Phosphoprotein Identification
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Phosphoprotein Identification is employed for identification of phosphorylation sites and phosphoproteins to provide key information for understanding their biological function. This technique greatly expands our knowledge of the diversity of phosphoproteins. With this advanced analysis technique, we could investigate entire phosphorylation-based signaling networks.

Benefits:

  • High accuracy: Capable of localizing a phosphorylation site to a specific amino acid
  • Large-scale：Thousands of phosphorylation sites can be detected in a single experiment
  • Multiple fragmentation methods: Provides CID, HCD, or ETD fragmentation methods

Services:

Single Phosphoprotein Identification

Single phosphoprotein identification aims to identify the phosphorylation-site and peptides on purified protein sample (concentration >80%). The protein samples are firstly digested by trypsin and then enriched by TiO2, followed by analysis using LC-MS/MS.

Phosphoproteome Profiling

Phosphoproteome profiling is mainly focused on organisms or tissues, and aimed at identifying all phosphoproteins as well as their corresponding sites and peptides. Proteins are digested and then enriched by TiO2, followed by analysis using LC-MS/MS and database search.

Identification and Analysis of Phosphorylation Status of Proteins in Dormant Terminal Buds of Poplar. BMC Plant Biology. 11:158 (2011).

To discover the role of protein phosphorylation in the process of dormancy regulation, mass spectrometry combined with TiO2 phosphopeptide-enrichment strategies was performed to investigate and identify 161 uniquephosphorylated sites in 161 phosphopeptides from 151 proteins. Moreover, 141 proteins have orthologs in Arabidopsis, while 10 proteins are unique to poplar. This study provides evidence about the significance of protein phosphorylation during dormancy and will be useful for similar studies on other woody plants.

Workflow:

Figure 1. Workflow of Phosphoproteomics Analysis

Bioinformatics:

Standard bioinformatics analysis

  • Data statistics
  • Phosphorylation site localization
  • Phosphopeptide and phosphoprotein identification
  • Phosphoprotein GO annotation (available for phosphoproteome profiling)
  • Phosphoprotein COG annotation (available for phosphoproteome profiling)
  • Phosphoprotein pathway analysis (available for phosphoproteome profiling)

Custom Bioinformatics Analysis

  • We can also perform customized analysis to meet specific project needs.

Sample Requirements:

Single Phosphoprotein Identification

  • Protein: amount >30ug; concentration of purified protein >80%
  • Gel band: ≥5 visible Coomassie stained gel bands

Phosphoproteome Profiling

Sample Type

Amount of Sample required

Comment(s)

Fresh animal tissue (wet weight)

≥ 100 mg

For tissue with high impurity or low amount of proteins such as plant root or phloem, 5g sample is needed.

Fresh plant tissue or fungus (wet weight)

≥ 1 g

Microorganism (e.g. bacteria)

≥ 200 mg

Cell

≥ 3-5×106 cells

For phosphorylated protein identification, the cell number should be more than 107.

Blood

Serum / plasma ≥ 500 µl

Whole blood ≥ 5 ml

We do not recommend sending whole blood sample because blood cells can be broken during transport process.

Please remove high-abundance protein before sending extracted proteins and attach gel images before and after the removal of high-abundance protein.

Body fluid (e.g. saliva and cerebrospinal fluid)

≥ 5 ml

/

Urine

≥ 50 ml

Centrifuge at 1000g for 5min to discard the precipitate before sending your sample to BGI Tech.

Protein extract

≥ 500 µg (concentration ≥1mg/ml)

Without enrichment

≥ 4mg

Enrichment without fraction

≥ 10mg

Enrichment with fraction

 

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