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Selective enrichment of newly synthesized proteins for quantitative secretome analysis
Jeroen Krijgsveld, Principal Investigator, EMBL
Cell-secreted proteins are crucial mediators of intercellular communication. They are fundamental in maintaining homeostasis e.g. in metabolism and immunity, and in conferring properties underlying various diseases, e.g. cancer metastasis. Here we introduce a novel approach for quantitative secretome analysis, allowing cells to be grown in the presence of serum, while preserving the capacity to identify a large repertoire of low-abundant secreted proteins.
The key aspect of our approach is the capacity to selectively enrich for secreted proteins from culture media by the metabolic incorporation of azidohomoalanine (AHA), an azide-bearing analogue of methionine, into proteins, followed by their covalent coupling to an alkyne-activated resin via click-chemistry. This was combined with pulse labeling with SILAC amino acids (pSILAC), using mass spectrometry for identification and quantification of captured proteins.
We demonstrate the successful application of the method by 1) comparing secretomes of cancer cells, 2) determining the effect of stress (serum starvation) on secretome composition and 3) determining secretion kinetics upon cellular stimulation. In each of these cases we quantified many low-abundant proteins of important families such as cytokines, chemokines, receptors, hormones and proteases.
The method gives access to a poorly-covered part of the proteome, with a strong potential in biomarker discovery.
Proteomic analysis of exosomes from urine in the search of new targets and biomarkers of diabetic nephropathy
Gloria Alvarez-Llamas, Senior Investigator, IIS-Fundacion Jimenez Diaz
Diabetic nephropathy (DN) is the leading cause of renal failure and it is detected once proteinuria is established. The aim of this study is to investigate the proteome of the urinary exosome related to DN in the search for disease biomarkers. Exosome isolation was optimized and confirmed by electron microscopy and Western Blot. To avoid disturbance from high abundance proteins (albumin and IgG), co-present in the exosomal fraction of patients, depletion strategies were set-up to equalize them in patients and healthy individuals, which otherwise would mask minor proteins candidate for biomarkers. Differential label free LC-MS/MS analysis was carried out comparing the urinary exosome proteome of 5 healthy donors and 5 DN patients. A total of 562 proteins were identified, 200 of which have been previously described in urinary exosomes, 94 in exosomes from different sources and a sub-set of 268 proteins which were not previously described in the exosome fraction. Four proteins were found to be significantly varied in ND patients, in view of the number of unique identified peptides in every sample which is related to protein abundance. Biomarkers candidates are validated in a new cohort or samples by Western Blot and Chip-LC-MS in SRM mode.
Multi-analyte biomarker research in early cancer detection with sensitive proximity probing assays
Simon Fredriksson, CEO, Olink
In our search for biomarkers to improve cancer detection, retrospective analysis of biobanked material finds us needing highly multiplexed and sensitive protein quantification metods. The proximity probing assay utilizes oligonucleotide-linked antibody pairs, which in a homogeneous reaction converts proteins in to real-time PCR amplicons. This very sensitive assay can quantify putative biomarkers in 1 micro litre sample volumes from sources such as serum, plasma, CSF, or tissue lysates. The assay can be performed in multiplex with low femto molar sensitivity by converting multiple putative biomarkers into identification encoded sequences, quantified in high throughput microfluidic qPCR. The assay is especially suited for screening bio-banked materials for novel biomarkers.
Data from multiplexed proximity probing in a colorectal cancer detection biomarker study will be presented quantifying 150 proteins in 2 micro litres of plasma with 5 log linear range with sensitivities down to low femto Molar. This study of 350 samples using a multivariate signature of these protein is one of the largest studies of low abundance putative biomarkers performed in one and the same sample collection.
Selective enrichment of newly synthesized proteins for quantitative secretome analysis
Jeroen Krijgsveld, Principal Investigator, EMBL
Cell-secreted proteins are crucial mediators of intercellular communication. They are fundamental in maintaining homeostasis e.g. in metabolism and immunity, and in conferring properties underlying various diseases, e.g. cancer metastasis. Here we introduce a novel approach for quantitative secretome analysis, allowing cells to be grown in the presence of serum, while preserving the capacity to identify a large repertoire of low-abundant secreted proteins.
The key aspect of our approach is the capacity to selectively enrich for secreted proteins from culture media by the metabolic incorporation of azidohomoalanine (AHA), an azide-bearing analogue of methionine, into proteins, followed by their covalent coupling to an alkyne-activated resin via click-chemistry. This was combined with pulse labeling with SILAC amino acids (pSILAC), using mass spectrometry for identification and quantification of captured proteins.
We demonstrate the successful application of the method by 1) comparing secretomes of cancer cells, 2) determining the effect of stress (serum starvation) on secretome composition and 3) determining secretion kinetics upon cellular stimulation. In each of these cases we quantified many low-abundant proteins of important families such as cytokines, chemokines, receptors, hormones and proteases.
The method gives access to a poorly-covered part of the proteome, with a strong potential in biomarker discovery.
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