Ciara A. McManus

Effect of phosphorylated hsp27 on proliferation of human endothelial and smooth muscle cells.

Recent studies have suggested a protective role of hsp27 against atherosclerosis and transplant graft vasculopathy. Here we have investigated the effects of over‐expression of wild‐type hsp27 and its phosphorylation mimics on proliferation of human endothelial cells (ECs) and smooth muscle cells (SMCs). ECs and SMCs cultured from human blood vessels or cells lines (human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC) were infected with adenovirus containing DNA from wild‐type hsp27, hyper‐phosphorylated hsp27 mimic (3D hsp27), hypo‐phosphorylated hsp27 mimic (3A hsp27) or anti‐sense hsp27, and proliferation measured over the next 5 days. Protein extracts from infected cells were subjected to proteomic analysis using 2‐D DIGE. Over‐expression of 3D hsp27 and anti‐sense hsp27 but not 3A hsp27 mimic caused significant inhibition of proliferation of ECs and SMCs. Proteomic analysis focussed on proteins that were significantly down‐regulated by the 3D hsp27 mutant. The cell cycling proteins stathmin, cofilin and ubiquitination enzymes fullfilled these criteria. 1‐D Western blots of infected human microvascular endothelial cell line and human telomerase reverse transcriptase subunit SMC confirmed down‐regulation of stathmin, cofilin and ubiquitination enzymes by 3D hsp27. The phosphorylation status of hsp27 is an important regulator of proliferation of human vascular ECs and SMCs; possibly contributing to cardiovascular protection.

A proteomic investigation of similarities between conventional and herbal antidepressant treatments

Abstract Increasing clinical evidence for the effectiveness of herbal antidepressants has led to investigations at the molecular level. Using two-dimensional gel electrophoresis, this study investigated similarities in protein expression between clomipramine, St John’s wort and a Chinese herbal formula, xiao-yao-san, often used in mood disorder treatment. HT22 cells, derived from a mouse hippocampal cell line, were treated for 24 h, and protein expression was compared with that of the untreated cells (n = 4/group). Forty-three protein spots were found to be significantly differentially expressed (P < 0.05) in more than one of the treatment groups. Twenty-nine of these were identified using mass spectrometry. The most affected proteins were those involved in the cytoskeleton and energy metabolism, and an up-regulation of vimentin by all three treatments was confirmed by Western blotting. This study provides preliminary evidence for multiple common molecular targets between conventional and alternative antidepressants, which appear to collectively affect neuronal plasticity.

CyDye immunoblotting for proteomics: co-detection of specific immunoreactive and total protein profiles.

The development of ECL‐Plex CyDye‐conjugated secondary antibodies allows the advancement of conventional Western blotting, opening up possibilities for highly sensitive and quantitative protein confirmation and identification. We report a novel proteomic method to simultaneously visualise the total protein profile as well as the specific immunodetection of an individual protein species by combining cyanine CyDye pre‐labelled proteins and antibody immunoblotting. This technique proposes to revolutionise both 2‐D immunoprobing and protein confirmation following MS analysis.

A 2-D gel reference map of the basic human heart proteome.

We have undertaken the identification of basic proteins (pH 6–11) of the human heart using 2‐DE. Tissue from the left ventricle of human heart was lysed and proteins were separated in the first dimension on pH 6–11 IPG strips using paper‐bridge loading followed by separation on 12% SDS polyacrylamide gels in the second dimension. Proteins were then identified by mass spectrometry and analysed using Proline, a proteomic data analysis platform that was developed in‐house. The proteome map contains 176 identified spots with 151 unique proteins and has been made available as part of the UCD‐2DPAGE database at http://proteomics‐portal.ucd.ie:8082. The associated mass spectrometry data have been submitted to PRIDE (Accession number ♯10098). This reference map, and the other heart reference maps available through the UCD‐2DPAGE database, will aid further proteomic studies of heart diseases such as dilated cardiomyopathy and ischaemic heart disease.

A fluorescent codetection system for immunoblotting and proteomics through ECL-Plex and CyDye labeling.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis has been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with the recently developed ECL-Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27-kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

Two‐dimensional reference map for the basic proteome of the human dorsolateral prefrontal cortex (dlPFC) of the prefrontal lobe region of the brain

We describe a 2‐DE proteomic reference map containing 227 basic proteins in the dorsolateral prefrontal cortex region of the human brain. Proteins were separated in the first dimension on pH 6–11 IPG strips using paper‐bridge loading and on 12% SDS‐PAGE in the second dimension. Proteins were subsequently identified by MS and spectra were analyzed using an in‐house proteomics data analysis platform, Proline. The 2‐DE reference map is available via the UCD 2‐DE Proteome Database (http://proteomics‐portal.ucd.ie:8082) and can also be accessed via the WORLD‐2DPAGE Portal (http://www.expasy.ch/world‐2dpage/). The associated protein identification data have been submitted to the PRIDE database (accession numbers 10018–10033). Separation of proteins in the basic region resolves more membrane associated proteins relevant to the synaptic pathology central to many neurological disorders. The 2‐DE reference map will aid with further characterisation of neurological disorders such as bipolar and schizophrenia.

Co-detection of Target and Total Protein by CyDye Labeling and Fluorescent ECL Plex Immunoblotting in a Standard Proteomics Workflow.

The qualitative and quantitative capabilities of 2-D electrophoresis and its use in widespread proteome analysis have been revolutionized over the past decade with the introduction of differential gel electrophoresis commonly known as DIGE. This highly sensitive CyDye protein labeling technique now attempts to advance conventional western blotting by the combination of DIGE labeling with ECL Plex CyDye conjugated secondary antibodies. The ability of this method to simultaneously visualize the total protein expression profile as well as the specific immunodetection of an individual protein species will significantly aid protein validation following 2-D gel separation by confirming the exact location of proteins of interest. This simple, rapid, and reproducible technique is demonstrated by 1-D and 2-D electrophoresis through the detection of the small 27 kDa heat shock protein (hsp 27), a protein known to be expressed in the human heart, from a complex cardiac protein extract.

1st Irish proteomics workshop April 17th 2008, University College Dublin, Conway Institute, Dublin, Ireland.

The workshop assembled an excellent collection of speakers from across Ireland and beyond who presented many interesting and diverse topical issues. Various proteomic applications were discussed throughout the day ranging from 2‐DE and 2‐D DIGE, to GeLC‐MS/MS, high density Protein and Antibody Arrays, with a particular focus on the importance of quantitative mass spectrometry in proteomics.