8 research outputs found
Complexity Reduction of Polymorphic Sequences (CRoPS™): A Novel Approach for Large-Scale Polymorphism Discovery in Complex Genomes
Application of single nucleotide polymorphisms (SNPs) is revolutionizing human bio-medical research. However, discovery of polymorphisms in low polymorphic species is still a challenging and costly endeavor, despite widespread availability of Sanger sequencing technology. We present CRoPS™ as a novel approach for polymorphism discovery by combining the power of reproducible genome complexity reduction of AFLP® with Genome Sequencer (GS) 20/GS FLX next-generation sequencing technology. With CRoPS, hundreds-of-thousands of sequence reads derived from complexity-reduced genome sequences of two or more samples are processed and mined for SNPs using a fully-automated bioinformatics pipeline. We show that over 75% of putative maize SNPs discovered using CRoPS are successfully converted to SNPWave® assays, confirming them to be true SNPs derived from unique (single-copy) genome sequences. By using CRoPS, polymorphism discovery will become affordable in organisms with high levels of repetitive DNA in the genome and/or low levels of polymorphism in the (breeding) germplasm without the need for prior sequence information
Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry
We show that parallel reaction monitoring
(PRM) can be used for
exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled
peptides. We have compared two different PRM approaches on a digest
of a U87 cell culture, namely, direct-PRM (tryptic digest measured
by PRM without any further sample preparation) and TiO<sub>2</sub>-PRM (tryptic digest enriched with TiO<sub>2</sub> cartridges, followed
by PRM measurement); these approaches are compared for the following
phosphorylation sites: neuroblast differentiation-associated protein
(AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type
II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor
(EGFR S1166-p). A reproducible percentage of phosphorylation could
be determined (CV 6–13%) using direct-PRM or TiO<sub>2</sub>-PRM. In addition, we tested the approaches in a cell culture experiment
in which U87 cells were deprived of serum. As a “gold standard”
we included immune precipitation of EGFR followed by PRM (IP-PRM).
For EGFR (S1166) and AHNAK (S5480) a statistical significant change
in the percentage of phosphorylation could be observed as a result
of serum deprivation; for EGFR (S1166) this change was observed for
both TiO<sub>2</sub>-PRM and IP-PRM. The presented approach has the
potential to multiplex and to quantify the ratio of phosphorylation
in a single analysis
Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry
We show that parallel reaction monitoring
(PRM) can be used for
exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled
peptides. We have compared two different PRM approaches on a digest
of a U87 cell culture, namely, direct-PRM (tryptic digest measured
by PRM without any further sample preparation) and TiO<sub>2</sub>-PRM (tryptic digest enriched with TiO<sub>2</sub> cartridges, followed
by PRM measurement); these approaches are compared for the following
phosphorylation sites: neuroblast differentiation-associated protein
(AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type
II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor
(EGFR S1166-p). A reproducible percentage of phosphorylation could
be determined (CV 6–13%) using direct-PRM or TiO<sub>2</sub>-PRM. In addition, we tested the approaches in a cell culture experiment
in which U87 cells were deprived of serum. As a “gold standard”
we included immune precipitation of EGFR followed by PRM (IP-PRM).
For EGFR (S1166) and AHNAK (S5480) a statistical significant change
in the percentage of phosphorylation could be observed as a result
of serum deprivation; for EGFR (S1166) this change was observed for
both TiO<sub>2</sub>-PRM and IP-PRM. The presented approach has the
potential to multiplex and to quantify the ratio of phosphorylation
in a single analysis
Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry
We show that parallel reaction monitoring
(PRM) can be used for
exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled
peptides. We have compared two different PRM approaches on a digest
of a U87 cell culture, namely, direct-PRM (tryptic digest measured
by PRM without any further sample preparation) and TiO<sub>2</sub>-PRM (tryptic digest enriched with TiO<sub>2</sub> cartridges, followed
by PRM measurement); these approaches are compared for the following
phosphorylation sites: neuroblast differentiation-associated protein
(AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type
II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor
(EGFR S1166-p). A reproducible percentage of phosphorylation could
be determined (CV 6–13%) using direct-PRM or TiO<sub>2</sub>-PRM. In addition, we tested the approaches in a cell culture experiment
in which U87 cells were deprived of serum. As a “gold standard”
we included immune precipitation of EGFR followed by PRM (IP-PRM).
