Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae

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Abstract

Background: Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae.Results: Protein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI > 7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to detect some highly acidic proteins. The advantage of NEPHGE is higher protein capacity with good reproducibility and quality of spots at high protein load.Conclusions: Comparison of broad range (pH 3-10) gradient-based 2DE methods suggests that NEPHGE-based method is preferable over IPG (Invitrogen) 2DE method for the analysis of basic proteins. Nevertheless, the narrow range (pH 4-7) IPG technique is a method of choice for the analysis of acidic proteins. © 2013 Slibinskas et al.; licensee BioMed Central Ltd.

Figures

  • Figure 1 2DE of yeast whole cell lysates using IPG (A-C) and NEPHGE from control cells (transformed with empty vector pFGG3; A, D) and MeH cells were loaded onto IPG strips (50 μg of total protein in each strip) and values are indicated below the gels (pH 3–10 gradient was used in both m which separates acidic (on the left, pI < 7) and basic (on the right, pI > 7) p IPG-based 2D gels, their masses are indicated at the right (kDa). Arrows po spots that were identified in our previous work [17], whereas dotted arrow analysis of each indicated protein spot is presented in Table 3.
  • Figure 2 2DE of yeast whole cell lysates using IPG (A-C) and NEPHGE (D-F) based methods at high protein load. The same concentrated samples from control cells (transformed with empty vector pFGG3; A, D) and MeH (pFGG3-MeH transformant; B, E) or MeN (pFGG3-MeN; C, F) expressing cells were loaded onto IPG strips (100 μg of total protein in each strip) and NEPHGE gels (100 μg of total protein in each gel). Original scan of one of the replicas is shown for comparison (six gels were being scanned in parallel at the same time). The references are the same as in Figure 1.
  • Table 1 Comparison of spot parameters in IPG- and NEPHGE-
  • Table 2 Comparison of spot parameters in IPG- and NEPHGE-based 2DE at high protein load
  • Table 3 Quantitative analysis of differentially expressed prote pH4-7 platform (previous work, ref. [17])
  • Figure 3 Verification of proteomic results by immunoblot. SDS-PAGE (A) and Western blot (B) analysis of crude yeast lysates are shown. Lysates were prepared from galactose-induced yeast cells of S. cerevisiae AH22 strain transformed with empty vector (control, lane C) or plasmids expressing MeH (lane H) or MeN (lane N). (A) Coomassie blue-stained gel. Solid arrows indicate bands of recombinant MeH (lane H) and MeN (lane N) proteins. Lane M - prestained protein ladder with molecular weights of bands indicated at the left. (B) Western blot analysis using the same samples transferred onto nitrocellulose membrane. The blots were probed with antibodies against yeast Kar2 and Sis1 proteins. GAPDH was used as loading control.
  • Table 4 General comparison of IPG- and NEPHGE- based 2DE methods

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Slibinskas, R., Ražanskas, R., Zinkevičiute, R., & Čiplys, E. (2013). Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae. Proteome Science, 11(1). https://doi.org/10.1186/1477-5956-11-36

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