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Co-affinity purification mass spectrometry (CoAP-MS) is a highly effective method for identifying protein complexes from a biological sample and inferring important interactions, but the impact of the solid support is usually not considered in design of such experiments. Affinity purification (AP) experiments typically utilize a bait protein expressing a peptide tag such as FLAG, c-Myc, HA or V5 and high affinity antibodies to these peptide sequences to facilitate isolation of a bait protein to co-purify interacting proteins. We observed significant variability for isolation of tagged bait proteins between Protein A/G Agarose, Protein G Dynabeads, and AminoLink resins. While previous research identified the importance of tag sequence and their location, crosslinking procedures, reagents, dilution, and detergent concentrations, the effect of the resin itself has not been considered. Our data suggest the type of solid support is important and, under the conditions of our experiments, AminoLink resin provided a more robust solid-support platform for AP-MS.

Profiling changes in the concentration of functionally related peptide hormones is critical to understanding the etiology of many diseases and therapies. We present novel data using nano liquid chromatography–mass spectrometry (LC–MS) to simultaneously measure a select group of vasoactive peptides (angiotensin, bradykinin, and related hormones) in 50-μl plasma samples, enabling repeated sampling in rodent models. By chromatographically resolving target peptides and using multiple reaction monitoring to enhance MS sensitivity, linear responses down to 10 −17 mol were achieved. Purification of plasma peptides by either methanol precipitation or off-line high-performance liquid chromatography (HPLC) fractionation enabled the detection of endogenous peptides and revealed approaches for enhancing recovery. As proof of principle, seven vasoactive peptides were profiled before, during, and after acute angiotensin-converting enzyme (ACE) inhibition in an anesthetized rat. Of note was an apparent 10-fold increase in vasodilatory bradykinin that reversed after drug infusion but relatively minor changes in angiotensin II levels. Targeted MS analysis used to profile functionally related peptides or other analytes will greatly enhance our ability to define the sequence of events regulating complex and dynamic physiological processes.