Fig 1: Analysis of OPN standard by LC-MRM-MS.The assay consists of four peptides with four fragments measured for each peptide for a total of 14 MRMs. In this example, a standard solution at 200 ng/ml in buffer was analysed. The use of multiple fragments provides high specificity.
Fig 2: Quantification of OPN in plasma of metastatic breast cancer patients.Plasma OPN measurements based on the most sensitive peptide AIPVAQDLNAPSDWDSR from plasma of metastatic breast cancer patients. A) Extracted ion chromatogram (XIC) of a processed plasma sample (10 μL) following the established workflow. Plasma OPN concentration (red trace) was calculated using the ratio of the endogenous peak area over the heavy AQUA chain (blue trace). B) Bar plot representation of the OPN concentrations calculated from the plasma samples (mean ± SD). The control sample is from pooled normal human plasma.
Fig 3: Workflow and optimization of peptide recovery using addition extraction step.Diagrammatic depiction of OPN extraction procedure using single-tube extraction, digestion process (top panel). Recovery was initially low (panel A) when the manufacturer’s protocol was used but increased significantly when an additional extraction step was introduced (panel B).
Fig 4: Serial OPN measurements and tumor size.Serial tumor size measurement by CT reveals rapid tumor growth accompanied by spike in plasma OPN concentration in Patient 02 suggesting change in OPN concentrations may be used to monitor disease progression.
Fig 5: Quantification of OPN spike in normal plasma.OPN was spiked into control plasma at 400 ng/ml and processed using the optimised precipitation protocol. Quantification of the spiked protein (red traces) was performed using isotope dilution with a heavy labelled peptide (blue traces) for the four target peptides. The sum of four MRM transitions is shown.
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