... The authors would like to express their sincere thanks to all the persons who kindly helped u... more ... The authors would like to express their sincere thanks to all the persons who kindly helped us with national information for this survey. Danish Building Research Institute, Aalborg University Energy and environment February 2009 Søren Aggerholm Head of department Page 7. ...
Biochimica Et Biophysica Acta Proteins Proteomics, Dec 30, 2014
ABSTRACT Human growth hormone (GH) is suspected to be widely and illegally used in sport to impro... more ABSTRACT Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes’ performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20 kDa GH proteoforms that demonstrate the administration of the synthetic hormone. Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20 kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH purification by protein precipitation. Using GH-MSIA we successfully quantified the 22 / 20 kDa GH ratio in post-exercise capillary plasma extracted from two individuals. This GH-MSIA is applicable to anti-doping and GH-related disease analysis.
Metastasis is the main cause of cancer-related deaths and remains the most significant challenge ... more Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic vs. non-metastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), L-lactate dehydrogenase A (LDHA), niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p=0.02, OS: p=0.04). CYB5R3 gene knock-down using siRNA in metastasizing cells lead to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFβ and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines were confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are available via ProteomeXchange with identifier PXD001391.
Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, ... more Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC (stable isotope labeling with amino acids in cell culture) labeling to track histone modifications and histone variants during DNA replication and across the cell cycle. We show that post-translational modifications (PTMs) are transmitted with parental histones to newly replicated DNA. Di- and trimethylation marks are diluted twofold upon DNA replication, as a consequence of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment of these marks extends over several cell generations. Together, our results reveal how hi...
Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extre... more Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extremely challenging due to the high protein concentration range, characterised by abundant proteins that suppress and mask other proteins of lower abundance. This situation is further aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers for breast cancer diagnosis. We describe a sensitive and scalable method to quantify these 5 proteins of medium and low abundance by selected reaction monitoring (SRM) LC-MS/MS analysis in blood samples. Our method requires 7μL of plasma and reaches a throughput of up to ca. 80 analyses per day. It includes an initial protein precipitation protocol optimised for extraction of low mass proteins from blood samples ...
Tumor cell adhesion, detachment, and aggregation play an important part in tumor invasion and met... more Tumor cell adhesion, detachment, and aggregation play an important part in tumor invasion and metastasis, and a variety of cell adhesion molecules have been found on tumor cells. Cell adhesion molecules, including those of the immunoglobulin superfamily, are associated with the development of metastatic behavior in cutaneous melanomas. The neural cell adhesion molecule (NCAM) belongs to this family. To investigate its possible role in the development metastatic behavior of uveal melanomas, the authors studied immunohistochemically the expression of NCAM by using an antibody that recognizes all three major isoforms of NCAM and an antibody that recognizes the HNK-1 epitope present on some isoforms of NCAM. The authors studied 32 primary uveal melanomas from 32 patients (among these, 12 were rapidly metastasizing and 16 slowly metastasizing) and 29 metastases from 19 patients. From 13 patients the primary, as well as the metastatic, tumors were available. With one exception, all HNK-1 ...
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2015
Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athlet... more Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes' performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20kDa GH proteoforms that demonstrate the administration of the synthetic hormone. Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH purification by protein precipitation. Using GH-MSIA we successfully quantified the 22/20kDa GH ratio in post-exercise capillary plasma extracted from two individuals. This GH-MSIA is applicable to anti-doping and GH-related disease analysis.
The spine journal : official journal of the North American Spine Society, 2010
Previous research has documented various psychosocial risk factors with influence on outcome in l... more Previous research has documented various psychosocial risk factors with influence on outcome in low back pain (LBP) patients, but the value of clinical predictors has been less well documented. To identify clinical and psychosocial risk factors at baseline influencing disability and pain at 1 year in LBP patients sick-listed 3 to 16 weeks, and to look for differences between nonspecific LBP and radiculopathy. Cohort study with 1-year follow-up based on a randomized clinical trial. Disability and change of disability, pain and change in pain at 1 year. In a randomized clinical study evaluating multidisciplinary versus brief intervention, 325 patients were followed for 1 year. At baseline, they completed a questionnaire and went through a clinical low back examination, including measure of forward flexion and side flexion as well as tender point examination, a method to estimate diffuse tenderness. Furthermore, degenerative changes on plain X-rays of the lumbar spine were quantified, ...
