A semi-virtual two dimensional gel electrophoresis: IF–ESI LC-MS/MS

Graphical abstract


Method details
High resolution separation of complex protein samples has allowed the 2DE technique to be a leading approach in the study of proteomics since its introduction in 1975 by O'Farrell [1]. Separation is a main function of 2DE and a first step towards obtaining the necessary information about proteins. Together with large scale identification methods, like mass spectrometry, 2DE was a work horse of proteomics for a long time [2,3]. As high tech mass spectrometry can execute some 2DE functions, it is possible to perform 2D analysis in non-classical way. One example is so called "Virtual 2-D Gel Electrophoresis" [4,5], where isoelectrofocusing in IPG strips is used as a first dimension, but the conventional second separation by SDS-PAGE is replaced by MALDI-MS (matrix assisted laser desorption ionization-mass spectrometry). Usage of ESI LC-MS/MS, instead of MALDI-MS, in combination with classical electrophoretic methods allows the revelation of more information; especially when very complex samples like mammalian cell extracts are analyzed [6][7][8][9]. In particular, in comparison to the classical shotgun analysis semi-virtual 2DE allows the acquisition of not just protein profiles but also proteoform profiles.

Chemicals and materials
All reagents used were obtained from "Sigma-Aldrich" (St. Louis, MO, USA), unless another manufacturer is specified. The remaining reagents were obtained from the following companies: 4. An IPG strip (Immobiline DryStrip 3-11 NL, 24 cm, 'GE Healthcare') was placed onto the sample, gel side down, and was passively rehydrated for 5 h at 4 C 5. Proteins were separated by IEF using IPGphor (GE Healthcare), which was programmed as follows: first step -500 V, 8 h, second stepgradient to 1000 V, 1 h, third stepgradient to 10000 V, 3 h, fourth step -10000 V, 4 h, temperature 20 C, thereafter maintained at the voltage 500 V 6. After IEF, the strip was cut into 48 equal sections using scissors, and each section was transferred to Eppendorf tube.
7. For complete reduction, 300 mL of 3 mM DTT, 100 mM ammonium bicarbonate was added to each sample.
[ ( F i g . _ 1 ) T D $ F I G ] 9. For alkylation 20 mL of 100 mM iodoacetamide (IAM) were added to the same tube. 10. Incubated in the dark at room temperature for 15 min.
13. Liquid was removed and replaced with 150 mL of 100% acetonitrile.
14. Incubated for 10 min or until all gel slices are white. 15. Liquid was removed and gel was desiccated in Speed Vac (Thermo Scientific) for 5 min. 16. For digestion, 0.1 mg/mL stock trypsin was diluted 1:10 into 25 mM ammonium bicarbonate.
17. 100 mL of diluted trypsin was added into each tube. 26. Tryptic peptides were separated using reversed phase nano-LC gradients and analyzed online by Orbitrap Q-Exactive Plus mass spectrometer. 27. Protein identification and relative quantification were performed using Mascot "2.4.1" (Matrix Science) and emPAI. 28. A table with information about all detected protein proteoforms was built (Table 1). All proteins detected in the same section were given the pI of this section. Accordingly, the same proteins detected in different sections were considered as different proteoforms. 29. Based on information from NeXtProt about Mw of detected proteins, a semi-virtual 2DE map was constructed. An example of such a map for [ 3 _ T D $ D I F F ] [ 1 _ T D $ D I F F ] major proteins expressed in HepG2 cells is shown in Fig. 1.

Method validation
The method was validated in our laboratory using several cell lines. After protein identification, a list of 32,652 proteoforms (products of 3962 genes) was generated from HepG2 cells. Only 1242 proteins were identified in parallel shotgun experiment.
Lanner is acknowledged for the editing assistance and Igor Vakhrushevfor providing HepG2 cells. We acknowledge the IBMC "Human Proteome" Core Facility for assistance with the generation of mass-spectrometry data.
All the authors declare that they have no conflict of interest.