General Strategy for Using DIA to Expedite Targeted Assay Development

We use narrow-window DIA to identify peptides for use as surrogate peptides in targeted assays. Our experiments identify the peptides that provide the sensitive, linear, and precise quantification of the analyte. The first step in the process is to digest a sample that has the protein of interest in high enough abundance to yield useful data. For the higher abundance proteins in serum and plasma, the specimen of choice is unadulterated human sample. For other proteins, such as insulin and c-peptide, a protein precipitation step enriches lower molecular weight proteins allowing for peptide selection. However, for some other proteins that are of lower abundance in serum and plasma, purified proteins will be needed to select peptides. Purified proteins can come from recombinant sources, or may be purified from a source other than plasma (e.g., retinol binding protein can be purified from urine, thyroglobulin can be purified from thyroid glands, and type III collagen and its fragments can be purified from ascites). Narrow-window DIA will be used (i.e., multiple injections using a 90-min gradient liquid chromatography method and MS instrument methods that have overlapping 4 m/z windows) to identify and quantify (as measured by peak area) possible surrogate peptides in a variety of different experimental conditions. These experimental conditions will help identify peptides that have reasonable lower limits of quantification, appear to be precise, are stable, are liberated quickly, and are linear. As for specificity, analytical sensitivity, and accuracy, a full assessment of the precision of each surrogate peptide in a production workflow will be performed during validation.

New! Poster from ASMS 2019 Describing the Approach