The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a wi... more The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a widely used technique for enrichment and sequencing of phosphopeptides. In the IMAC method, negatively charged phosphate groups interact with positively charged metal ions (Fe3+, Ga3+, and Al3+) and this interaction makes it possible to enrich phosphorylated peptides from rather complex peptide samples. Phosphopeptide enrichment by IMAC is sensitive and specific for peptide mixtures derived from pure proteins or simple protein mixtures. The selectivity of the IMAC method is, however, limited when working with peptide mixtures derived from highly complex samples, e.g., whole-cell extracts, where sample prefractionation is advisable. Furthermore, lowering the pH value of the sample loading buffer reduces nonspecific binding to the IMAC resin significantly, thereby improving the selectivity of IMAC for phosphopeptides. The retained phosphopeptides are released from the IMAC resin by using alkaline buffers (pH 10-11), EDTA, or phosphate-containing buffers. We have described a detailed and robust protocol for IMAC for phosphopeptide enrichment from semi-complex mixtures.
... The authors would like to express their sincere thanks to all the persons who kindly helped u... more ... The authors would like to express their sincere thanks to all the persons who kindly helped us with national information for this survey. Danish Building Research Institute, Aalborg University Energy and environment February 2009 Søren Aggerholm Head of department Page 7. ...
Biochimica Et Biophysica Acta Proteins Proteomics, Dec 30, 2014
ABSTRACT Human growth hormone (GH) is suspected to be widely and illegally used in sport to impro... more ABSTRACT Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes’ performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20 kDa GH proteoforms that demonstrate the administration of the synthetic hormone. Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20 kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH purification by protein precipitation. Using GH-MSIA we successfully quantified the 22 / 20 kDa GH ratio in post-exercise capillary plasma extracted from two individuals. This GH-MSIA is applicable to anti-doping and GH-related disease analysis.
Metastasis is the main cause of cancer-related deaths and remains the most significant challenge ... more Metastasis is the main cause of cancer-related deaths and remains the most significant challenge to management of the disease. Metastases are established through a complex multistep process involving intracellular signaling pathways. To gain insight to proteins central to specific steps in metastasis formation, we used a metastasis cell line model that allows investigation of extravasation and colonization of circulating cancer cells to lungs in mice. Using stable isotopic labeling by amino acids in cell culture and subcellular fractionation, the nuclear, cytosol and mitochondria proteomes were analyzed by LC-MS/MS, identifying a number of proteins that exhibited altered expression in isogenic metastatic vs. non-metastatic cancer cell lines, including NADH-cytochrome b5 reductase 3 (CYB5R3), L-lactate dehydrogenase A (LDHA), niemann-pick c1 protein (NPC1), and nucleolar RNA helicase 2 (NRH2). The altered expression levels were validated at the protein and transcriptional levels, and analysis of breast cancer biopsies from two cohorts of patients demonstrated a significant correlation between high CYB5R3 expression and poor disease-free and overall survival in patients with estrogen receptor-negative tumors (DFS: p=0.02, OS: p=0.04). CYB5R3 gene knock-down using siRNA in metastasizing cells lead to significantly decreased tumor burden in lungs when injected intravenously in immunodeficient mice. The cellular effects of CYB5R3 knock-down showed signaling alterations associated with extravasation, TGFβ and HIFα pathways, and apoptosis. The decreased apoptosis of CYB5R3 knock-down metastatic cancer cell lines were confirmed in functional assays. Our study reveals a central role of CYB5R3 in extravasation/colonization of cancer cells and demonstrates the ability of our quantitative, comparative proteomic approach to identify key proteins of specific important biological processes that may also prove useful as potential biomarkers of clinical relevance. MS data are available via ProteomeXchange with identifier PXD001391.
Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, ... more Epigenetic states defined by chromatin can be maintained through mitotic cell division. However, it remains unknown how histone-based information is transmitted. Here we combine nascent chromatin capture (NCC) and triple-SILAC (stable isotope labeling with amino acids in cell culture) labeling to track histone modifications and histone variants during DNA replication and across the cell cycle. We show that post-translational modifications (PTMs) are transmitted with parental histones to newly replicated DNA. Di- and trimethylation marks are diluted twofold upon DNA replication, as a consequence of new histone deposition. Importantly, within one cell cycle, all PTMs are restored. In general, new histones are modified to mirror the parental histones. However, H3K9 trimethylation (H3K9me3) and H3K27me3 are propagated by continuous modification of parental and new histones because the establishment of these marks extends over several cell generations. Together, our results reveal how hi...
Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extre... more Biomarker analysis of blood samples by liquid chromatography (LC) mass spectrometry (MS) is extremely challenging due to the high protein concentration range, characterised by abundant proteins that suppress and mask other proteins of lower abundance. This situation is further aggravated when using fast high-throughput methods, which are necessary for analysis of hundreds and thousands of samples in clinical laboratories. The blood proteins IGF1, IGF2, IBP2, IBP3 and A2GL have been proposed as indirect biomarkers for detection of GH administration and as putative biomarkers for breast cancer diagnosis. We describe a sensitive and scalable method to quantify these 5 proteins of medium and low abundance by selected reaction monitoring (SRM) LC-MS/MS analysis in blood samples. Our method requires 7μL of plasma and reaches a throughput of up to ca. 80 analyses per day. It includes an initial protein precipitation protocol optimised for extraction of low mass proteins from blood samples ...
Tumor cell adhesion, detachment, and aggregation play an important part in tumor invasion and met... more Tumor cell adhesion, detachment, and aggregation play an important part in tumor invasion and metastasis, and a variety of cell adhesion molecules have been found on tumor cells. Cell adhesion molecules, including those of the immunoglobulin superfamily, are associated with the development of metastatic behavior in cutaneous melanomas. The neural cell adhesion molecule (NCAM) belongs to this family. To investigate its possible role in the development metastatic behavior of uveal melanomas, the authors studied immunohistochemically the expression of NCAM by using an antibody that recognizes all three major isoforms of NCAM and an antibody that recognizes the HNK-1 epitope present on some isoforms of NCAM. The authors studied 32 primary uveal melanomas from 32 patients (among these, 12 were rapidly metastasizing and 16 slowly metastasizing) and 29 metastases from 19 patients. From 13 patients the primary, as well as the metastatic, tumors were available. With one exception, all HNK-1 ...
Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2015
Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athlet... more Human growth hormone (GH) is suspected to be widely and illegally used in sport to improve athletes' performance. For the detection of GH abuse, blood samples are screened for abnormal ratios between the 22 and 20kDa GH proteoforms that demonstrate the administration of the synthetic hormone. Current detection methods are based on classical immunoassays as they provide sufficient sensitivity for the detection of GH proteoforms. These antibody based methods, however, suffer from unclear selectivity and potential cross-reactivity towards similar proteins. For unambiguous GH detection, we report a Mass Spectrometry ImmunoAssay (MSIA) that first enriches GH from plasma with an antibody of relatively low specificity, and subsequently quantifies the 22 and 20kDa proteoforms by Selected Reaction Monitoring (SRM) LC-MS/MS analysis. This method proved superior to an antibody-free strategy based on GH purification by protein precipitation. Using GH-MSIA we successfully quantified the 22/20kDa GH ratio in post-exercise capillary plasma extracted from two individuals. This GH-MSIA is applicable to anti-doping and GH-related disease analysis.
The spine journal : official journal of the North American Spine Society, 2010
Previous research has documented various psychosocial risk factors with influence on outcome in l... more Previous research has documented various psychosocial risk factors with influence on outcome in low back pain (LBP) patients, but the value of clinical predictors has been less well documented. To identify clinical and psychosocial risk factors at baseline influencing disability and pain at 1 year in LBP patients sick-listed 3 to 16 weeks, and to look for differences between nonspecific LBP and radiculopathy. Cohort study with 1-year follow-up based on a randomized clinical trial. Disability and change of disability, pain and change in pain at 1 year. In a randomized clinical study evaluating multidisciplinary versus brief intervention, 325 patients were followed for 1 year. At baseline, they completed a questionnaire and went through a clinical low back examination, including measure of forward flexion and side flexion as well as tender point examination, a method to estimate diffuse tenderness. Furthermore, degenerative changes on plain X-rays of the lumbar spine were quantified, ...
The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a wi... more The combination of immobilized metal affinity chromatography (IMAC) and mass spectrometry is a widely used technique for enrichment and sequencing of phosphopeptides. In the IMAC method, negatively charged phosphate groups interact with positively charged metal ions (Fe3+, Ga3+, and Al3+) and this interaction makes it possible to enrich phosphorylated peptides from rather complex peptide samples. Phosphopeptide enrichment by IMAC is sensitive and specific for peptide mixtures derived from pure proteins or simple protein mixtures. The selectivity of the IMAC method is, however, limited when working with peptide mixtures derived from highly complex samples, e.g., whole-cell extracts, where sample prefractionation is advisable. Furthermore, lowering the pH value of the sample loading buffer reduces nonspecific binding to the IMAC resin significantly, thereby improving the selectivity of IMAC for phosphopeptides. The retained phosphopeptides are released from the IMAC resin by using alkaline buffers (pH 10-11), EDTA, or phosphate-containing buffers. We have described a detailed and robust protocol for IMAC for phosphopeptide enrichment from semi-complex mixtures.
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