For EGFR (S1166) and AHNAK (S5480) a statistical significant change
in the percentage of phosphorylation could be observed as a result
of serum deprivation; for EGFR (S1166) this change was observed for
both TiO<sub>2</sub>-PRM and IP-PRM. The presented approach has the
potential to multiplex and to quantify the ratio of phosphorylation
in a single analysis
Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry
We show that parallel reaction monitoring
(PRM) can be used for
exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled
peptides. We have compared two different PRM approaches on a digest
of a U87 cell culture, namely, direct-PRM (tryptic digest measured
by PRM without any further sample preparation) and TiO<sub>2</sub>-PRM (tryptic digest enriched with TiO<sub>2</sub> cartridges, followed
by PRM measurement); these approaches are compared for the following
phosphorylation sites: neuroblast differentiation-associated protein
(AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type
II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor
(EGFR S1166-p). A reproducible percentage of phosphorylation could
be determined (CV 6–13%) using direct-PRM or TiO<sub>2</sub>-PRM. In addition, we tested the approaches in a cell culture experiment
in which U87 cells were deprived of serum. As a “gold standard”
we included immune precipitation of EGFR followed by PRM (IP-PRM).
For EGFR (S1166) and AHNAK (S5480) a statistical significant change
in the percentage of phosphorylation could be observed as a result
of serum deprivation; for EGFR (S1166) this change was observed for
both TiO<sub>2</sub>-PRM and IP-PRM. The presented approach has the
potential to multiplex and to quantify the ratio of phosphorylation
in a single analysis
Determination of Site-Specific Phosphorylation Ratios in Proteins with Targeted Mass Spectrometry
We show that parallel reaction monitoring
(PRM) can be used for
exact quantification of phosphorylation ratios of proteins using stable-isotope-labeled
peptides. We have compared two different PRM approaches on a digest
of a U87 cell culture, namely, direct-PRM (tryptic digest measured
by PRM without any further sample preparation) and TiO<sub>2</sub>-PRM (tryptic digest enriched with TiO<sub>2</sub> cartridges, followed
by PRM measurement); these approaches are compared for the following
phosphorylation sites: neuroblast differentiation-associated protein
(AHNAK S5480-p), calcium/calmodulin-dependent protein kinase type
II subunit delta (CAMK2D T337-p), and epidermal growth factor receptor
(EGFR S1166-p). A reproducible percentage of phosphorylation could
be determined (CV 6–13%) using direct-PRM or TiO<sub>2</sub>-PRM. In addition, we tested the approaches in a cell culture experiment
in which U87 cells were deprived of serum. As a “gold standard”
we included immune precipitation of EGFR followed by PRM (IP-PRM).
For EGFR (S1166) and AHNAK (S5480) a statistical significant change
in the percentage of phosphorylation could be observed as a result
of serum deprivation; for EGFR (S1166) this change was observed for
both TiO<sub>2</sub>-PRM and IP-PRM. The presented approach has the
potential to multiplex and to quantify the ratio of phosphorylation
in a single analysis
Intercenter reproducibility of binary typing for Staphylococcus aureus
The reproducibility of the binary typing (BT) protocol developed for epidemiological typing of Staphylococcus aureus was analyzed in a biphasic multicenter study. In a Dutch multicenter pilot study, 10 genetically unique isolates of methicillin-resistant S. aureus (MRSA) were characterized by the BT assay as presented by van Leeuwen et al. [J. Clin. Microbiol. 2001 39 (1) 328]. The BT assay, including a standardized DNA extraction protocol was performed in duplicate in eleven medical microbiology laboratories. Two different hybridization detection procedures were applied and a prelabeled DNA sample as process control was included. Only three laboratories accurately identified all strains. Divergence in technical procedures resulted in misinterpretation due to an increasing number of faint or absent hybridization signals in combination with high background staining. The binary type of the process control was determined correctly by all participating laboratories. The feasibility of the BT protocol was related directly to the skill of the laboratory personnel. On the basis of the national study, we concluded that the DNA extraction protocol needed modification to improve DNA yield and purity. Subsequently, seven European laboratories participated in an international study to determine the reproducibility of the modified BT protocol. Each center was asked to analyze 10 DNA samples previously extracted from 10 MRSA strains (phase 1) and, additionally, to analyze 10 MRSA strains, using the standardized or their in-house DNA isolation protocol (phase 2). A prelabeled DNA process control sample was included again. The binary types of all DNA samples were identified correctly by all but one laboratories. This latter laboratory diverged from the protocol by adding an excess of labeled DNA to the hybridization mixture, resulting in a high background and, therefore, noninterpretable BT results. All centers produced identical BT results for the process control. Five of the seven centers correctly identified the binary types of all 10 MRSA strains in phase 2 of the international study. Three of these centers used their in-house DNA extraction protocol. Divergence from the standard BT protocol in the remaining two centers resulted in no interpretable BT data for the 10 MRSA strains. The study demonstrated that each center that followed the BT protocol to the letter could generate reproducible results, irrespective whether or not an in-house DNA isolation protocol was used. The current BT protocol thus represents a simple method generating robust, reproducible genotype data for S. aureus strains. © 2002 Elsevier Science B.V. All rights reserved.SCOPUS: ar.jinfo:eu-repo/semantics/publishe