Jules A. Westbrook

A modified silver staining protocol for visualization of proteins compatible with matrix‐assisted laser desorption/ionization and electrospray ionization‐ mass spectrometry

The growing availability of genomic sequence information, together with improvements in analytical methodology, have enabled high throughput, high sensitivity protein identification. Silver staining remains the most sensitive method for visualization of proteins separated by two‐dimensional gel electrophoresis (2‐D PAGE). Several silver staining protocols have been developed which offer improved compatibility with subsequent mass spectrometric analysis. We describe a modified silver staining method that is available as a commercial kit (Silver Stain PlusOne; Amersham Pharmacia Biotech, Amersham, UK). The 2‐D patterns abtained with this modified protocol are comparable to those from other silver staining methods. Omitting the sensitizing reagent allows higher loading without saturation, which facilitates protein identification and quantitation. We show that tryptic digests of proteins visualized by the modified stain afford excellent mass spectra by both matrix‐assisted laser desorption/ionization and tandem electrospray ionization. We conclude that the modified silver staining protocol is highly compatible with subsequent mass spectrometric analysis.

Zooming-in on the proteome: very narrow-range immobilised pH gradients reveal more protein species and isoforms.

Two‐dimensional gel electrophoresis (2‐DE) enables separation of complex mixtures of proteins on a single polyacrylamide gel according to isoelectric point, molecular weight, solubility, and relative abundance. For this reason, 2‐DE together with mass spectrometry (MS) has become a key technology in proteome analysis. The introduction of immobilised pH gradients (IPGs) for isoelectric focusing of proteins affords improved reproducibility and permits full‐scale proteome analyses to be undertaken. Whilst broad‐range IPGs are useful for investigating simple proteomes (e.g. Mycoplasma genitalium) it is becoming clear that additional resolving power is needed for separating the more complex proteomes of eukaryotic organisms. The use of narrow‐range and very narrow‐range IPGs provides the means with which to dissect a complex proteome. We have compared very narrow‐range IPGs (3.5–4.5L, 4–5L, 4.5–5.5L, 5–6L, and 5.5–6.7L) with broad‐ (3–10NL) and narrow‐range IPGs (4–7L and 6–9L) for the visualisation of the human heart proteome. The superior ability of very narrow‐range IPGs to separate different protein species and isoforms, compared with 3–10NL and 4–7L 2‐D gels is demonstrated. The results are supported by MS identifications which further show that reduction of the number of comigrating protein species results in less ambiguous and more reliable database search results.

Upregulation of the Bcl-2 family of proteins in end stage heart failure

OBJECTIVES To elucidate the pattern of expression of four members of the Bcl-2 family of proteins and to correlate this with terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling (TUNEL) and DNA fragmentation. BACKGROUND Apoptosis has been implicated as a possible mechanism in the development of heart failure. However, the mechanisms involved remain unclear. METHODS We have studied the expression of four members of the Bcl-2 family that are involved in the regulation of apoptosis and analyzed DNA fragmentation as a marker of apoptosis and as a biochemical criterion to distinguish between apoptosis and necrosis in dilated cardiomyopathy (DCM), ischemic heart disease (IHD) and normal donors. RESULTS Western blot analysis and immunocytochemistry of the proapoptotic and antiapoptotic Bcl-2 proteins demonstrated significantly higher levels of all these proteins in the diseased groups compared with normal donors. Additionally, Bax was significantly higher in the IHD group compared with DCM. Terminal deoxynucleotidyl transferase [TdT]-mediated dUTP nick end labelling analysis demonstrated a significantly higher percentage of TUNEL-positive cells in the diseased groups compared with the control. Genomic DNA extraction of ventricular myocardial tissue showed no demonstrable DNA laddering for any of the groups. CONCLUSIONS The significant increases in the levels of the proapoptotic proteins Bak and Bax and the higher percentage of TUNEL-positive cells in both diseased groups suggests the presence of ongoing apoptosis. However, increases in the antiapoptotic proteins, Bcl-2 and Bcl-xL, suggest a possible concomitant, compensatory antiapoptotic mechanism in patients with heart failure.

Androgens target prohibitin to regulate proliferation of prostate cancer cells

Proteins involved in the growth response of prostate cancer cells to androgen were investigated by comparing the proteomes of LNCaP cells treated with vehicle or androgen. Whole-cell lysates were separated by two-dimensional PAGE, and HPLC-MS/MS was used to identify androgen-regulated proteins. Prohibitin, a protein with cell-cycle regulatory activity, was shown to be downregulated by 50% following androgen stimulation. Western blot and reverse transcription–PCR experiments confirmed the result and showed that regulation occurs at the level of transcription. To determine the importance of prohibitin in androgen-stimulated growth, we used transient transfection to overexpress the protein and RNA interference to knock down the protein. Subsequent FACS analysis showed that cells with reduced levels of prohibitin showed a slight but reproducible increase in the percentage of population in cell cycle, while cells with increased prohibitin levels showed a clear reduction in the percentage entering cell cycle, following dihydrotestosterone stimulation, when compared to untransfected controls. Confocal microscopy showed localization of prohibitin in the nucleus as well as the mitochondria of LNCaP cells. It therefore seems that the regulation of prohibitin is a vital part of the cellular growth response to androgen stimulation in LNCaPs and prohibitin may have a nuclear regulatory role in cell-cycle progression.

