Systems Biology · Quiz Five

Separation and LC-MS

1. What is 2D PAGE? What is the first dimension and what is the second dimension? Can you revise the order? Please explain.

Two-dimensional gel electrophoresis, abbreviated as 2-DE or 2-D electrophoresis, is a form of gel electrophoresis commonly used to analyze proteins. Mixtures of proteins are separated by two properties in two dimensions on 2D gels.
isoelectric point (pI); molecular weight (MW)
No. IEF must be performed first in order to make protein migrate according to their MW at the second stage instead of charge; pI can be predicted.

2. What are purposes of SDS in the denaturing gel for protein separation?

Shield the original charge and shape of the protein and make it migrate strictly according to molecular mass.

3. Please list at least three major disadvantages of using gel-based separation for proteomic research.

  • Incomplete peptides recovery from gel
  • Manual handling and time consuming
  • Proteins with extreme values of pl or MW are excluded
  • 溶解度问题

4. For peptide HTSGPPFKTNR, after demethylation with D13CDO and NaBH3CN, what is the mass difference between heavy and light peptides?


5. In the reverse phase chromatograph, peptide samples are usually dissolved in 5% acetonitrile in water to load on the RPLC column. Peptides are eluted off the column when increasing organic solvent in the mobile phase. There is a new type of LC called HILIC based on hydrophilic-hydrophilic interaction. Please explain how peptides are dissolved and loaded on the HILIC column and how peptides are eluted off.

HILIC is characterized by the use of a hydrophilic stationary phase and a hydrophobic mobile phase. Separation is mainly accomplished by the hydrophilic interaction between the stationary phase and the solute. The hydrophilic stationary phase can enrich the water molecules in the buffer to form an aqueous layer on the surface of the stationary phase. The hydrophilic solute is distributed in this aqueous layer and the hydrophobic eluent. In HILIC, the mobile phase is an organic solvent containing a small amount of water / polar solvent, the organic component is a weak eluent, and the aqueous phase is a strong eluent. The final separation is through electrostatic interaction between the eluent and the stationary phase or hydrogen bonding with the stationary phase to compete for the binding site of the hydrophilic solute and the stationary phase, so that the solute is eluted.

6. Please explain why nanoflow LC-MS is much more sensitive than regular and micro flow LC-MS.

In nanoflow LC (nanoLC) chromatographic separations are performed using flow rates in the range of low nanoliter per minute, which result in high analytical sensitivity due to the large concentration efficiency afforded by this type of chromatography.

7. What are the advantages and disadvantages of UHPLC?


  • Increased resolution (higher peak capacity)
  • Increased sensitivity
  • UHPLC use much less of solvents
  • In proteomics, increased capacity with longer column


  • Not suitable for dirty or unfiltered samples
  • High pressure environment


8. Using your own words please explain why SRM/MRM is more sensitive than regular MS/MS. What is transition in SRM/MRM? Why you preferably choose Y ions in tryptic peptides for the measurement?

Ignoring all other ions that flow into the mass spectrometer, this technique gains sensitivity while maintaining accuracy.
The specific precursor ion selected by the first mass analyzer is fragmented by CAR to generate the product ion with a specific m/z value.
It’s easier to get large fragment ion with strong signal.

9. Why PRM is more specific than MRM? What instrument can perform PRM?

In PRM all product ions are monitored providing high confidence of peptide identification. The high resolution mass analyzer increases specificity (narrower mass window) compared to a MRM. 
The PRM assay is performed in a high resolution and high mass accuracy mode on a mass spectrometerMS-MS(such as TOF, FT-ICR and Orbitrap).

10. What are advantages and challenges of DIA compared with DDA?


  • Does not require prior knowledge of the protein composition of the sample
  • Less biased as all peptides are included in the analysis
  • Allows greater temporal resolution, which is an advantage for certain analyses (e.g., looking at changes in protein expression or post-translational modifications over time within the same tissue)
  • Can quantify proteins in complex mixtures over a large dynamic range, thereby overcoming the challenge of undersampling when using DDA
  • Offers higher precision and better reproducibility than DDA
  • Best approach for discovery proteomics as no assumptions are made (e.g., comparison of large sample cohorts to see differences in protein expression)
  • DIA data can be retrospectively analyzed with an improved algorithm to generate even better results


  • Amount of data generated is much larger, so can place a high demand on computational resources
  • Data analysis is challenging because of the multiplexed nature of the MS2 spectra
  • The robust database-based search methods used for DDA cannot be applied directly
  • Further improvements are required in the tools and software used to deconvolute the complex spectra produced
  • De novo search algorithms used in DDA are usually iterative and may not always converge around the same answers
  • Fragment ions in MS2 spectra cannot be traced back to their precursors as they can potentially result from multiple precursor ions
  • Tends to be more expensive than DDA
  • In terms of quantification, DIA has lower sensitivity than DDA as the complete spectrum must be scanned, reducing the acquisition time per data point
  • De novo search algorithms are not as good at quantification as database search algorithms, which can also reduce quantification sensitivity
  • Algorithms need to control the false discovery rate among the identified peptides while also identifying as many of the real peptides as possible