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

Abstract

BackgroundTwo-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.ResultsProtein 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.ConclusionsComparison 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.

A tetrad apparatus for protein gel casting, electrophoresis, staining, and scanning techniques with dual sensors for automatic detection of gel polymerization and protein migration

“…c) Representative gel staining tray/apparatus: 5 Routine gel staining tray (Polypropylene staining tray; #S96421 series, Fishers scientific); 6 Dodeca Stainer (# 1653401, Biorad); and 7 Pierce TM Power Stainer (#22833, Thermo Fisher). d) Representative gel imaging systems: 8 Safe Image TM 2.0 Blue light transilluminator (#G6600, Thermo Fisher Scientific); 9 Transilluminator UV-white light (#Z363839, Sigma-Aldrich); and 10 Dual wavelength white light-UV transilluminator, (#UVSTS20W/L;Topac Inc., MA, USA). exposed gel during staining process which can be adjusted by simple mechanism to regulate the grip.…”

“…Electrophoresis run time is efficiently optimized with precast gels using programmable electrophoresis power supply unit due to the use of high-purity and strictly quality-controlled reagents for consistent protein separation. But many limitations with precast gels like ambiguity in market availability, inaccessibility in distant countries, and requirement of dedicated instrument sometimes affect their use [8]. However, the electrophoresis over-run cannot be avoided owing to uncertainties in electrophoresis run conditions.…”

High-level secretion of native recombinant human calreticulin in yeast

“…Tryptic peptide mass fingerprinting was carried out at the Proteomics Center in the Institute of Biochemistry of Vilnius University (Lithuania). S. cerevisiae -secreted CRT was analyzed by MALDI-TOF/TOF tandem MS/MS (mass spectrometry) as described previously [ 43 ]. Trypsin digestion of P. pastoris -secreted CRT was done according to a modified FASP protocol as described by Wisniewski et al [ 44 ].…”

Proteomic analysis to identify markers of exposure to cadmium sulphide quantum dots (CdS QDs)

“…For protein extraction for 2D-gel separation, the cells were resuspended in 300 µl of cold denaturing IEF buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 1% ampholytes (pH 3-10, GE Healthcare), 75 mM DTT (added just before use) containing a protease inhibitor cocktail (Sigma, cat # P8215). (80) For iTRAQ analysis, cells were resuspended in 250 µl extraction buffer containing 7 M urea, 2 M thiourea, 2% CHAPS, 20 mM Tris, and the protease inhibitor cocktail. Acid-washed glass beads were added in order to mechanically lyse the cells using a Thermo Savant FastPrep® Cell Disrupter (Qbiogene Inc. Carlsbad, CA).…”

“…iTRAQ labelling of the peptides from the different biological replicates was performed in the same conditions, except that the labels were inverted to reduce bias between samples.Strong cation exchange (SCX) fractionation of the iTRAQ-labelled peptidesThe dried iTRAQ-labelled peptides were resuspended in 3 ml of sample-loading buffer (10 mM ammonium formate, 20% acetonitrile, pH 3.0) and loaded on a 1-ml NuviaTMS cartridge prepared according to the manufacturer's instructions (BioRad) at 0.5 ml min − 1 using a syringe pump. After sample loading, the cartridges were washed with 5 ml of sample loading buffer at 0.5 ml min − 1 and peptide elution was performed at the same flow rate with consecutive 1.5-ml ammonium formate salt plugs at pH 3.0(30,50,80, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, and 400 mM in 20% acetonitrile). The eluent from each salt plug was dried using a SpeedVac centrifugal vacuum concentrator, and the peptides were purified on a PepClean C-18 column (Thermo Fischer Scientific, USA) prior to MS analysis.Nano-LC-MS-MS analysis of the strong cation exchange fractionsPeptide analysis was performed by reverse-phase LC-electrospray ionization-MS-MS using a nanoACQUITY Ultra Performance Liquid Chromatography system coupled to a Q-TOF mass spectrometer (Xevo Q-TOF, Waters, Milford, USA).…”