Microscale solution IEF combined with 2‐D DIGE substantially enhances analysis depth of complex proteomes such as mammalian cell and tissue extracts

Abstract

Current gel-based protein profiling methods such as 2-DE and fluorescent 2-D difference in gel electrophoresis (DIGE) evaluate small portions of complex proteomes. Hence, sample prefractionation is essential for more comprehensive proteome coverage and detection of low-abundant proteins. In this study, we describe the combination of DIGE labeling with microscale solution IEF (MicroSol-IEF) fractionation and subsequent analysis on slightly overlapping narrow pH range 2-D gels. By fluorescently tagging and mixing samples and controls prior to prefractionation, complications resulting from minor run-to-run variations during MicroSol-IEF separations of multiple samples are avoided. This greatly improves the reliability of quantitative comparisons. To illustrate its utility, this 3-D DIGE strategy was applied to analysis of human melanoma cells and mouse lung tissue extracts. Approximately 1000 reproducible spots can be obtained from narrow range 2-D gels of individual MicroSol-IEF fractions, and approximately 6000 spots can be obtained from entire proteomes. Quantitative changes in closely related samples could be more reliably detected and the method has a greatly increased capacity to distinguish between closely related protein isoforms. Thus the 3-D DIGE strategy produces a powerful method for more comprehensive and more reliable quantitative comparisons of protein profiles of very complex proteomes.

A systems biology analysis of metastatic melanoma using in‐depth three‐dimensional protein profiling

“…Each unique sequence is highlighted with a yellow background in Supplemental Table 2. Consistent with our previous study, these results show that, in some cases, the 3-D DIGE method can effectively separate and distinguish between multiple closely-related splice forms [12]. While many of the observed changes in spot intensities are likely due to changes in abundance of the identified protein, some of the detected changes are most likely due to changes in levels of PTMs that result in detectable shifts of either the pI or apparent size of the protein, such as phosphorylation or glycosylation (see Supplemental Table 2).…”

“…Typically, a Cy3-and a Cy5-labeled sample were mixed and prefractionated using MicroSol-IEF, essentially as previously described [12], using partition membrane disks (ZOOM disks) with pH values of 3.0, 4.6, 5.4, 6.2, 7.0, 9.1, and 12 in a ZOOM IEF Fractionator (Invitrogen, Carlsbad, CA). A total of 1.5 mg of mixed Cy-labeled proteins was separated using maximum limits of 1 mA and 1 W until a maximum voltage of 1200 V was reached.…”

“…Fractions then were applied to the desired narrow pH range Immobiline DryStrips (GE Healthcare), using either cup-loading or in-gel rehydration following the manufacturer's protocol in an IPGphor (GE Healthcare) using five phases of stepped voltages from 200 to 8000 V, with total focusing of 60 kV·h. To improve throughput, 18-cm narrow pH range IPG strips were trimmed to fit Criterion gel cassettes (Bio-Rad, Hercules, CA) by removing the pH range of the IPG strip outside the pH range of the MicroSol-IEF fraction, as shown in Table 1 and as previously described [12]. The trimmed IPG strips were run on 10% Tris-Tricine gels at 165 mA constant current with cooling.…”

“…In the current study, an in-depth comparative proteome analysis of two genetically, veryclosely-related melanoma cell lines was performed using 3-D difference in gel electrophoresis (3-D DIGE), which we recently showed could substantially enhance proteome coverage when analyzing melanoma cell or lung tissue extracts [12]. We performed a systematic comparison of the primary human melanoma cell line (WM793) with a highly metastatic variant of the WM793 cell line that was selected in athymic mice [13].…”

Bactericidal properties of the antimicrobial peptide Ib‐AMP4 from Impatiens balsamina produced as a recombinant fusion‐protein in Escherichia coli

“…Samples for the MS/MS analysis were prepared as described previously 21. Briefly, protein spots were excised and destained by incubation in 30 mM potassium ferricyanide and 65 mM sodium thiosulfate for 10 min.…”