes
  • Protein solution: total protein of 5-10 mg
  • Body fluid samples: urine of 15 mL × 4 tubes (centrifuge at 1000 x g for 5 minutes and discard sediment); or other body fluids (saliva, amniotic fluid, cell culture supernatant, etc.) > 15 mL

Reference
1. Amy F Roth, Junmei Wan, Aaron O Bailey, Beimeng Sun, Jason A Kuchar, William N Green, Brett S Phinney, John R Yates 3rd, Nicholas G Davis Global analysis of protein palmitoylation in yeast. Cell 2006;125(5):1003-13.

* For Research Use Only. Not for use in diagnostic procedures.
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