Immuno-affinity enrichment of peptides coupled to targeted, multiple reaction monitoring-mass spectrometry (immuno-MRM) enables precise quantification of peptides. peptide assays, immunoaffinity enrichment, antibody, mass spectrometry Graphical Abstract Intro Targeted proteomics has the potential for common impact in medical, biopharmaceutical, and PHA 291639 fundamental biological studies. Targeted mass spectrometry methods, like multiple reaction monitoring (MRM), have found broad applicability in targeted proteomic quantification with several advantages over traditional immunoassays, including the ability to multiplex, the relative ease of development, and inter-laboratory transferability.1,2 Peptide immunoaffinity enrichment can be coupled with MRM to improve level of sensitivity for low abundance and/or modified peptides, reduce upstream sample handling requirements, and improve throughput.3C5 The producing immuno-MRM assays have shown utility for precise, specific, reproducible, and sensitive measurements in a number of sample types and matrices.6C13 Most work to date has used affinity-purified polyclonal antibodies, although there are several examples of monoclonal antibodies8,9,14C18 and recombinant antibody fragments.19 Polyclonal antibodies are relatively inexpensive and can be generated in a few months; however, the yield of such reagents can vary20, and they are limited in supply. Once worn out, re-immunization of new animals is required to obtain more antibodies, and the immune response of different animals can be highly variable. Thus, the one-time nature of polyclonals limits their use to preliminary studies of limited capacity. Transforming reagents to renewable monoclonal antibodies has been successful14C17, but requires a considerable expense in time and money, and thus only the most well characterized and desired assays are chosen for monoclonal development. Extending the use of the polyclonal antibody resource would enable larger studies to fully evaluate assay targets and PHA 291639 also reduce the long-term cost of using the assays prior to investing in generating a monoclonal reagent. Based on the experience that antibodies can be denatured and re-natured without loss of activity21,22, affinity reagents are regenerated and reused in many applications, including commercially available columns and preparations (e.g. protein A/G columns, affinity depletion columns, etc.) and peptide enrichment using an in line bead trap device.23 Thus, we predicted that antibodies used in solution phase immuno-MRM assays (run in a 96-well format) could be reused, lowering per sample assay cost and increasing the number of samples that can be analyzed. In this study, we evaluate the removal of bound peptides and the overall performance of regenerated antibodies/beads in capturing a multiplexed panel of phosphorylated and non-modified peptides and demonstrate that analytical overall performance of the multiplexed panel is consistent for at least ten occasions of washing and re-use. The findings are significant because per PHA 291639 sample costs are reduced, and the number of samples that can be analyzed is usually greatly expanded PHA 291639 using the recycling approach. EXPERIMENTAL Reagents Dimethyl pimelimidate dihydrochloride (DMP #80490) was purchased from Sigma-Aldrich (St. Louis, MO). Trypsin (#V511X) was obtained from Promega (Madison, MI). Synthetic peptide (light) and stable isotope-labeled peptide requirements (SIS) were synthesized by New England Peptide (Gardner, MA). For stable isotope-labeled peptides, the C-terminal arginine or lysine was labeled with [13C] and [15N] labeled atoms. Peptide stock concentration was determined by amino acid analysis performed at New England Peptide. Affinity purified polyclonal antibodies were generated by Epitomics, an AbCam Organization (Burlingame, CA). Dynabeads? protein G beads (MyOne? #109150) were purchased from Invitrogen (Grand Island, NY). Cell culture The human mammary epithelial cell collection MCF10A was obtained from the ATCC (Manassas, VA) and produced at 37C and 5% CO2 in DMEM/F12 1:1 (Invitrogen #11320) supplemented with 5% horse serum (Invitrogen), 10 g/mL of insulin (Sigma #I6634), 20 ng/mL of EGF (PeproTech #AF-100-15), 0.5 mg/mL of hydrocortisone (Sigma #H-0888), 100 ng/mL of cholera toxin (Sigma #C-8052), 100 units/mL of penicillin, and 100 g/mL streptomycin. Cells were produced to 80% confluency in 100 mm plates. After incubation, growth medium was removed, and cells were rinsed in 0.25% trypsin/EDTA solution (Gibco #25200-056) and lifted off of the plates by incubation in a fresh aliquot of 0.25% trypsin/EDTA solution at 37C, 5% CO2. When cells experienced lifted from your plate, the trypsin was quenched SPP1 by the addition of 3 volumes of DMEM/F12 with 5% horse serum. Cell lysis, digestion, and desalting Cells were harvested in pre-chilled tubes, aliquots were removed for counting, and cells were spun.