Services

a. Gel-based protein identification by MALDI-TOF/TOF
b. Protein mixture identification by LC-MS/MS (Q-TOF)
c. Protein/peptide mass determination
d. PTM identification

a. 2-dimensional gel electrophoresis
b. iTRAQ

a. Sample clean up (Desalting)
b. Sypro Ruby staining and Typhoon gel imaging

• a. Gel-based protein identification by MALDI-TOF/TOF
  1. The identification of proteins obtained from conventional protein purification, affinity chromatography, or immunoprecipitation. Proteins are resolved by 1D or 2D SDS-PAGE and visualized by staining the gel with either coomassie blue or varies fluorescent staining techniques.
  2. Selected protein spots from stained gels are picked and transfered into Eppendorf tubes manually using spot picker. Big spots or 1D gel bands are cut with scalpel.
  3. A band or spot containing the candidate protein is excised, processed, and digested with a protease such as trypsin using the Tecan ProTeam system.
  4. After digestion, the peptides will be desalted by ZipTip ® and Analysed by MALDI-TOF/TOF.
• b. Protein mixture identification by LC-MS/MS (Q-TOF)
1. Integrated LC-MS-MS method for online peptide separation and protein identification with high throughput and excellent resolution and accuracy.
2. Protein quantitation from whole cell lysates or tissue extraction analysis for biomarker discovery.
• c. Protein/peptide Mass determination
• d. PTM identification
  1. Phosporylation site
  2. Disulfide bond identification
  3. Acetylation, methylation site identification
  4. Nitrosylation site identification

• BioRad Citerion 2D-Gel
In CAPR, by using BioRad PROTEAN IEF Cell and BioRad Criterion Dodeca cell, a total of twelve 2-D gels can be run simultaneously.

• 2D DIGE
  Fluorescence 2-D difference gel electrophoresis (2D DIGE) uses molecular weight- and pI-matched, spectrally resolvable dyes (Cy2, Cy3 and Cy5) to label protein samples prior to 2-D electrophoresis. By using different dyes to separately label proteins isolated from normal and diseased tissues, multiple samples (up to three) can be co-separated and quantitated by three different sets of wavelengths. This approach overcomes many of the disadvantages of the traditional 2-D analysis by eliminating the requirement for spot matching.
  The fully optimized system (including CyDye fluorescent dyes, imager, and DeCyder Differential Analysis Software) offers increased throughput, ease of use, reproducibility, and accurate quantitation of protein expression differences. Reduced system variability enables accurate study of protein expression differences against a baseline of biological variation. Using DeCyder, the 2D DIGE platform is capable of routine detection of > 95% statistical confidence, within minutes.

Example of DIGE analysis of proteins isolated from heart left ventricles (normal, left ventricle hypertrophy and heart failure animal)

• This method facilitates a multiplexed comparison of proteins from up to 4 samples in a single experiment. The labeling reagent consists of a quantification group (N-methylpiperazine), a balance group (carbonyl), and a hydroxyl succinimide ester group that reacts with primary amines. Peptide quantification is based on the relative abundance of the four reporter ions, m/z 114.1, 115.1, 116.1 and 117.1 produced following MS/MS fragmentation of isobaric iTRAQ labeled peptide mixture. These peptides share similar chromatographic properties, allowing both peptide identification and quantification to be derived from the same MS/MS spectrum. iTRAQ chemistry offers more reliable quantification than other shotgun approaches that require highly reproducible chromatography runs. [Ref 2, 3]

• Remove the salt and detergent from protein/peptides using C18 or strong cation exchange ZipTip ®

Compared to Coomassie Blue and other staining methods, Sypro Ruby stain can detect as low as 1 ng of protein and has linear dynamic range from 1-1000 ng. It is also compatible with downstream mass spectrometric analysis.

1. Yan, L., Ge, H., Li, H., Lieber, SC., Natividad, F., Resuello, R., Kim, SJ., Akeju, S., Sun, A., Loo, K., Peppas, A., Rossi, F., Lewandowski, D., Thomas, A., Vatner, SF., Vatner, D. (2004) Gender-Specific Proteomic Alterations in Glycolytic and Mitochondrial Pathways in Aging Monkey Hearts. J. Mol. Cell. Cardiol. 37, 921-9.
2. Jun Hu, Jin Qian, Oleg Borisov, Sanqiang Pan, Yan Li, Tong Liu, Longwen Deng, Kenneth Wannemacher, Michael Kurnellas, Christa Patterson, Stella Elkabes* and Li, H* (2006). Optimized poteomic analysis of a mouse model of cerebellar dysfunction using amine-specific isobaric tags. Proteomics. 15, 4321-34. * Equal contribution.
3. Tong Liu, Veera D’mello, Longwen Deng, Jun Hu, Michael Ricardo, Sanqiang Pan, Xiaodong Lu, Scott Wadsworth, John Siekierka, Raymond Birge and Li, H. (2006) Proteome analysis of neurite outgrowth: A comparative study with nerve growth factor and an immunophilin ligand reveals divergent patterns. J. Neurosci. Method. 158, 22-9.