PhosphoProteomics

PhosphoProteomics Identification and Quantification Services

Phosphorylation is one of the most important PTMs in a biological system. This service can be used to study the phosphorylated proteins in your biological samples. Phosphorylated proteins/peptides will be enriched and then subject to downstream proteome identification or quantification through Mass Spectrometry Analysis.

Total phosphorylation proteome (through TiO2 enrichment) and specific targeted phosphorylation proteome (through enrichment using anti-Phospho-Tyrosine or anti-Phospho-(Ser/Thr) Phe antibodies) can be analyzed to identify the phosphorylated proteins in your sample, and the difference in phosphorylation levels of proteins in your samples when combining with TMT or iTRAQ labeling strategy.

Your samples (several test groups and a control group) will be digested with Trypsin, and peptides will be enriched using an appropriate method, followed by chemical labeling by either iTRAQ or TMT labeling method if a quantitative analysis is desired. Enriched (with or without labeling) peptideswill be cleaned using SCX HPLC and a bRPLC fraction to further generate the 2nd dimansion of separation which has been proved to significantly increase the coverage and depth of the Mass Spectrometry Analysis.

1) Sample preparation – Cell or tissue lysis, biofluid (plasma or serum) depletion; protein quantification on all samples.

2) Sample digestion – Reduction and alkylation followed by in-solution trypsin digestion and C18 cleaning before labeling.

3) Phospho-peptide enrichment – TiO2 or specific antibody enrichment according to your choice. We will apply our patented Mass Spectrometry friendly technique, My-IPElu, to fully recover your phospho-peptides.

4) (Optional)Sample labeling – Reagents are available that allow the labeling of 2 through 10 samples individuallysimultaneously.

5) (Optional)Sample pooling – Equal amounts (according to total protein quantification) of labeled samples are pooled together to allow unbiased downstream purification and Mass Spectrometry analysis.

6) SCX cleaning – HPLC cleaning and fractionation of pooled sample through off-line SCX.

7) 2D fractionation – bRPLC separation of the SCX cleaned samples into 96 fractions, and further pooled into 12 or 24 fractions based on the rule of avoiding co-elution.

8) Nano LC/MS/MS – 50 nL/min liquid chromatography to enable super high ionization efficiency for downstream Mass Spectrometry Analysis using the state-of-the-art Orbitrap Mass Spectrometer.

9) Protein profiling – Protein database searching with Mascot, Sequest HT, PEAKS de novo approaches, etc.

10) Data analysis – Data validation, visualization and quantification using Omics Technologies' unique R package and scripts.

11) Report generation – Report will be sent to you in Excel format as well as a summary in pdf format. We will also provide you any detail you need for your papers' MATERIALS AND METHOD section. We will make sure you understand your result.

1, 2D gel spots, SDS-PAGE bands

2, Cell Lysates and Tissue Lysates

3, Biofluids, such as plasma*, serum*, saliva, tear, etc.

*We provide High Abundance Protein Depletion Service to significantly (100-500 folds) increase the depth of your biofluid proteomics analysis by removing top abundant proteins from your samples. Read more for details​.

4, FFPE slide/ FFPE extract

5, Customized sample types (please contact us to discuss)

Please refer to our Omics Technologies Proteomics Sample Preparation and Shipping Guidelines for instructions on how to prepare your samples with less contamination for Mass Spectrometry Analysis.