NIH/NIDDK – Mass Spectrometric Assays for the Reliable and Reproducible Detection of Proteins/Peptides of Importance in Obesity Research (U01 Clinical Trial Not Allowed)

Many assays in basic and clinical science research rely exclusively on antibodies. However, there are no widely accepted guidelines or standardized methods to determine the validity of these reagents. Furthermore, many recent publications have highlighted the limitations of commercial antibodies, including failure to detect the intended target. The rigor and reproducibility of many assays commonly used by researchers in the obesity research field could be substantially improved by applying Mass Spectrometry (MS) instead of relying only on antibodies.

For several analytes and peptide/protein hormones, mass spectrometric assays that are more specific and less variable than previous measurements have been already developed, and some are already commonly used in high-throughput clinical chemistry laboratories. Despite the capability of several common mass spectrometers to analyze many analytes at once (e.g., by using Multiple Reaction Monitoring), most of these assays have not been multiplexed. There is an opportunity to combine simple and inexpensive separation methods such as solid-phase extraction (SPE) and µElution with Liquid Chromatography MS for developing multiplexed assays of interest to the obesity research community. The use of highly reproducible antibodies, such as the recombinant monoclonal antibodies, only for the enrichment of the proteins and peptides of interest in combination with MS assays for developing targeted Immuno-MRM assays, is also an option. However, when feasible it would be advantageous to develop multiplexed assays that require only simple fractionation/enrichment steps prior to the analysis by MS.

Purpose and Research Objectives

This FOA invites applications proposing the development of targeted mass spectrometric assays for proteins and peptides of primary interest to the obesity research community (e.g., Adiponectin, Leptin, Resistin, Neuropeptide Y, Alpha-melanocyte-stimulating hormone, Peptide YY, Glucagon-like peptide 1, Ghrelin, Adrenocorticotropin, Corticotropin-releasing hormone, Gastrin, Cholecystokinin, Secretin, Vasoactive intestinal peptide, gastric-inhibitory peptide, gastrin-releasing peptide, motilin, pancreatic polypeptide, RBP4, myostatin, FGF21). These assays should be validated quantifying the endogenous proteins and peptides of interest in human plasma or serum and should use isotopically labeled internal standards. These assays should be qualified demonstrating accurate quantification, precision, specificity, analytical sensitivity including Limit Of Detection (LOD), Upper Limit Of Quantification (ULOQ), and Lower Limit Of Quantification (LLOQ) (see also CPTAC guidelines). Ideally, these assays would have a small number of enrichment/fractionation steps prior to the MS analysis and would not use an antibody for enriching the analyte of interest. The goal should be to develop assays that are easily transferable to any laboratory that has appropriate expertise and access to a mass spectrometry facility. For this purpose, the use of easily accessible and inexpensive separation steps such as SPE and µElution is encouraged.

The development of a single multiplexed assay that includes all proteins and peptides of potential interest to the obesity research community would be optimal but might not be feasible due to the different chemical physical characteristics of several of the proteins/peptides of interest. Thus, the development of several multiplexed assays that target specific subsets of proteins/peptides should be considered. It should be noted that even the development of a highly reproducible and well qualified mass spectrometric assay for some specific analytes that requires special conditions is desirable.

Adiponectin, Leptin, Resistin, Neuropeptide Y, Alpha-melanocyte-stimulating hormone, Peptide YY, Glucagon-like peptide 1, Ghrelin, Adrenocorticotropin, Corticotropin-releasing hormone, Gastrin, Cholecystokinin, Secretin, Vasoactive intestinal peptide, gastric-inhibitory peptide, gastrin-releasing peptide, motilin, RBP4, myostatin, FGF21, and pancreatic polypeptide should be considered only as examples of potential proteins and peptides of interest to the obesity research community. The applicants should include in their application a list of proteins and peptides that are present in human plasma or serum and that are of potential interest to the obesity research community. The rationale used for compiling this list should be described. This could be achieved by, for example, assembling a group of experts in obesity research that express interest for well qualified assays of specific proteins and peptides.

The NIDDK will also work together with the awardees in facilitating the identification and prioritization of the proteins and peptides of interest in obesity research. For example, the NIDDK might publish a Request for Information (RFI) soliciting feedback from the scientific community for identifying proteins and peptides of primary interest to the obesity research community.

The NIDDK envisions that a successful application is likely to require an interdisciplinary team that includes expertise in clinical chemistry, obesity, and targeted mass spectrometry assay development.

Cooperative relationships and data sharing

The awardees are expected to work closely and collaboratively with the NIDDK in achieving the goals stated in the FOA.

A plan for data sharing and disseminating the successfully developed assays to the scientific community is required. This plan should include the deposition in a public portal, such as the CPTAC, of the assays characterization/validation data and of the Standard Operating Procedures (SOPs) to perform the assays.

The NIDDK will appoint an NIDDK Project Scientist and an External Scientific Panel (ESP), consisting of two to three independent expert scientists. All Principal Investigators must participate in a bimonthly teleconference with the NIDDK assigned Project Scientist. The ESP and Project Scientist will participate in an annual site visit. Awardees are required to participate in this site visit as well, as to budget for the travel and participation of the ESP.

Deadline:  March 17, 2019 (letters of intent); April 17, 2019 (full proposals)

URL:  https://grants.nih.gov/grants/guide/rfa-files/RFA-DK-19-001.html