Analytical characteristics of ICAT-LC-MS/MS for quantitative proteomics studies

AbstractRecent studies have employed isotope-coded affinity tags (ICAT) in concert with tandem mass spectrometry (MS/MS) to assess differential expression of proteins on a proteomic scale and perform unbiased quantitative comparisons between samples from different physiological or disease states. We sought to determine the performance characteristics and analytical profile of ICAT-based quantitative proteomics studies using proteins released by follicular lymphoma (FL)-derived cells as a model system

Simultaneous detection of lung fusions using a multiplex RT-PCR next generation sequencing-based approach:A multi-institutional research study

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Analytical Characteristics of Cleavable Isotope-Coded Affinity Tag-LC-Tandem Mass Spectrometry for Quantitative Proteomic Studies

Quantitative proteomic studies using cleavable isotope-coded affinity tags (cICAT) in concert with tandem mass spectrometry (MS/MS) permit unbiased comparisons between biologically distinct samples. We sought to determine the analytical characteristics of cICAT-based studies by examining the cumulative results of multiple, separate cICAT-based experiments involving human lymphoma-derived cells. We found that the number of identified proteins increased with larger numbers of fractions analyzed. The majority of proteins were identified by single peptides. Only 24 to 41% of the peptides contained cysteine residues, but 85% of the cysteine-containing peptides yielded quantification data. Approximately 28% of all identified proteins yielded quantification data, with 57% of these being differentially expressed by at least 1.5-fold. The quantification ratios of peptides for proteins with multiple quantified peptides were concordant in trend in 87% of instances. cICAT-labeled peptides identified proteins in all subcellular compartments without significant bias. Analysis of the flow-through fraction did not increase the number of peptides identified per protein. Our studies indicate that cICAT-LC-MS/MS yields quantifications primarily based on single peptides, and analysis of flow-through peptides does not contribute signifi-cantly to the results. Nevertheless, identifications based on single cICAT-labeled peptides with tryptic ends provide sufficiently reliable protein identifications and quantification information in cICAT-LC-MS/MS-based proteomic studies

Confirmation of Single Exon Deletions in MLH1 and MSH2 Using Quantitative Polymerase Chain Reaction

Deletions of one or more exons in the mismatch repair genes MLH1 and MSH2 have been implicated in a significant fraction of hereditary nonpolyposis colorectal cancer (HNPCC or Lynch syndrome). Multiplex ligation-dependent probe amplification (MLPA) detection of deletions of multiple sequential exons is widely accepted; however, there is concern over the reliability of MLPA results showing single exon deletions. Given the clinical implications of a diagnosis of Lynch syndrome, it is important to use an alternative technique to confirm single exon deletions. To verify single exon deletions, we developed a SYBR Green-based quantitative polymerase chain reaction (PCR) assay. Clinical DNA samples containing deletions in 33 of the 35 exons in MLH1 and MSH2, previously screened by MLPA, were evaluated by quantitative PCR. Gene dosage ratios were determined by both the relative standard curve method and by the 2−ΔΔCT method. Deleted exons had gene dosage ratios of 0.4 to 0.6, while nondeleted exons exhibited ratios of 0.8–1.3. We found 100% concordance between the quantitative PCR and MLPA results, including confirmation of all single exon deletions. The 2−ΔΔCT method was as accurate as using standard curves for the calculation of ratios. Single exon deletions in MLH1 and MSH2 can be verified using quantitative PCR. Assays using this method are simple to design and easy to perform, making them ideal for confirmatory testing