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Peptide Modifications: Phosphorylation

Phosphorylation plays essential roles in nearly every aspect of cell life. Protein kinases regulate signalling pathways and cellular processes that mediate every aspects of cellular communication and functions. However abnormal phosphorylation is a cause or consequence of many diseases. Mutations in particular protein kinases and phosphatases gives rise to a number of disorders and many naturally occurring toxins and pathogens exert their effects by altering the phosphorylation states of intracellular proteins.

Peptide phosphorylation

  • Methods
  • Introduction
  • Case Studies

Methods for Detecting Protein Phosphorylation

Peptide phosphorylation in bacteria

Radiolabel studies suggest that approximately 30% of proteins in eukaryotic cells are subject to phosphorylation. A classical method of measuring protein phosphorylation involves 2D gel electrophoresis or incubation of cells with radiolabeled 32P-orthophosphate, the generation of cellular extracts, separation of proteins by SDS-PAGE, and then exposure to film. These methods are labor-intensive and requires radioisotopes. The following summary provides a brief description of several methodologies currently used to assess phosphorylation.

  • Kinase Activity Assays
  • Kinase activity is usually measured in vitro by incubating the immunoprecipitated kinase with a substrate in the presence of ATP. Measurement of the phosphorylated substrate can be assessed by reporter systems including colorimetric, radioactive, or fluorometric detection.

  • Phospho-Specific Antibody Development
  • The development of phosphorylation-dependent antibodies has been used by many researchers. LifeTein developed many phospho-specific antibodies for researchers. Phospho-specific peptides representing the amino acid sequence surrounding the phosphorylation site of the target protein were first synthesized and then conjugated to keyhole limpet hemocyanin (KLH) for immunization. The immune sera will then be applied to a peptide affinity column to generate a highly specific immunoreagent. The successful detection is dependent on the specificity and affinity of the antibody for the phospho-protein of interest.

  • Western Blot
  • Many phospho-specific antibodies are very sensitive and can readily detect the phosphorylated protein in a routine sample. Both chemiluminescent and colorimetric detection methods are common, and molecular weight markers are also generally used to provide information about protein mass.

  • Enzyme-Linked Immunosorbent Assay (ELISA)
  • ELISAs generally provide an indirect measurement of kinase activity and is more quantitative than Western blotting. The phospho-specific ELISA technique can easily quantify the results by utilizing a calibrated standard. Using of two antibodies specific for the target protein employed together in the sandwich format will render high specificity. In addition, the microplate-based format of ELISAs allows for high throughput and smaller sample volumes and the detection of low abundance proteins.

  • Cell-Based ELISA
  • Analyzing protein phosphorylation within intact cells may more accurately represent the status of specific signaling networks. Usually phospho-specific antibodies are used to assess phosphorylation status using fluorometric or colorimetric detection systems.

  • Intracellular Flow Cytometry and ICC/IHC
  • Flow cytometry is advantageous because it allows for rapid, quantitative, single cell analysis.Cells are usually stimulated and fixed with formaldehyde or paraformaldehyde to cross-link the phospho-proteins and stabilize them for analysis. The fixed cells must then be permeabilized to allow for entry of phospho-specific antibodies into the cells.

  • Mass Spectrometry
  • Mass spectrometry (MS) techniques are useful tools for identification of phospho-proteins and phosphopeptides and sequencing of the phosphorylated residues. MS can be used with excellent sensitivity and resolution to identify a single protein or peptide. Although signals from phosphopeptides are generally weaker, new technologies have beed developed to enrich the MS signals. The enrichment strategies include immobilized metal affinity chromatography, phosphospecific antibody enrichment, chemical-modification-based methods and replacement of the phosphate group with biotinylated moieties.

  • Multi-Analyte Profiling
  • Multi-Analyte profiling involves the use of phospho-specific antibodies and include microplate-based and membrane-based detection formats. These assays provide more data while requiring very little sample volume. However these assays are less sensitive than the conventional methods due to potential antibody cross-reactivity.

Background Introduction

Phosphorylation of Ser, Thr and Tyr is a reversible protein modification involved in regulating a myriad of cellular events including receptor signaling, protein association and compartmentalization, activation and inhibition of protein function, and cell survival. Phosphates are negatively charged (with each phosphate group carrying two negative charges) so that their addition to a protein will change the characteristics of the protein. This change is often a conformational one, causing the protein to change how it is structured. The protein switches back to its original conformation when the phosphorus is removed. If these two conformations provide the protein with different activities, phosphorylation of the protein will act as a molecular switch, turning the activity on or off.

Many hormones can adapt the activity of specific enzymes by increasing their phosphorylation state of Ser or Thr residues. Growth factors (like insulin) can trigger phosphorylation of Tyr. The phosphate groups on these amino acids can be quickly removed, thus Ser, Thr and Tyr function as molecular switches during regulation of cellular processes (e.g. cancer proliferation).

Synthetic peptides have played a useful role in studies of protein kinase substrates and interaction domains. Several factors have hindered or limited the applicability of various techniques for phosphorylated peptide synthesis, such as the inability of achieving full automation using solid phases, or the lack of convenient interfacing with standard analytical platforms. LifeTein's process for synthesis of peptides and phosphopeptides on PeptideSynTM platform overcomes previous limitations and demonstrates its scalability by increasing the synthesis efficiency. The PeptideSynTM platform is well suited to the study of protein kinase substrates, antigens, binding molecules, and inhibitors.

  • We offer phosphorylation on pSer, pTyr, pThr or D-pSer, D-pTyr, D-pThr.
  • Phosphorylation is available at two sites, three sites, four sites and five sites, for example, NDEpSpTDYEpSERQpTD.

Peptide modification: phosphorylation

Case Study: Solid-phase synthesis of a peptide with 4 phosphorylation sites.

A 14 amino acid peptide (molecular weight: 1981.53) with 4 phosphorylation sites at 95% purity was delivered in 3 weeks: xxxpSpTxxxpSxxxpTx

HPLC Results:

Peptide synthesis: Phosphorylation-HPLC

MS Results:

Peptide synthesis: Phosphorylation-MS

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