Hydrophilic Interaction Liquid Chromatography (HILIC)

Immobilized metal affinity chromatography (IMAC) is an affinity purification technique and has been widely used to enrich biological samples for phosphopeptides. Creative Proteomics specializes in peptide separation enrichment for MS-based PTM analysis based on a broad range of separation and enrichment methods, including IMAC.

Immobilized metal affinity chromatography (IMAC) for phosphopeptide enrichment

IMAC has been commonly exploited to enrich phosphorylated proteins and peptides. This affinity purification technique utilizes trivalent metal ions immobilized on a stationary phase to selectively chelate negatively charged phosphate groups of phosphoproteins or phosphopeptides based on the affinity of negatively charged phosphate groups and positively charged metal ions. Currently, a variety of various metal ions are available for phosphorylated peptide enrichment, such as Fe³⁺, Zr⁴⁺, Ga³⁺, and Ti⁴⁺. IMAC enrichment of phosphopeptides is characterized by high selectivity and sensitivity. However, this enrichment technique has the disadvantage that highly acidic non-phosphorylated peptides have an affinity for metal ions and can interact with IMAC resin. While low pH can reduce the binding of non-phosphorylated peptides, it can also reduce the binding of specific phosphorylated peptides. Therefore, enormous effort has been made to improve the efficiency of IMAC-based phosphopeptide enrichment.

Fig. 1 Schematic process for recombinant protein purification using IMAC affinity columns. IMAC, Immobilized metal affinity chromatography.(Velarde-Salcedo, Aída Jimena, et al, 2021)

Advantages of IMAC for phosphopeptide enrichment

• The column resin of IMAC shows high chemical stability and exhibits high tolerance toward a wide range of reducing agents.
• The system is compatible with commonly used detergents.
• The technique is cost-effective.

The service offering at Creative Proteomics

In general, the IMAC-based phosphopeptide enrichment process consists of three main steps, including incubation of the trypsin digest on an IMAC column, washing the resin with IMAC binding buffer, and enrichment of phosphopeptides at high pH or with phosphate salt conditions. To improve the efficiency of the IMAC approach, we use Ti⁴⁺-based resin. Since Ti⁴⁺ is highly acidic and signifies a higher affinity toward oxygen. This type of resin can prevent the binding of acidic peptides and is superior to the use of other metal ion-based phosphopeptide enrichment methods. In addition, it is also worth mentioning that a combination of enrichment procedures and appropriate fractionation strategies is necessary in order to reduce the complexity of the samples and increase the depth of PTM coverage. We also offer sequential elution from IMAC (SIMAC) as the complementary strategy for IMAC enrichment. The SIMAC approach combines IMAC and TiO₂ enrichment strategies to enrich phosphopeptide in a consecutive manner, which can greatly facilitate the identification of more phosphorylation sites.

Major benefits of our service

• Effective and affordable enrichment methods for phosphoproteins or phosphopeptides.
• Improved methods with high sensitivity are suitable for the treatment of limited samples.
• Customized enrichment strategies according to specific experimental objectives.
• Experienced protein enrichment specialists.

Related services

• Enrichment of Modified Peptides Using Strong Anion/Cation Exchange Chromatography (SAX, SCX)
• Enrichment of Modified Peptides Using Hydrophilic Interaction Liquid Chromatography (HILIC)
• Enrichment of Modified Peptides Using Reverse Phase Liquid Chromatography (RPLC)
• Enrichment of Modified Peptides Using Metal Oxide Affinity Chromatography (MOAC)
• Enrichment of Modified Peptides Using Immunoaffinity Chromatography
• Enrichment of Modified Peptides Using Multilectin Affinity Chromatography (M-LAC)

In addition to phosphopeptide enrichment service, we also offer acetylated peptide enrichment service, ubiquitinated peptide enrichment service, glycopeptide enrichment service, and histone isolation and enrichment service. Do you have any questions or comments or would like a free quote? Do not hesitate to contact us.

References

1. Chang, Y-Y., H. Li, and H. Sun. "Immobilized metal affinity chromatography (IMAC) for metalloproteomics and phosphoproteomics." Inorganic and Organometallic Transition Metal Complexes with Biological Molecules and Living Cells. Academic Press, 2017. 329-353.
2. Ke, Mi, et al. "Identification, quantification, and site localization of protein posttranslational modifications via mass spectrometry-based proteomics." Modern proteomics–sample preparation, analysis and practical applications (2016): 345-382.
3. Velarde-Salcedo, Aída Jimena, et al. "Novel technologies in bioactive peptides production and stability." Biologically Active Peptides. Academic Press, 2021. 47-74.

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