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A solid phase extraction-based platform for rapid phosphoproteomic analysis.

ABSTRACT: Protein phosphorylation is among the most common and intensely studied post-translational protein modification. It plays crucial roles in virtually all cellular processes and has been implicated in numerous human diseases, including cancer. Traditional biochemical and genetic methods for identifying and monitoring sites of phosphorylation are laborious and slow and in recent years have largely been replaced by mass spectrometric analysis. Improved methods for phosphopeptide enrichment coupled with faster and more sensitive mass spectrometers have led to an explosion in the size of phosphoproteomic datasets. However, wider application of these methods is limited by equipment costs and the resultant high demand for instrument time as well as by a technology gap between biologists and mass spectrometrists. Here we describe a modified two-step enrichment strategy that employs lysC digestion and step elution from self-packed strong cation exchange (SCX) solid phase extraction (SPE) columns followed by immobilized metal ion affinity chromatography (IMAC) and LC-MS/MS analysis using a hybrid LTQ Orbitrap Velos mass spectrometer. The SCX procedure does not require an HPLC system, demands little expertise, and because multiple samples can be processed in parallel, can provide a large savings of time and labor. We demonstrate this method in conjunction with stable isotope labeling to quantitate peptides harboring >8000 unique phosphorylation sites in yeast in 12h of instrument analysis time and examine the impact of enzyme choice and instrument platform.

SUBMITTER: Dephoure N

PROVIDER: S-EPMC3156280 | biostudies-literature | 2011 Aug

REPOSITORIES: biostudies-literature

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A solid phase extraction-based platform for rapid phosphoproteomic analysis.

Dephoure Noah N Gygi Steven P SP

Methods (San Diego, Calif.) 20110401 4

Protein phosphorylation is among the most common and intensely studied post-translational protein modification. It plays crucial roles in virtually all cellular processes and has been implicated in numerous human diseases, including cancer. Traditional biochemical and genetic methods for identifying and monitoring sites of phosphorylation are laborious and slow and in recent years have largely been replaced by mass spectrometric analysis. Improved methods for phosphopeptide enrichment coupled wi ...[more]

PMID: 21440633

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Project description:Cell-free DNA (cfDNA) is a liquid biopsy marker that can carry signatures (i.e., mutations) associated with certain pathological conditions. Therefore, the extraction of cfDNA from a variety of clinical samples can be an effective and minimally invasive source of markers for disease detection and subsequent management. In the oncological diseases, circulating tumor DNA (ctDNA), a cfDNA sub-class, can carry clinically actionable mutations and coupled with next generation sequencing or other mutation detection methods provide a venue for effective in vitro diagnostics. However, cfDNA mutational analyses require high quality inputs. This necessitates extraction platforms that provide high recovery over the entire ctDNA size range (50 ? 150 bp) with minimal interferences (i.e., co-extraction of genomic DNA), and high reproducibility with a simple workflow. Herein, we present a novel microfluidic solid-phase extraction device (?SPE) consisting of a plastic chip that is activated with UV/O3 to generate surface-confined carboxylic acid functionalities for the ?SPE of cfDNA. The ?SPE uses an immobilization buffer (IB) consisting of polyethylene glycol and salts that induce cfDNA condensation onto the activated plastic microfluidic surface. The ?SPE consists of an array of micropillars to increase extraction bed load (scalable to loads >700 ng of cfDNA) and can be produced at low-cost using replication-based techniques. The entire ?SPE can be fabricated in a single molding step negating the need for adding additional extraction supports to the device simplifying production and keeping device and assay cost low. The ?SPE allowed for recoveries >90% of model cfDNA fragments across a range of sizes (100-700 bp) and even the ability to extract efficiently short cfDNA fragments (50 bp, >70%). In addition, the composition of the IB allowed for reducing the interference of co-extracted genomic DNA. We demonstrated the clinical utility of the ?SPE by quantifying the levels of cfDNA in healthy donors and patients with non-small-cell lung and colorectal cancers. ?SPE extracted cfDNA from plasma samples was also subjected to a ligase detection reaction (LDR) for determining the presence of mutations in the KRAS gene for colorectal and non-small cell lung cancer patients.

Dataset Information

A solid phase extraction-based platform for rapid phosphoproteomic analysis.

Publications

A solid phase extraction-based platform for rapid phosphoproteomic analysis.

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