A combined radiolabelling and silver staining technique for improved visualisation, localisation, and identification of proteins separated by two‐dimensional gel electrophoresis

Two‐dimensional gel electrophoresis (2‐DE) remains the method of choice for the Separation of protein mixtures whilst mass spectrometry (MS) is rapidly becoming the premier tool for protein identification. When combined, 2‐DE and MS form the current operating paradigm for classical proteomics. One of the key challenges of proteome research is that of detecting and identifying all of the elements (proteins) of a proteome. Silver staining and radiolabelling, e.g. with 35S‐methionine ([35S]‐met), represent two sensitive methods used to visualise many of the constitutive and synthesised elements of a proteome, respectively. The latter method allows a very low total protein loading on a two‐dimensional (2‐D) gel and challenges protein identification using current MS‐based technology. Therefore, it is necessary to refer to and locate a radiolabelled spots cognate on a preparatively loaded stained gel, or Western blot, and use that protein spot for identification. Unfortunately, the images of autoradiographs and preparative gels or blots, even of the same sample, often do not correspond making it difficult to accurately locate and select spots of interest by visual comparison. We have established a technique that permits the unambiguous localisation of radiolabelled proteins on the same silver stained 2‐D gel. Protein identification of superimposed spots is described by peptide mass fingerprinting and database searching using matrix‐assisted laser desorption/ionization‐time of flight mass spectrometry and by peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time of flight MS (Q‐TOF).

Preserving the yeast proteome from sample degradation

Sample degradation is a common problem in all types of proteomic analyses as it generates protein and peptide fragments that can interfere with analytical results. An important step in preventing such artefacts is to preserve the native, intact proteome as early as possible during sample preparation prior to proteomic analysis. Using the budding yeast Saccharomyces cerevisiae, we have evaluated the effects of trichloroacetic acid (TCA) and thermal treatments prior to protein extraction as a means to minimise proteolysis. TCA precipitation is commonly used to inactivate proteases; thermal stabilisation is used to heat samples to approximately 95°C to inactivate enzyme activity. The efficacy of these methods was also compared with that of protease inhibitors and lyophilisation. Sample integrity was assessed by 2‐D PAGE and a selection of spots was identified by MS/MS. The analysis showed that TCA or thermal treatment significantly reduced the degree of degradation and that these pre‐treatment protocols were more effective than treatment with either protease inhibitors or lyophilisation. This study establishes standardised sample preparation methods for the reproducible analysis of protein patterns by 2‐D PAGE in yeast, and may also be applicable to other proteomic analyses such as gel‐free‐based quantitation methods.

Assessing the use of thermal treatment to preserve the intact proteomes of post‐mortem heart and brain tissue

Protein degradation that occurs in tissue during post‐mortem interval or sample preparation is problematic in quantitative analyses as confounding variables may arise. Ideally, such artefacts should be prevented by preserving the native proteome during sample preparation. We assessed the efficacy of thermal treatment (TT) to preserve the intact proteome of mouse heart and brain tissue in comparison to standard snap‐freezing with liquid nitrogen (LN). Tissue samples were collected, either snap frozen (LN), subjected to TT, or snap frozen followed by thermal treatment, and subsequently analysed by 2‐DE. In heart tissue, following quantitative image analysis, we observed 77 proteins that were significantly altered across the three treatment groups (ANOVA, p<0.05). Principal component and clustering analyses revealed LN and TT to be equally beneficial. These findings were confirmed by MS identification of the significantly altered proteins. In brain tissue, 189 proteins were significantly differentially expressed across the three treatment groups (ANOVA, p<0.05). Brain tissue appeared to be more responsive to TT than heart and distinct clusters of differentially expressed proteins were observed across treatments. Overall, TT of brain tissue appears to have beneficial effects on protein stabilisation during sample preparation with preservation of high‐molecular‐weight proteins and reduction in protein fragmentation.

A Combined Radiolabeling and Silver Staining Technique for Improved Visualization and Localization of Proteins on Two-Dimensional Gels

Two-dimensional gel electrophoresis (2-DGE) remains the method of choice for the separation of complex mixtures of proteins, whereas mass spectrometry (MS) is rapidly becoming the key tool for protein identification. When combined, 2-DGE and MS form the current operating paradigm for classical proteomics. One of the key challenges of proteome research is that of detecting and identifying all the elements (proteins) of a proteome. Silver staining and radiolabeling, e.g., using [35S]methionine ([35S]met), represent two sensitive methods that are used to visualize many of the constitutive and synthesized elements of a proteome, respectively. The latter method allows a very low total protein loading on a 2-D gel and, consequently, challenges protein identification using current MS-based technology. That is, identifying 2-D gel-resolved radiolabeled proteins is problematic because the amounts of radiolabeled protein present on the gel are insufficient for excision, digestion, and identification using current MS technology. Therefore, it is necessary to refer to and locate a radiolabeled spot’s equivalent on a preparatively loaded silver-or dye-stained gel (or dye-stained Western blot) and to use that protein spot for identification. Unfortunately, the images of autoradiographs and preparative gels or blots, even of the same sample, are often dissimilar, making it difficult to locate and select spots of interest accurately by visual comparison. A technique that permits the visualization and unambiguous localization of radiolabeled proteins to their silver-stained (MS-compatible) equivalents on the same 2-D gel is described in this chapter. Subsequent protein identification of superimposed spots by, e.g., peptide mass profiling using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS and peptide sequencing using tandem MS by hybrid quadrupole/orthogonal acceleration time-of-flight (Q-TOF) MS is possible.