Protein Post-translational Modification Analysis

Disulfide Bond

Disulfide bond is formed due to post-translational modifications in proteins formed between the sulfur atoms of two cysteine residues during the biosynthesis of the proteins in the cell. Disulfide bonds are important in protein folding, they play a significant role in both protein structure and function. Therefore, the analysis of disulfide bond in proteins is of great significance to reveal the higher structure and biological functions of proteins. In addition, incorrect disulfide bond formation or exchange can cause malfunctioning of protein, so understanding the disulfide bonding is critical for protein characterization.


  • Available for protein analysis at the single protein level and proteomics level.
  • Highly versatile. We can analyze various forms of samples such as tissue extracts, whole cell lysates, subcellular fractions and so on.
  • High throughput, high degree of automation, and strong separation ability.

Applications of disulfide bond analysis service:

  • Study unknown disulfide bonds in novel proteins.
  • Analyze disulfide bonds in refolded proteins to test whether a protein is correctly folded.
  • Confirmation of correct disulfide bond linkage in protein therapeutics.

Glycosylation Analysis of Protein

Glycosylation is a common post-translational modification. There are two main types of protein glycosylation: N-glycosylation, in which the glycan is attached to an Asn residue present in the tripeptide consensus sequon Asn-X-Ser/Thr (where X can be any amino acid except Pro), and O-glycosylation, in which the glycan is attached to a Ser or Thr residue. Glycans are implicated in a wide range of intracellular, cell–cell and cell–matrix recognition events and are therefore of great biological interest1,2. To correlate functional features with defined structural parameters, detailed structural analyses of glycan chains are required. The most common manner of characterizing protein glycosylation involves the following steps: first, an enzymatic or chemical release of the attached glycans; second, derivatization of the released glycans via reductive amination with aromatic or aliphatic amines or permethylation; third, analysis of the glycans. The complete structural elucidation of glycans requires the determination of the sugar composition, sugar sequence, monosaccharide branching, interglycosidic linkages and anomeric configuration. In addition, glycoproteins may carry several different glycans, often a mixture of N- and O-linked glycans, and these glycans may occur in variable amounts at glycosylation sites, with varying degrees of site occupancy. As a consequence, glycan analysis requires the sequential employment of several analytical techniques.


Phosphorylation modification has mainly two regulatory mechanisms for the target proteins:

  1. Phosphorylation may cause a conformational change in the structure of modified proteins, such as enzymes and receptors, turning “on” or “off” the function.
  2. Phosphorylation changes the proteins’ affinity to their effector, by doing so, phosphorylated proteins can recruit or release their downstream effectors. Hence, it is conceivable that phosphorylation modification regulates a broad range of biological activities, such as cell growth, cell metabolism, cell division, and so on. Phosphorylation may occur on serine, threonine, tyrosine, and histidine residues in eukaryotic organisms. Among these four types of phosphorylation, tyrosine phosphorylation is most rare, but very crucial. The most famous tyrosine kinase receptor is in the very upstream of the MAPK pathway, and regulates Ras and other kinase, initiating the phosphorylation cascade.

Workflow of our Phosphorylation analysis service:

  • In gel or in solution digestion of proteins (depends on type of samples you send us)
  • Concentration of phosphor-peptides (optional; depends on the abundance of phosphor-peptide)
  • HPLC separation, followed by MALDI-TOF MS/MS analysis
  • Mass spectrometry data interpretation


Similar to phosphorylation, S-Nitrosylation is a reversible precess. The denitrosylation is an enzymatic catalyzing process that reverses the S-Nitrosylation process. However, S-nitrosylation is not a random event, and only specific cysteine residues are S-nitrosylated. Under physiologic conditions, protein S>-nitrosylation and SNOs provide protection preventing further cellular oxidative and nitrosative stress. Aberrant S-Nitrosylation may lead to protein misfolding, synaptic damage, and apoptosis.

Methylation, Acetylation

Protein methylation is a process of post-translational modification (PTM), in which highly specific enzymes called methyltransferases are responsible for the addition of methyl groups to a targeted molecule and S-adenosyl methionine (SAM) as the primary donor of methyl group. Protein methylation commonly found on arginine, lysine, histidine, proline, and carboxyl groups. Protein methylation plays an important role in modulating cellular and biological processes, including transcriptional regulation, RNA processing, metabolism and signal transduction.

Sample Requirements

  • Plant roots, xylem, phloem, etc.: 5g or more
  • Animal tissues: wet weight >200 mg
  • Microorganisms: wet weight > 2 g
  • Body fluids (saliva, amniotic fluid, cerebrospinal fluid, etc.): 10 mL or more
  • Serum: 500 μL or more
  • Urine: 50 mL or more
  • Protein extract: concentration > 2 mg/mL, total not less than 1 mg. In order to ensure the test results, please inform the buffer components, whether it contains thiourea, SDS, or strong ion salts. In addition, the sample should not contain components such as nucleic acids, lipids, and polysaccharides, which will affect the separation effect.