1. FAQ (tech support)

A1: When doing proteome profiling, there are two methods for protein fractionation such as SDS-PAGE and strong cation exchange (SCX), both of which are usual methods in our company.

These two methods have their own advantage and shortage. For fractionating by using SDS- PAGE, protein with lower solubility such as hydrophobic protein could be dissolved and fractionated well, while protein with extremely small or large molecular weight could be lost. When using SCX, the losing of protein with extreme molecular weight could be avoided. However the protein with lower solubility could be lost.

A2. Staining methods that do not cause irreversible modification of proteins, including Coomassie Brilliant Blue, are acceptable. Conventional silver staining methods include glutaraldehyde or high concentrations of formaldehyde, which may cause irreversible modification and crosslinking of proteins. These modifications interfere with protein identification.
A3. Keratin, a protein found in hair and skin, is ubiquitous and a common contaminant of lab equipment and reaction tubes because of electrostatic charging. All glassware used for staining should be thoroughly washed with 0.2 µm filtered Milli-Q water before use. Additionally, we highly recommend a clean bench and work environment to prevent direct contamination of protein gels by dust and skin particles.
A4: Phosphatase can remove phosphate group(s) from phosphopeptides, yielding a mass shift relative to the original mass of the phosphopeptide. Depending on the number of phosphate groups, the mass shift is -80 Da (HPO3=79.966) or multiples (-80 × n Da, where n is the number of phosphate groups). Based on this principle, we can determine the phosphorylation sites.
A5. Each project is specific. Generally, we monitor 1~2 peptides per protein, and 3~5 mass transitions (precursor ion product ion) per peptide. We recommend monitoring about 300 transitions per run, resulting in detection of 30 or more proteins.
A6. The sample quantity required for MRM is based on the abundance of target protein. Generally, detection can be done for proteins with 500 attomole or above (10-18). If the abundance of the target protein is low, the amount of the sample required will be higher. Micrograms of sample are needed for sample preparation. Milligrams of sample are needed for chromatography and nanograms are needed for MS.
A7. High sensitivity, suitable for studying the low abundant protein and eliminate the noise signal; High throughput, could quantify the proteins in a large of samples;
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3.Case Study
  1. Qing, Dongjin, Yang, Zhu, Li, Mingzhe, Wong, Wai Shing, Guo, Guangyu, Liu, Shichang, Guo, Hongwei, Ning Li* (2016) Quantitative and Functional Phosphoproteomic Analysis Reveals that Ethylene Regulates Water Transport via the C-terminal Phosphorylation of Aquaporin PIP2;1 in Arabidopsis. Mol Plant. 9, 158–174.

  2. Yang, Zhu and Li, Ning (2015) Absolute quantitation of protein posttranslational modification isoform. Methods Mol Biol 1306:105-19. doi: 10.1007/978-1-4939-2648-0_8

  3. Qin Hu, Guangyu Guo, Zhu Yang, Yaojun Li, Yiji Xia and Ning Li* (2014) Stable Isotope Metabolic Labeling-Based Quantitative Thiol Redox Proteomic Analysis of Hydrogen Peroxide-treated Arabidopsis plant. Journal of Proteomics & Bioinformatics. 7: 121-133.

  4. Zhu Yang, Guangyu Guo, Manyu Zhang, Claire Y. Liu, Qin Hu, Henry Lam,Han Cheng, Yu Xue, Jiayang Li, and Ning Li * (2013) Stable Isotope Metabolic Labeling-Based Quantitative Phosphoproteomic Analysis of Arabidopsis Mutants Reveals Ethylene-Regulated Time-Dependent Phosphoproteins and Putative Substrates of CONSTITUTIVE TRIPLE RESPONSE 1 Kinase. Mol Cell Proteomics. 12, 3559-3582.

  5. Lin Zhu, Dandan Liu, Yaojun Li, and Ning Li* (2013) Functional Phosphoproteomic Analysis Reveals That a Serine-62-Phosphorylated Isoform of Ethylene Response Factor110 Is Involved in Arabidopsis Bolting. Plant Physiol. 161: 904-917.

  6. Zhu, Lin and Li, Ning*. (2013) Quantitation, networking and function of protein phosphorylation in plant cell Frontiers in plant proteomics. 3, 302. doi: 10.3389/ fpls.2012.00302

  7. Li Y, Shu Y, Peng C, Zhu L, Guo G, Li, Ning* (2012) AQUIP: Absolute Quantitation of Isoforms of Post-translationally modified proteins in transgenic organism. Mol Cell Proteomics. 2012 11: 272-285.

  8. Li, Ning (2012) Quantitative Measurement of Phosphopeptides and Proteins via Stable Isotope Labeling in Arabidopsis and Functional Phosphoproteomic Strategies. Methods Mol Biol., 2012. v. 876, 2012, p. 17-32.

  9. Guo GY and Ning Li* (2011) Relative and Accurate Measurement of Protein Abundance in (15)N Stable Isotope Labeling in Arabidopsis (SILIA). Phytochemistry. 72, 1028-1039.

  10. Li, H, Wong WS, Zhu, L, Guo, HW, Ecker, J and Ning LI* (2009) Phosphoproteomics analysis of ethylene-regulated protein phosphorylation in etiolated seedlings of Arabidopsis mutant ein2 using 2-D-separations coupled with a hybrid Q-TOF mass spectrometry. Proteomics. 9, 1646-1661.

  11. Mehta, R A, Cassol, T, Li, Ning, Ali,N, Handa, AK, and Mattoo, AK (2002) Engineered polyamine accumulation in tomato enhances phytonutrient content, juice quality and vine life. Nature Biotechnology. 20, 613-618.