Development of a selected reaction monitoring mass spectrometry-based assay to detect asparaginyl endopeptidase activity in biological fluids.

Cancer Biomarkers have the capability to improve patient outcomes. They have potential applications in diagnosis, prognosis, monitoring of disease progression and measuring response to treatment. This type of information is particularly useful in the individualisation of treatment regimens. Biomarkers may take many forms but considerable effort has been made to identify and quantify proteins in biological fluids. However, a major challenge in measuring protein in biological fluids, such as plasma, is the sensitivity of the assay and the complex matrix of proteins present. Furthermore, determining the effect of proteases in disease requires measurement of their activity in biological fluids as quantification of the protein itself may not provide sufficient information. To date little progress has been made towards monitoring activity of proteases in plasma. The protease asparaginyl endopeptidase has been implicated in diseases such as breast cancer, leukaemia and dementia. Here we describe a new approach to sensitively and in a targeted fashion quantify asparaginyl endopeptidase activity in plasma using a synthetic substrate peptide protected from nonspecific hydrolysis using D-amino acids within the structure. Our selected reaction monitoring approach enabled asparaginyl endopeptidase activity to be measured in human plasma with both a high dynamic range and sensitivity. This manuscript describes a paradigm for future development of assays to measure protease activities in biological fluids as biomarkers of disease.

Extracellular matrix remodelling in response to venous hypertension: proteomics of human varicose veins.

AIMS: Extracellular matrix remodelling has been implicated in a number of vascular conditions, including venous hypertension and varicose veins. However, to date, no systematic analysis of matrix remodelling in human veins has been performed. METHODS AND RESULTS: To understand the consequences of venous hypertension, normal and varicose veins were evaluated using proteomics approaches targeting the extracellular matrix. Varicose saphenous veins removed during phlebectomy and normal saphenous veins obtained during coronary artery bypass surgery were collected for proteomics analysis. Extracellular matrix proteins were enriched from venous tissues. The proteomics analysis revealed the presence of >150 extracellular matrix proteins, of which 48 had not been previously detected in venous tissue. Extracellular matrix remodelling in varicose veins was characterized by a loss of aggrecan and several small leucine-rich proteoglycans and a compensatory increase in collagen I and laminins. Gene expression analysis of the same tissues suggested that the remodelling process associated with venous hypertension predominantly occurs at the protein rather than the transcript level. The loss of aggrecan in varicose veins was paralleled by a reduced expression of aggrecanases. Chymase and tryptase ß1 were among the up-regulated proteases. The effect of these serine proteases on the venous extracellular matrix was further explored by incubating normal saphenous veins with recombinant enzymes. Proteomics analysis revealed extensive extracellular matrix degradation after digestion with tryptase ß1. In comparison, chymase was less potent and degraded predominantly basement membrane-associated proteins. CONCLUSION: The present proteomics study provides unprecedented insights into the expression and degradation of structural and regulatory components of the vascular extracellular matrix in varicosis.

An assessment of peptide enrichment methods employing mTRAQ quantification approaches.

The human plasma peptidome has potential in biomarker discovery not least because the plasma proteome is a challenging matrix due to its complexity and dynamic range. However, methods to significantly reduce the amount of protein present in plasma while retaining the less abundant peptides present in plasma samples has been a major issue. Here, we present a novel strategy which has been employed to assess the effectiveness of removing interfering proteins while retaining peptides of interest. To monitor peptide retention, a spiked in digested protein, in this case a synthetic QconCAT protein, was employed. This enabled a variety of target analytes (peptides) to be monitored for their retention in liquid phase, providing a broader picture of peptide loss from each method assessed. The incorporation of mTRAQ labeling allowed the presence of each peptide to be monitored, and accurate peptide losses to be determined in a Selected Reaction Monitoring (SRM) assay, thus, enabling an objective semiquantitative conclusion to be drawn regarding the suitability of each method for protein removal and peptide retention. We also assessed a range of methods for retaining nontryptic peptides in a plasma peptidomics workflow. From these data, we determined an optimal workflow for removing intact protein, while retaining peptides for MS-based analyses.

BCR/ABL modulates protein phosphorylation associated with the etoposide-induced DNA damage response.

BCR/ABL expression is the key characteristic of chronic myeloid leukaemia (CML). The progression of CML is associated with genomic instability. Systematic analysis of DNA damage signalling in the presence of BCR/ABL thus offers opportunities to identify mechanisms of leukaemic progression. We therefore undertook a quantitative phosphoproteomics study to test whether BCR/ABL expression could globally affect the response to genotoxic stress signalling. Etoposide-induced DNA damage was chosen and the concentration and time of exposure determined such that apoptosis was not associated with the study. More than 1100 phosphoentities were identified. BCR/ABL was shown to significantly alter the response to etoposide in many cases. These include sites on MDC1, a key component of DNA repair, and Hemogen. Hemogen is a transcriptional target of HOXB4 and GATA1, two transcription factors involved in haemopoietic development, and is overexpressed in acute myeloid leukaemia. To validate Hemogen phosphorylation, absolute quantification using an isotopomeric standard and selected reaction monitoring was performed. This revealed a strong correlation with isobaric tagging data and offering quantification at about 10 fmol per million cells. Furthermore we found that multiple protein phosphorylation changes mediated by BCR/ABL were connected to the increased activation of NFκB, a key survival transcription factor, after etoposide exposure.